JAK-STAT Signaling in

Diseases

JAK-STAT Signaling in

Diseases

Edited by

Ritobrata Goswami

CRC Press
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Contents

Preface........................................................................................................................ vii

Editor.......................................................................................................................... ix

Contributors ................................................................................................................. xi

1. Regulation of Cytokine Signaling by the JAK-STAT Pathway ........................................... 1

Nicolette Nadene Houreld

2. The Structure-Function Bonhomie of JAK-STAT Molecules.............................................. 9

Ritobrata Goswami

3. MicroRNA-Mediated Regulation of JAK-STAT Signaling in Non-Cancerous

Human Diseases ..................................................................................................... 35

Chandra S. Boosani, Wanlin Jiang, Taylor Burke, and Devendra K. Agrawal

4. JAK-STAT Signaling in Asthma and Allergic Airway Inflammation.................................. 49

Amina Abdul Qayum, Tristan Hayes and Mark H. Kaplan

5. Role of JAK-STAT Signaling in Atopic Dermatitis........................................................ 69

Radomir M. Slominski and Matthew J. Turner

6. JAK-STAT Signaling Pathway and Gliosis in Neuroinflammatory Diseases........................ 83

Han-Chung Lee, Kai-Leng Tan, Pike-See Cheah and King-Hwa Ling

7. JAK-STAT Signaling in Cardiovascular Disease ..........................................................103

George W. Booz, Raffaele Altara and Sean P. Didion

8. Diabetes and Obesity: Abnormal JAK-STAT Signaling .................................................123

Marcia J. Abbott

9. JAK-STAT Signaling in Liver Fibrosis.......................................................................143

Marwa K. Ibrahim and Noha G. Bader El Din

10. Renal Disorders: Involvement of JAK-STAT Pathway...................................................159

Yuji Nozaki

11. JAK-STAT Signaling in Hematologic Malignancies .....................................................177

Thomas Pincez and Thai Hoa Tran

12. Aberrant JAK-STAT Signaling in Hematopoietic Malignancies ......................................225

Parvis Sadjadian

v
vi Contents

13. Immunodeficiency: Consequences of Mutations in JAK-STAT Signaling ...........................241

Daniel Silberger and Duy Pham

14. Targeting JAK-STAT Pathway for Various Inflammatory Diseases and

Viral Infections ......................................................................................................257

Christina Gavegnano and Raymond F. Schinazi

Index .................................................................................................................. 269

Preface

JAK-STAT (Janus kinase-Signal Transducers and Activators of Transcription) signaling is one of

the few conserved signaling cascades transmitting multiple signals required for homeostasis in
animals and humans. In JAK-STAT signaling, extracellular ligand binding to its cognate receptor
leads to pathway activation and signal transduction. This pathway is critical for hematopoiesis,
immune system development, and other events. Activation of JAK leads to induction of cell
proliferation, cell migration, differentiation, and apoptosis. Mutations in JAK-STAT pathway can
either lead to inflammatory diseases or may impede regular homeostasis.
JAK-STAT pathways can cross-talk with other signaling pathways. JAK-STAT pathway has
been targeted to develop drugs to downregulate the immune response. Few of the JAK inhibitors
are in Phase III of clinical trial and have been approved by FDA. Tofacitinib that targets JAK3
has shown its effect to treat rheumatoid arthritis, psoriasis. OPB-51602, which targets STAT3, is
in phase I to treat nasopharyngeal carcinoma. JAK inhibitors have an advantage over cytokine
receptor blocking drugs in that they that can be taken orally as they are small molecule drugs.
JAK-STAT pathway is a very well-studied signaling pathway. The book has consolidated
information on the role of JAK-STAT signaling in autoimmune and inflammatory diseases
including asthma, atopic dermatitis, hepatic fibrosis, diabetes and obesity, cancer, cardiovascular
disease, immunodeficiency, renal disorders, neuro-inflammatory disorders, among others. This
book is unique as it encompasses different aspects of JAK-STAT signaling and how dysregulation
of this signaling pathway is associated with various disorders that are prevalent worldwide leading
to morbidity and mortality associated with increased expenditure on healthcare and loss of
manpower. This book has an update on the therapeutic implication of JAK-STAT inhibitors
different phases of clinical trial. I sincerely believe this book will stimulate interest in graduate
students, academicians and scientific community. This book will be good reference point for
industry professionals who are involved in translational research leading to develop products to
various diseases. Positive feedback of this book from the readers will allow us to expand the
disease repertoire that is associated with dysregulated JAK-STAT signaling.
Editor

Ritobrata Goswami, PhD, is presently working as an Assistant Professor in the

School of Bioscience, IIT Kharagpur since 2016. Prior to his present affiliation,
Dr Goswami was associated with the Division of Biological and Life Sciences,
Ahmedabad University (2014-16). He received his bachelor (2005) and mas­
ters’ degree (2007) in Biotechnology from West Bengal University of Tech­
nology and Birla Institute of Technology & Science, Pilani; respectively.
Dr Goswami obtained his PhD at Indiana University, Indianapolis, USA
(2012) followed by post-doctoral training at Icahn School of Medicine at
Mount Sinai, New York, USA (2012-14). He is a life member of the Indian
Immunology Society. Dr Goswami has extramural projects funded by the Government of India.
Dr Goswami’s research interests include the role of nutrients and hormones in the development
and function of T helper cells to regulate inflammatory disorders. He is keen on identifying
a regulating network of transcription factors involved in autoimmune and inflammatory disorders so
that better therapeutics can be developed.

ix
Contributors

Marcia J. Abbott Pike-See Cheah

Department of Health Sciences Department of Human Anatomy, Faculty of
Crean College of Health and Behavioral Medicine and Health Sciences
Sciences, Chapman University Universiti Putra Malaysia
Orange, California Seri Kembangan, Malaysia

Devendra K. Agrawal Sean P. Didion

Department of Clinical and Translational Science Department of Pharmacology and Toxicology
Creighton University School of Medicine The University of Mississippi Medical Center
Omaha, Nebraska Jackson, MS

Raffaele Altara Christina Gavegnano

Institute for Experimental Medical Research Center for AIDS Research, Laboratory of
Oslo University Hospital and University of Biochemical Pharmacology, Department of
Oslo Pediatrics
Oslo, Norway Emory University
Atlanta, GA
Noha G. Bader El Din
Department of Microbial Biotechnology Ritobrata Goswami
Genetic Engineering and Biotechnology School of Bioscience
Division Indian Institute of Technology Kharagpur
National Research Centre Kharagpur, India
Dokki, Egypt
Tristan Hayes
Chandra S. Boosani Department of Pediatrics and Herman B Wells
Department of Clinical and Translational Center for Pediatric Research
Science Indiana University School of Medicine
Creighton University School of Medicine Indianapolis, Indiana
Omaha, Nebraska
Nicolette Nadene Houreld
George W. Booz Laser Research Centre, Faculty of
Department of Pharmacology and Toxicology Health Sciences
The University of Mississippi University of Johannesburg
Medical Center Johannesburg, South Africa
Jackson, MS
Marwa K. Ibrahim
Taylor Burke Department of Microbial Biotechnology,
Department of Clinical and Translational Genetic Engineering and Biotechnology
Science Division
Creighton University School of Medicine National Research Centre
Omaha, Nebraska Dokki, Egypt

xi
xii Contributors

Wanlin Jiang Parvis Sadjadian

Department of Clinical and Translational University Clinic for Hematology, Oncology,
Science Hemostaseology and Palliative Care,
Creighton University School of Medicine Johannes Wesling Medical Center
Omaha, Nebraska Minden
University of Bochum
Mark H. Kaplan Minden, Germany
Department of Pediatrics and Herman B Wells
Center for Pediatric Research Raymond F. Schinazi
Indiana University School of Medicine Center for AIDS Research, Laboratory of
Indianapolis, Indiana Biochemical Pharmacology
Department of Pediatrics, Emory
Han-Chung Lee University
Laboratory Centre Atlanta, GA
Xiamen University Malaysia
Sepang, Malaysia Daniel Silberger
Department of Pathology
King-Hwa Ling University of Alabama at
Department of Biomedical Sciences, Birmingham
Faculty of Medicine and Health Sciences Birmingham, Alabama
Universiti Putra Malaysia
Seri Kembangan, Malaysia Radomir M. Slominski
Department of Dermatology
Yuji Nozaki Indiana University School of
Department of Hematology and Medicine
Rheumatology Indianapolis, Indiana
Kindai University School of Medicine
Osaka-Sayama, Japan Kai-Leng Tan
Institute of Biomedical and Pharmaceutical
Duy Pham Sciences
Department of Pathology Guangdong University of Technology
University of Alabama at Birmingham Guangzhou, China
Birmingham, Alabama
Thai Hoa Tran
Thomas Pincez Division of Pediatric
Division of Pediatric Hematology-Oncology Hematology-Oncology
Charles-Bruneau Cancer Center, Ste-Justine Charles-Bruneau Cancer Center,
Hospital Ste-Justine Hospital
Quebec, Canada Quebec, Canada

Amina Abdul Qayum Matthew J. Turner

Department of Pediatrics and Herman B Wells Department of Dermatology
Center for Pediatric Research Indiana University School of
Indiana University School of Medicine Medicine
Indianapolis, Indiana Indianapolis, Indiana
1
Regulation of Cytokine Signaling by the
JAK-STAT Pathway

Nicolette Nadene Houreld

Laser Research Centre, Faculty of Health Sciences
University of Johannesburg
Johannesburg, South Africa

1.1 Introduction
The Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway is a prompt
pleiotropic cytoplasmic to nuclear signaling pathway used to transduce a variety of signals, activated
by cytokines, hormones, and growth factors, for development and homeostasis. The JAK-STAT
pathway is responsible for controlling signals of over fifty cytokines, growth factors, and hormones
(Morris, Kershaw, and Babon 2018; Hammaren et al. 2019), while negative regulation is through
suppressor of cytokine signaling (SOCS) proteins (bind to and inactivate JAK3), and the protein
inhibitors of activated STATs (PIAS; bind to STAT dimers thereby preventing DNA binding).
Cytokines are glycoproteins (ligands) secreted by cells and operate as intercellular messengers,
inducing differentiation, proliferation, growth, and apoptosis of their target cells.
Signaling via the JAK-STAT pathway is instigated by binding of a ligand to its receptor. Binding
results in dimerization, oligomerization, and/or conformational changes of the receptor complex,
which allow JAK proteins to bind to the receptor complex intracellular domains inducing trans­
autophosphorylation of the tyrosine residues (JH1), converting the receptor into a tyrosine kinase.
Phosphorylated chains serve as docking sites for SH2 domain-containing signaling molecules such as
STATs. Receptor-bound STATs are phosphorylated by JAK on a specific tyrosine in the C-terminal
tail, allowing them to form homo- and heterodimers, which rapidly translocate into the nucleus. In the
nucleus, they associate with proteins and produce transcriptional complexes/factors with extensive
effects on regulation of transcription and epigenetics (Hammaren et al. 2019).

1.2 The JAKs

Janus (kinase) comes from the Roman mythological two-faced god, who looks to the future and
the past. JAK relates to the two faces due to the presence of two kinase domains, namely the
pseudokinase domain (JAK homology 2, JH2) and a catalytically active kinase domain (JH1).
They also contain an N-terminal band, and a four-point-one, ezrin, radixin, moesin (FERM)­
domain, which mediate the interaction of JAKs with their receptors. JAKs combine with the
proline-rich, membrane-proximal box1/box2 domain on cytokine receptors. They also contain an
Src homology 2 (SH2)-like domain, of unknown function, which lies between the pseudokinase
and FERM domains (Figure 1.1a) (Schindler, Levy, and Decker 2007; Hammaren et al. 2019).
JH2 has significant regulatory functions and is a source of numerous mutations that is the cause
of various diseases and disorders, including hematopoietic malignancies (JAK2 mutations),

1
2 JAK-STAT Signaling in Diseases

FIGURE 1.1 Structural organisation of JAK-STAT proteins. (a) JAKs share seven conserved homology domains:
JH1 serves as the catalytic, kinase domain, while JH2 represents the pseudokinase domain. JH3 and half of JH4
include the nonfunctional SH2-domain, and half of JH4 to JH7 includes a FERM domain. (b) STATs share seven
domains: the amino-terminal domain (NH2), the coiled-coil domain, the DNA-binding domain, the linker domain, the
SH2 domain, the tyrosine-activation domain, and the transactivation domain.

leukemia and lymphomas (all JAKs), and cancer (JAK1, JAK3) (Hammaren et al. 2019). There
are four JAKs in mammals (JAK1, JAK2, JAK3, and TYK2). JAK1, JAK2, and TYK2 are
ubiquitously expressed and relatively constitutive in their expression, while the expression of
JAK3 is mostly confined to cells of hematopoietic origin, and its expression is more inducible.
JAK1 associates with type I (IFN-α/β), type II (IFN-γ), IL-2, and IL-6 receptors. JAK2
interacts with single-chain receptors (i.e., EPOR, GH-R, and PRL-R) and IL-3 (IL-3R, IL-5R,
and GM-CSFR) cytokine families, as well as the IFN-γ receptor. Leukocyte-specific JAK3
exclusively associates with the IL-2 receptor γ-chain, and Tyk2 associates with receptors for
IFN-I, IL-6, IL-10, and IL-12/23 cytokine families (Schindler, Levy, and Decker 2007).

1.3 The STATs

The STAT family includes STAT1, STAT2, STAT3, STAT5A/B, and STAT6. STAT proteins
consist of seven well-defined, conserved domains: an N-terminal conserved domain (NH2, critical
for STAT function); a coiled-coil domain (involved in receptor binding, and associates with
regulatory proteins); a DNA-binding domain (DBD, cooperate in binding to the promoters of
target genes); a linker region (LK, spacer to maintain proper conformation between the dimeriza­
tion and DNA binding domains); an SH2 domain (critical for the recruitment of STATs to
activated receptor complexes and for the interaction with JAK and Src kinases); a tyrosine-
activation domain (Y); and a C-terminal transactivation domain (TAD, modulates the transcrip­
tional activation of target genes and vary considerably among STAT family members) (Jatiani
et al. 2010) (Figure 1.1b).

1.4 Cytokine Receptors

Cytokines function by binding to their associated transmembrane receptor, which triggers
intracellular signaling events and pathways that result in the alteration of gene expression. Most
Cytokine Signaling Regulation by JAK-STAT 3

of these receptors consist of a unique ligand-binding subunit and a signal-transducing subunit.

Often the signal transducing or cytoplasmic subunits are structurally similar to other cytokine
receptors, particularly in regions labeled as box 1 or the proline-rich motif and the box-2 motif,
and this is critical for proper receptor functioning and mediating of mitogenic signals. The ligand-
binding subunit, or membrane distal region, remains uniquely different to ensure differentiation
(Jatiani et al. 2010). Cytokine binding results in receptor tyrosine phosphorylation.
Cytokine receptors are divided into type I and type II receptors. Type I cytokine receptors bind
to and react to cytokines with four α-helical strands and share an amino acid motif (WSXWS).
Type II cytokine receptors are similar to type I, but lack the WSXWS motif. Cytokine receptors
signal through the JAK-STAT pathway and other pathways that typically trigger activation of the
mitogen-activated protein (MAP) kinase cascade. Different types of cells and tissues express well-
defined and diverse receptor combinations that respond to cytokine combinations unique to their
microenvironment. Thus, at any particular time, a single cell may respond to signals from multiple
cytokine receptors (Murray 2007). Different receptor classes preferentially associate with one JAK
family member, or a JAK combination.
Typically, receptors required for hematopoietic cell development and proliferation prefer JAK2;
common γ-chain receptors utilize JAK1 and JAK3, while other receptors use only JAK1 (Murray
2007). All interferons (IFNs), which are essential mediators of innate immunity against bacterial
and viral infection, as well as the interleukin(IL)-10 family (IL-10, IL-19, IL-20, IL-22, IL-24, IL­
26), anti-inflammatory cytokines, function through type II cytokine receptors, which dimerize in
multiple combinations to generate distinct downstream effects. JAK1 is imperative for signaling
through these type II receptor complexes (Ferrao et al. 2016).

1.5 Activation of JAK-STAT Pathways by Cytokines

Cytokine signaling through the JAK-STAT pathway regulates numerous cellular responses, including
proliferation, differentiation, motility, and cell survival. JAKs mediate signaling of around fifty to
sixty different hormones, cytokines, and growth factors ranging from regulators of the immune
system and hematopoiesis, such as IFNs, ILs, thrombopoietin (TPO), and erythropoietin (EPO), to
regulators of development and metabolism, such as growth hormone (GH) and prolactin (PRL)
(Table 1.1) (O’Shea and Plenge 2012).
Ligand binding results in receptor dimerization/oligomerization, which in turn results in the
juxtapositioning of JAKs through homodimeric or heterodimeric interactions. This leads to
the autophosphorylation and/or transphosphorylation of JAK, which causes the phosphoryla­
tion of receptor target tyrosine residues that serve as docking sites and allow the binding of
other SH2 domain–containing signaling molecules such as STAT (Jatiani et al. 2010)
(Figure 1.2). In unstimulated cells, STATs are idle, unphosphorylated cytosolic proteins.
Cellular stimulation via cytokines induces phosphorylation of receptor tyrosine residues that
serve as docking sites for STATs via their SH2 domains. Once phosphorylated, dimerized
STATs translocate to the nucleus and drive the transcription of cytokine inducible genes.
STAT activation is swift, with a maximum accumulation of phosphorylated STAT1 in the
nucleus within 30 min (Lim and Cao 2006).
Recently, a number of biophysical and protein engineering studies have presented new data,
which emphasizes the intricacy and complexity of signaling sparked by a cytokine–cytokine
receptor complex. This permits cytokines to produce diverse biological responses in spite of
using a marginal set of surface receptors and effector signaling molecules (Gorby et al. 2018).
Cytokine receptors will interact with the JAK-STAT pathway though its interaction with a
combination of JAKs. Cytokine-receptor interactions result in various JAK activation that exists
in association with cytokine receptors, for which activation is essential for the activation of
STATs. On the other hand, activated JAKs do not seem to exhibit specificity for a particular
STAT, as different receptors stimulate a common STAT even though they activate distinctively
4 JAK-STAT Signaling in Diseases

TABLE 1.1
Type I and Type II Cytokine Receptors and their Corresponding JAKs and STATs. Associated JAK­
STAT Proteins for which the Data is Weaker are Shown in Brackets (adapted from (Hammaren et al.
2019)).
Cytokine JAKs STATs

Type I Cytokine IL-6 JAK1, JAK2, TYK2 STAT3, STAT1

Receptors IL-11 JAK1, JAK2, TYK2 STAT3, STAT1
LIF JAK1, JAK2, TYK2 STAT3, STAT1
CNTF JAK1, (JAK2, TYK2) STAT3, (STAT1)
CLCF1 JAK1, (JAK2) STAT3, STAT1
CT-1 JAK1, (JAK2, TYK2) STAT3
OSM JAK1, (JAK2, TYK2) STAT3, STAT1
IL-31 JAK1, (JAK2) STAT3, STAT5,STAT1
G-CSF JAK1, (JAK2) STAT3
Leptin JAK2 STAT3
IL-12 TYK2, JAK2 STAT4
IL-23 TYK2, JAK2 STAT3, STAT4, STAT1
IL-27 JAK1, JAK2 STAT1, STAT3, STAT4, (STAT5)
IL-35 JAK1, JAK2 STAT1, STAT4
IL-2 JAK1, JAK3, (JAK2) STAT5, (STAT3)
IL-4 JAK1, JAK3 STAT6
IL-7 JAK1, JAK3 STAT5, (STAT3)
IL-9 JAK1, JAK3 STAT5, STAT3
IL-15 JAK1, JAK3 STAT5, (STAT3)
IL-21 JAK1, JAK3 STAT3, STAT5, (STAT1)
TSLP JAK1, JAK2 STAT1, STAT3, STAT4, STAT5, STAT6
IL-13 JAK1, JAK2, TYK2 STAT6, (STAT3)
IL-3 JAK2, JAK1 STAT5, STAT3
IL-5 JAK2 STAT5, STAT1, STAT3
GM-CSF JAK2 STAT5
EPO JAK2 STAT5
GH JAK2 STAT5, (STAT3)
PRL JAK2 STAT5
TPO JAK2 STAT5
Type II Cytokine IFN-I JAK1, TYK2 STAT1, STAT2, STAT3, STAT4,
Receptors (type I) (STAT5, STAT6)
IFN-γ JAK1, JAK2 STAT1
(type II)
IL-28a, JAK1, TYK2 STAT1, STAT2, STAT3, STAT5
IL-28b
IL-29 JAK1, TYK2 STAT1, STAT2, STAT3, STAT5
IL-10 JAK1, TYK2 STAT3, STAT1
IL-19 JAK1, JAK2 STAT3, STAT1
IL-20 JAK1, JAK2 STAT3, STAT1
IL-24 JAK1, JAK2 STAT3, STAT1
IL-22 JAK1, TYK2 STAT3, STAT1, (STAT5)
IL-26 JAK1, TYK2 STAT3, STAT1
Interleukin (IL); Leukemia inhibitory factor (LIF); Ciliary neurotrophic factor (CNTF); Cardiotrophin-like cytokine
factor 1 (CLCF1); Cardiotrophin-1 (CT-1); Oncostatin M (OSM); Granulocyte-colony stimulating factor (G-CSF);
Thymic stromal lymphopoietin (TSLP); Granulocyte-macrophage colony-stimulating factor (GM-CSF); Erythropoietin
(EPO); Growth hormone (GH); Prolactin (PRL); Thrombopoietin (TPO).
Cytokine Signaling Regulation by JAK-STAT 5

FIGURE 1.2 Overview of the JAK-STAT signaling pathway. Ligand binding (1) results in receptor dimerization, JAK
activation, and receptor phosphorylation (2). STAT binds to the phosphorylated receptor (3), which becomes
phosphorylated by JAK (4). STAT dimers form (5), which translocates into the nucleus and stimulates gene
transcription (6). Negative regulation of JAK-STAT signaling is through suppresser of cytokine signaling (SOCS)
proteins, which directly bind to and inactivate JAKs, and protein inhibitors of activated STATs (PIAS) that bind to
phosphorylated STAT dimers, averting DNA binding.

different JAKs. It would appear that the specificity for the activation of STAT lies with the STAT
docking sites on the receptors themselves (Jatiani et al. 2010).
Once activated STAT has translocated to the nucleus, it binds to a consensus DNA-recognition
motif known as gamma-activated sites (GAS) in the promoter region of cytokine-inducible genes and
activates transcription. These GAS-like elements act as ligands for a variety of STAT family members.
There are three key features of STAT-binding elements (SBE), which include the core motif (with
sequence TT-(X)n-AA; where X is a G/C rich palindromic sequence in length n), the core spacing
between the palindromic A/T residues, and composition of the sequence between palindromic A/T
residues. Generally, there is a preference for a specific core spacing for each class of cytokine­
responsive genes, for example, GAS elements consistently occupy five-base pair core spacing, whereas
genes that respond to IL-6 occupy a four-base core spacing (Wilks and Harpur 1994). STAT1 and
STAT5 prefer sites with a three-base pair spacer; however, STAT5 has also been shown to bind weakly
to a four base pair spacer. STAT6 prefers sites with a four-base pair spacer, and STAT4 prefers the
palindromic sequence (T/A)TTCC(C/G)GGAA(T/A) where the first and last T/A sites outside of the
usual motif are also necessary for binding (Morris, Kershaw, and Babon 2018).

1.6 JAK-STAT Regulation

A characteristic feature of JAK-STAT signaling is its rapid onset and subsequent decay. Activated
STATs accumulate within a few hours in the nucleus, however, the signal promptly decays and the
6 JAK-STAT Signaling in Diseases

STATs are re-exported back to the cytoplasm for the next round of signaling. This decline in
activity involves down-regulation of both receptors and JAK proteins, as well as STAT-transcriptional
activity (Schindler, Levy, and Decker 2007). Three well-defined mechanisms of signal decay
include dephosphorylation by protein phosphotyrosine phosphatases, nuclear export, and feed­
back inhibition via SOCS, as well as inhibition by PIAS. Post-translational modifications (PTMs)
of STAT proteins institute another important regulatory mechanism. STATS also undergo post-
translational covalent modifications such as ubiquitination (post-translational modification by
adding ubiquitin to the protein sequence).
Protein phosphotyrosine phosphatases (PTPs) negatively regulate the JAK-STAT pathway by
dephosphorylating tyrosine residues. There are six PTPs that regulate JAK-STAT. Receptor and
JAK dephosphorylation has been carried out by phosphatases, and include SHP-1, SHP-2, and
CD45, while STAT dephosphorylation is carried out by SHP-2, PTP1B, TC-PTP, and PTP-BL
(Schindler, Levy, and Decker 2007; Seif et al. 2017). Since PTPs are constitutively expressed, they
tend to restrain the amplitude of the signaling cascade rather than controlling its duration
(Morris, Kershaw, and Babon 2018). The process of nuclear translocation is complex and there
is a balance between STAT nuclear import and export. This seems to be controlled by multiple
nuclear export sequence (NES) and nuclear localization sequence (NLS) elements. During activa­
tion of cellular signaling, there is a shift toward STAT accumulation in the nucleus. This balance
is shifted toward nuclear export when there is signal decay (Schindler, Levy, and Decker 2007).
Proteolytic processing of STAT via cleavage of the C-terminal acts as a general mechanism for the
negative regulation of STAT protein function (Hendry and John 2004). This may occur in the
nucleus or in the cytoplasm, and this protease may cleave activated (phosphorylated) or inactivated
STAT. STAT are rapidly inactivated by dephosphorylation with a half-life of phosphorylated
STAT1 at less than 15 min (Lim and Cao 2006).
Post-translational modifications regulate every aspect of transcription factor function and
coordinate access of RNA polymerases to promoter templates. Site-specific, DNA-binding
transcription factors repress, activate, enhance, or silence complexes and associated enzymatic
activities (Filtz, Vogel, and Leid 2014). Cytokine signal transduction is dependent on the ability of
individual receptors to recruit signal transcription factors (STFs), which in turn activate the
transcription of the genes which are related to that particular cytokine (Wilks and Harpur 1994).
This process is very complex and relies on affinity differences of STFs for GAS elements. There
are two main subclasses of DNA response elements in gene promoters, and include interferon-
stimulated response element (ISRE) and GAS-like sequences. ISRE is located upstream of
interferon alpha/beta (IFN-α/β) inducible genes, while GAS is located upstream of IFN gamma
(IFN-γ)-inducible genes. GAS-like elements, which bind to STFs induced by other cytokines, all
consist of the same basic DNA motif with slight changes in individual genes (Wilks and Harpur
1994). Owing to activation of other transcription factors and crosstalk between different signal
transducing pathways, determination of the complete set of genes upregulated by STAT proteins
is difficult. However, it is known that several hundred to thousands of genes can be upregulated or
down-regulated in response to each STAT protein (Morris, Kershaw, and Babon 2018).
The SOCS family members are the primary drivers of signal attenuation and are negative-
feedback inhibitors of signaling induced by cytokines and other stimuli that act via the JAK­
STAT pathway. There are eight SOCS proteins: SOCS1 to 7 and CIS. These proteins all contain
an SH2 domain and a short, C-terminal domain, known as the SOCS box. The SOCS box is
associated with an adapter complex, elongin BC, which allows for the recruitment of an E3
ubiquitin ligase scaffold (Cullin5) to catalyse and ubiquitinate signaling intermediates recruited by
their SH2 domains (Liau et al. 2018). Ubiquitination is mediated by an ubiquitin-activating
enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which mediates
between E2 and the substrate. The resulting poly-ubiquitinated conjugates are rapidly identified
and degraded by 26S proteasome. SOCS proteins form E3 ubiquitin ligase complexes with Cullin­
2, Elongin B, Elongin C, and Rbx1 and ubiquitinate JAK and receptor molecules, followed by
their degradation and internalization, respectively (Hatakeyama 2012). SOCS1 and SOCS3 are
capable of directly binding to and inhibiting the kinase activity of JAK, and is due to a short
Cytokine Signaling Regulation by JAK-STAT 7

motif, kinase inhibitory region (KIR), upstream of the SH2 domain. SOCS1 and SOCS3 also
have the ability to directly inhibit JAK1, JAK2, and TYK2 (but not JAK3) by blocking the
substrate-binding groove on JAK, thereby acting as a pseudosubstrate. SOCS1 is the most
influential SOCS family member, and is the principal regulator of numerous cytokines involved in
the immune response, in particular IFN-γ (Liau et al. 2018). Genetic deletion of SOCS1 is fatal, and
its down-regulation plays a role in the progression of cancer. Each SOCS protein is also specific for
only a subset of cytokines; for example, SOCS3 is prompted by IL-2, -3, -4, -6, -7, -9, -10, -11, -12,
-13, -21, and -22, as well as granulocyte-colony stimulating factor (G-CSF), granulocyte-macro­
phage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), PRL, IFN-α, IFN-γ,
GH, EPO, thyroperoxidase, oncostatin M (OSM), calcitonin-1 (CT1), ciliary neurotrophic factor
(CNTF), and leptin (Morris, Kershaw, and Babon 2018).
There are also members of the PIAS family, which are able to inhibit the JAK-STAT pathway
by binding to and inhibiting STAT dimers, preventing DNA binding and gene transcription.
However, the exact mechanism by which PIAS proteins do their regulative functions remains
unrevealed. There are several PIAS family members, including PIAS1, PIAS3 (KchAP), PIASy,
and PIASx (ARIP3) (O’Shea and Plenge 2012). PIAS family members possess several domains,
including a serine/threonine rich domain located at the C-terminus (responsible for target binding),
a Zn-binding RING–finger-like domain (RLD, responsible for SUMO transfer), and a conserved-
SAP domain (scaffold attachment factor A/B, Acinus, PIAS) near the N-terminus (important part for
target binding via scaffold/matrix attachment regions, S/MARs) (Seif et al. 2017).

1.7 Failure of JAK Regulation Leads to Disease

Deregulation of cytokines and/or their downstream signaling pathways are at the root of many
human disorders and diseases, including asthma, severe combined immunodeficiency (SCID), and
various cancers (Gorby et al. 2018; Hammaren et al. 2019). A number of these disorders are because
of point mutations, as in the case of SCID (mutation and loos in function of JAK3). Oncogenic JAK2
rearrangements to multiple fusion gene partners have been identified in acute lymphoblastic leukemia
(ALL), atypical chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloprolifera­
tive neoplasms (MPN), and/or Hodgkin lymphoma (Hammaren et al. 2019).
A number of gain-of-function mutations that have been identified in JAK1, JAK2, and JAK3
are responsible for approximately 20% of T-ALL cases and to a lesser extent in B-ALL or
hepatocellular adenoma (Hammaren et al. 2019). In some cases, mutations in other components
and JAK-STAT regulatory molecules can be the cause of the disease, as is the case in 1%–5% of
primary myelofibrosis (PMF) and essential thrombocythemia (ET). In these cases, there is a point
mutation in the thrombopoietin receptor (TPOR) gene MPL, which enables ligand-independent
activation of JAK2 signaling (Leroy et al. 2016). Frameshift mutations are also frequent,
accounting for approximately 13% of MPN cases, causing thrombopoietin-independent TPOR–
JAK2 signaling. In this instance, a frameshift mutation in calreticulin (CALR), an endoplasmic
reticulum protein, results in a highly charged protein capable of binding to TPOR, thus activating
the receptor (Hammaren et al. 2019). Apart from mutations resulting in JAK-STAT hyperactivity,
JAK-STAT loss-of function mutations have also been described. Loss-of-function mutations in
JAK3 or JAK3-associated receptors IL-7R and common gamma chain (IL-2Rγ) cause autosomal
recessive SCID. TYK2 deficiency has been reported in primary immunodeficiency (PID) (Hammaren
et al. 2019).

1.8 Conclusion
There is a shared interaction between external actions and internal reactions that enables a cell to
remain viable and survive. Binding of extracellular cytokines and growth factors to their receptor
8 JAK-STAT Signaling in Diseases

sets off a cascade of responses and activates cellular signaling pathways, leading to gene transcription
and ultimately cellular proliferation, differentiation, activation/inhibition, and survival/apoptosis. The
JAK-STAT pathway plays a fundamental role in the transfer of extracellular signals from membrane
receptors to the nucleus. Over fifty cytokines make use of the JAK-STAT pathway by binding to type
I or type II receptors to carry out their effects. Several regulators modulate the function of the JAK­
STAT pathway, such as protein tyrosine phosphatases, SOCS, and PIAS family members. There is still
a lack of knowledge concerning the complete molecular understanding of JAK activation and
inhibition. Any dysregulation in the JAK-STAT pathway may lead to various pathologies. Better
knowledge of these mechanisms may be the answer in the development of therapeutic strategies to
target the JAK-STAT pathway to treat various immunological disorders and cancers.

REFERENCES
Ferrao, R., H. J. Wallweber, H. Ho, et al. 2016. The structural basis for class II cytokine receptor
recognition by JAK1. Structure 24 (6):897–905.
Filtz, T. M., W. K. Vogel, and M. Leid. 2014. Regulation of transcription factor activity by interconnected
post-translational modifications. Trends Pharmacol Sci 35 (2):76–85.
Gorby, C., J. Martinez-Fabregas, S. Wilmes, and I. Moraga. 2018. Mapping determinants of cytokine
signaling via protein engineering. Front Immunol 9:2143.
Hammaren, H. M., A. T. Virtanen, J. Raivola, and O. Silvennoinen. 2019. The regulation of JAKs in
cytokine signaling and its breakdown in disease. Cytokine 118:48–63.
Hatakeyama, S. 2012. Ubiquitin-mediated regulation of JAK-STAT signaling in embryonic stem cells.
JAKSTAT 1 (3):168–75.
Hendry, L., and S. John. 2004. Regulation of STAT signalling by proteolytic processing. Eur J Biochem
271 (23–24):4613–20.
Jatiani, S. S., S. J. Baker, L. R. Silverman, and E. P. Reddy. 2010. Jak/STAT pathways in cytokine signaling
and myeloproliferative disorders: approaches for targeted therapies. Genes Cancer 1 (10):979–93.
Leroy, E., J. P. Defour, T. Sato, et al. 2016. His499 regulates dimerization and prevents oncogenic
activation by asparagine mutations of the human thrombopoietin receptor. J Biol Chem 291
(6):2974–87.
Liau, N. P. D., A. Laktyushin, I. S. Lucet, et al. 2018. The molecular basis of JAK/STAT inhibition by
SOCS1. Nat Commun 9 (1):1558.
Lim, Cheh Peng, and Xinmin Cao. 2006. Structure, function, and regulation of STAT proteins. Mol
Biosyst 2 (11):536–50.
Morris, R., N. J. Kershaw, and J. J. Babon. 2018. The molecular details of cytokine signaling via the JAK/
STAT pathway. Protein Sci 27 (12):1984–2009.
Murray, P. J. 2007. The JAK-STAT signaling pathway: input and output integration. J Immunol 178
(5):2623–9.
O’Shea, J. J., and R. Plenge. 2012. JAK and STAT signaling molecules in immunoregulation and immune-
mediated disease. Immunity 36 (4):542–50.
Schindler, C., D. E. Levy, and T. Decker. 2007. JAK-STAT signaling: from interferons to cytokines. J Biol
Chem 282 (28):20059–63.
Seif, F., M. Khoshmirsafa, H. Aazami, M. Mohsenzadegan, G. Sedighi, and M. Bahar. 2017. The role of
JAK-STAT signaling pathway and its regulators in the fate of T helper cells. Cell Commun Signal
15 (1):23.
Wilks, A. F., and A. G. Harpur. 1994. Cytokine signal transduction and the JAK family of protein
tyrosine kinases. Bioessays 16 (5):313–20.
2
The Structure-Function Bonhomie of
JAK-STAT Molecules

Ritobrata Goswami
School of Bioscience
Indian Institute of Technology Kharagpur
Kharagpur, India

2.1 Introduction
The existence of life requires the ability to comprehend and respond to external signals. These
signals are intercepted by numerous cell-surface receptors that utilize a range of intracellular
signaling pathways to communicate with the nucleus at the cellular level. The culmination of these
signaling ensue an appropriate response mediated by peripheral sensory organs via the central
nervous system. The Janus kinase (JAK)-signal transducers and activators of transcription
(STAT) pathway used by plants and animals, including flies, is one of the few signal transduction
pathways that transduce a multitude of signals required for development and homeostasis
(Rawlings, Rosler, and Harrison 2004). Activation of JAK induces various physiological events
including, but not limited to, cell proliferation, migration, differentiation, and apoptosis. JAK­
STAT pathway’s association is evident in 12 major cancers (Vogelstein et al. 2013). JAK-STAT
pathway is one of the major signaling pathways employed by multiple growth factors and
cytokines (Liongue and Ward 2013). These cellular events are critical to hematopoiesis, develop­
ment of immune system, and subsequent functions. JAK-STAT signaling is dependent upon
tyrosine phosphorylation and harbors comparatively simple signal transduction pathway requir­
ing few components. Activation of JAK-STAT pathway ensues when growth factors or cytokines
bind to their cognate receptors leading to multimerization of receptor subunits (Rawlings, Rosler,
and Harrison 2004). The multimerization of receptor subunits can form homodimers that lead to
close proximity of the receptor-associated JAK molecules. Via transphosphorylation, JAKs
phosphorylate each other on tyrosine residues, a cue for their activation. Activated JAKs then
phosphorylate on tyrosine residues of cytokine or growth-factor receptors paving the way for
binding of proteins bearing SH2 domains. With the help of SH2 domains, STAT molecules bind
to the phosphorylated tyrosine residues on the cognate receptor. STAT molecules dissociate from
the receptor after being tyrosine-phosphorylated by JAKs. Following activation, STAT molecules
form either homo or hetero-dimers and SH2 domain of each STAT molecule binds to the
phosphotyrosine residue of its corresponding STAT. Subsequently, the STAT dimer translocate
to the nucleus, bind to the genes bearing consensus STAT-binding sequence, and activate gene
transcription (Figure 2.1). Thus, the JAK-STAT signaling cascade is a direct mechanism to
translate an extracellular signal into a transcriptional response. The canonical JAK-STAT
proteins are inactivated by negative regulators, including SH2-containing protein tyrosine phos­
phatase (SHP) and suppressor of cytokine signaling (SOCS) proteins (Liongue et al. 2012). While
phosphorylation can be denoted as an “on” switch (binary 1), dephosphorylation can be
attributed as an “off” switch (binary 0). In this chapter, the structure–function relationship of
JAK-STAT signaling has been discussed.

9
10 JAK-STAT Signaling in Diseases

FIGURE 2.1 Brief overview of JAK-STAT domain structure. The JAK-STAT signaling pathway has been shown in
between the domain structures JAK (shown on top) and STAT (shown at the bottom).

2.2 The Beginning

The journey of JAK-STAT pathway started with the discovery of “interferons” way back in 1957
by Issacs and Lindenmann (Isaacs and Lindenmann 1957). As the moniker suggests, interferons
(IFNs) “interfered” with the replication process of virus. Three decades later, studies involving the
induction of mRNA and translation of proteins by interferons staged the set for identifying
members of JAK-STAT signaling pathway. JAK family has four members: JAK1, JAK2, JAK3,
and TYK2. STAT family has seven members: STAT1, STAT2, STAT3, STAT4, STAT5A,
STAT5B, and STAT6. During 1984–1988, IFN-α-induced transcriptional stimulation of specific
genes was characterized and IFN-dependent promoters were identified (Stark and Darnell 2012).
The first members of the JAK family were identified in 1989, while cDNA clones for the first
STAT family member was discovered in 1992 (Wilks 1989, Shuai et al. 1992). The Janus of Janus
kinase comes from two-faced Roman God of doorways, “Janus”, since JAK possesses two near-
identical phosphate-transferring domains (Wilks 1989). During 1989–1991, JAK1, JAK2, and
TYK2 were identified, while JAK3 sequencing was completed in 1994 (Stark and Darnell 2012).
Sequencing of STAT3, 4, 5A, 5B, and 6 were completed in the same year (Larner et al. 1993,
Ruff-Jamison, Chen, and Cohen 1993, Sadowski et al. 1993, Silvennoinen et al. 1993, Akira et al.
1994, Zhong, Wen, and Darnell 1994a, 1994b, Jacobson et al. 1995). The functional and
structural domains of STATs were described during 1995–1998 (Hoey and Schindler 1998).
STATs were first crystalized in 1998 followed by identification of target genes (Becker, Groner,
and Muller 1998). The onset of twenty-first century was when the underlying importance of the
signaling pathway was linked to several disorders that were associated with JAK-STAT mutations
and gained momentum.
JAK-STAT Structure Function 11

2.3 Structure and Activation of JAK

Cytokine or growth-factor receptors lack intrinsic kinase domain, thus depending on JAK for
transmitting signal to the cytoplasmic components. Structurally, JAKs have seven domains known
as JAK homology regions (JH1-7) (Bousoik and Montazeri Aliabadi 2018) (Figure 2.1). Func­
tionally, JAKs have been shown to possess four domains: catalytically active tyrosine kinase
domain, the pseudotyrosine kinase domain, the Src homology 2 (SH2) domain, and the FERM
domain (Bousoik and Montazeri Aliabadi 2018). JH1 encodes the kinase domain, while JH2 has
a pseudo-kinase domain that controls the kinase activity of JH1 (Manning et al. 2002, LaFave
and Levine 2012). The JH2 domain activity has been observed specifically for JAK2 and has been
suggested to possess auto-inhibitory function (Ungureanu et al. 2011). The SH2 domain is
encoded by JH3 and JH4 regions, while JH5-7 is responsible for receptor binding (Cornejo,
Boggon, and Mercher 2009). The JH3-7 regions form the N-terminal of JAKs, while JH1-2 forms
the C-terminus of the domain (Yamaoka et al. 2004). JAK activation occurs upon ligand-
mediated receptor multimerization because two JAKs are brought into close proximity, allowing
trans-phosphorylation. Binding of the ligand to cytokine receptor revamps the receptor–JAK
dimers, which brings the JAKs close enough to transphosphorylate the partner JAK in the dimer
at JH1. The activated JAKs subsequently phosphorylate additional targets, including both the
receptors and the major substrates, STATs (Yamaoka et al. 2004). The specificity of ligand-
mediated STAT activation is dependent on the interactions between SH2 domain and phospho­
tyrosine residues. The current understanding of JAK domains will be elaborated in the subsequent
sections.

2.4 Structure of STAT and Translocation to the Nucleus

STATs function both as signal transducers and transcription factors. STATs are latent transcrip­
tion factors that reside in the cytoplasm until activated. Like JAKs, STATs also possess distinct
conserved domains: the N-terminal coiled-coil domain, DNA-binding domain, a linker, SH2
domain, and C-terminal transactivation domain (Figures 2.1 and 2.2). The transactivation
domain interacts with multiple partners forming transcriptional complexes. Tyrosine at position
705 is important when it comes to STAT activation (Yu and Jove 2004). Other than STAT2 and
STAT6, a conserved serine at position 727 is another site of phosphorylation that controls STAT
transcriptional activity (Decker and Kovarik 2000). The DNA-binding domain is the central
region that controls DNA-binding selectivity for each STAT molecule. Between position 600 and
700 amino acid residues, the SH2 domain is located that aids the dimer formation between two
activated STAT molecules via SH2-phosphotyrosine interaction (Horvath, Wen, and Darnell
1995). The linker region is located between the amino acid residues 500 and 575 (Subramaniam
et al. 2013).
After activation by tyrosine phosphorylation, STATs become dimerized and translocate into the
nucleus, where they act as transcription factors. While most STATs form homodimers, evidences
support heterodimer formation of STAT1/2, STAT1/3, and STAT5A/B (Delgoffe and Vignali
2013). STAT1 has been reported to exist as pre-formed homodimers, and phosphorylation
induces reorientation. STAT molecules face restricted movement while translocating from cyto­
plasm to nucleus by the nuclear envelope owing to molecular weight >40 kDa (Mertens et al.
2006). Thus, STATs require facilitated transport mediated by specific receptor molecules that are
known as importins (adaptor proteins) bearing α and β subunits. Using nuclear localization
sequence (NLS), STATs bind to importin α (Gorlich and Mattaj 1996, Jans, Xiao, and Lam
2000). Following interaction of both the importin subunits, STAT–importin complex gets docked
at the nuclear pore complex (NPC) (Gorlich and Mattaj 1996). The NPCs form high-order
octagonal channels that are composed of proteins called nucleoporins, some of which contain
hydrophobic phenylalanine/glycine (FG)-rich repeat motifs (Wolf and Mofrad 2008). The energy
12 JAK-STAT Signaling in Diseases

FIGURE 2.2 STAT domain structure. Minor differences in various human STAT molecules has been depicted.

for the translocation is provided by Ran, a GTPase. The intracellular RanGTP/RanGDP gradient
essentially drives this active transport, which allows the accumulation of cargo against
a concentration gradient. In the cytosol, the concentration of the GTP form of Ran is low due
to nucleotide hydrolysis by RanGAP, which is cytoplasmically localized by RanGTPase-activating
protein (Reich 2013). A differential requirement of importin α has been attributed for STAT
translocation (Goldfarb et al. 2004). Out of the six human importin α molecules, STAT1 requires
importin α5, while STAT5 requires importin α3 (Sekimoto et al. 1997, Liu, McBride, and Reich
2005). One study even argued that STATs may not be having functional NLS (Subramaniam,
Torres, and Johnson 2001). Furthermore, it has also been suggested that NLS-binding site on
STAT1 and STAT3 differs from its binding site in other proteins. STAT3, 5, and 6 could also be
translocated to the nucleus in unphosphorylated form (Reich 2013). Carrier-dependent export of
unphosphorylated STATs requires the specific export receptor, CRM1 (Fornerod et al. 1997).
Translocation of unphosphorylated STATs requires direct interaction with nucleoporins and is
carrier and energy-free, devoid of any help from importins. This process also does not require any
activation by cytokines.

2.5 JAK Domains

As mentioned previously, JAKs contain specific domains. In this section, we discuss about the
domains found in JAKs.

2.5.1 Protein Tyrosine Kinase Domain

The JH1 domain is the PTK domain of the JAK protein. Similar to other protein tyrosine
kinases, JAKs possess an “activation loop” that regulates kinase activity, which is a major site of
autophosphorylation (Toms et al. 2013). Using crystal structures of JAK2 and JAK3, it has been
JAK-STAT Structure Function 13

demonstrated that the PTK has an open conformation representing the active conformation of
the protein. JAK2 and JAK3 crystal structures have been solved at 2A° and 2.5A°, respectively
(Boggon et al. 2005, Lucet et al. 2006). JAK2 PTK domain has a small N-terminal lobe,
comprised of one α-helix and five anti-parallel β strands, and large C-terminal lobe comprised of
eight α-helices (Taylor et al. 1992). The JAK2 kinase domain is relatively an “open” conforma­
tion, but the ATP-binding site is relatively “closed” when compared to other kinases (Wilks 2008).
The two lobes of the JAK2 kinase domain are slightly twisted with respect to other kinases. The
ATP-binding site is relatively less accessible as a consequence. A loop structure located between
amino acids 1056–1078, termed the JAK2 kinase insertion loop, is rather a unique feature not
observed in any other kinases (Wilks 2008). JAK3 has ~60% sequence identity with JAK2 and
most of the amino acid residues are conserved in the ATP-binding cleft (Boggon et al. 2005).

2.5.2 Pseudokinase Domain

JAKs are named after Roman God “Janus” as they have a kinase and “kinase-like” pseudokinase
domain. The pseudokinase domain has been suggested to have critical positive and negative
regulatory roles (Saharinen, Takaluoma, and Silvennoinen 2000, Saharinen, Vihinen, and Silvennoi­
nen 2003). Despite lacking key residues that are shown to be important for catalytic activity, this
domain has retained the capability to bind nucleotides in the presence of divalent cations and
mediates essential functions in regulating catalytic activity. The pseudokinase domain shares high
degree of sequence similarity with the kinase domain, but residues required for phosphotransferase
activity are modified from the canonical motifs. Among the four JAK molecules, only the JAK2
pseudokinase domain possesses weak catalytic activity and autophosphorylates itself in cis on two
auto-inhibitory phosphorylation sites, Ser523 and Tyr570 (Argetsinger et al. 2004, Ishida-Takahashi
et al. 2006, Ungureanu et al. 2011). Evidence of JAK2-specific mechanism of activation comes from
the fact that those residues are not conserved among the other JAKs. Two models of JAK activation
have been hypothesized. The pseudokinase domain binds and inhibits the kinase domain either in cis
or in trans (Babon et al. 2014). The JAK pseudokinase domains act as protein interaction modules,
whose primary function is to bind and inhibit the JH1 domain activation. However, many studies
have reported conflicting findings on the mechanism of inhibition. Furthermore, it is suggested that
nucleotide binding to these pseudokinase domains primarily function to modulate the overall
conformation of the JAK (Hammaren et al. 2015). The amino acid residues forming the “activation
loop” in the JAK2, JAK1, and TYK2 pseudokinase domain form a well-defined conformation that
resembles inactive protein kinase structures. These structures have been hypothesized to block the
putative substrate binding site, subverting the fact that a rearrangement of JAK2 pseudokinase
activation loop is required for phosphoryl-transfer activity. The importance to arrange a specific
conformation to allow intermolecular interactions is evident from crucial stabilization mediated by
ATP binding on purified recombinant JAK1 and JAK2 pseudokinase domains (Hammaren et al.
2015). In polycythemia vera (PV) patients, mutations in the JAK2 pseudokinase-SH2 linker region,
including K539I, were recently characterized that reportedly led to constitutively activated JAK2
(Zhao et al. 2009). Two distinct mutational hotspots within the JAK2 pseudokinase domain have
been identified based on functional studies of activation (Babon et al. 2014). Close to V617F,
mutations point to the N-terminal lobe of the domain. The pseudokinase-SH2 linker region was
found to confer factor-independent growth. However, there was no detectable increase in the
catalytic activity (Babon et al. 2014). Interestingly, the crystal structure of the JAK2 pseudoki­
nase domain possessing the activating mutant, V617F, indicates toward increased structural
integrity within this domain leading to pathogenesis (Babon et al. 2014). In contrast, another
structure of the JAK1 pseudokinase domain demonstrated that the αC region length can be
variable regardless of the presence of the subsequent activating mutation, V658F (Toms et al.
2013). Examples exist to support JAK phosphorylation on other sites that appear to be
important for regulation of catalytic activity. For example, Y785 in JAK3 and Y813 in JAK2
denote other important sites of autophosphorylation (Kurzer et al. 2004). These sites recruit
SH2-domain-containing adapter molecules such as SH2-Bb. For JAK2, this could be a mechanism
14 JAK-STAT Signaling in Diseases

to enhance catalytic activity. APS, another related adapter, also binds JAK2, but it negatively
regulates the activity in contrast (O’Brien, O’Shea, and Carter-Su 2002).

2.5.3 SH2 Domain

A sequence-specific phosphotyrosine-binding module, the SH2 domain is present in many signaling
molecules. In humans, SH2 domains constitute the largest class of phosphotyrosine-specific recogni­
tion domains (Kaneko et al. 2012). The SH2 domain controls cellular localization, substrate
recruitment, and regulation of kinase activity in proximity to the SH1 domain located at the
N-terminal of cytoplasmic tyrosine kinases (Filippakopoulos, Muller, and Knap 2009). The position­
ing of an N-terminal SH2 domain followed by a kinase domain is strongly conserved in all family
members of cytoplasmic tyrosine kinases (Filippakopoulos, Muller, and Knap 2009). Evolutionary,
this might have resulted in an invariant signaling unit associated with the occurrence of tyrosine
phosphorylation. In eukaryotic cells, protein kinase activity is tightly regulated. Majority of the
kinases are kept in an inactive state that can be rapidly activated by interaction with regulatory
elements such as interacting with proteins or domains located outside the catalytic domain to ensure
specific propagation of cellular signals (Wagner et al. 2013). It is suggested that this specific domain
arrangement are meant to target kinases to their substrates. Initial structural studies indicated that
SH2 domain stabilized the inactive state of Src family members (Filippakopoulos, Muller, and Knap
2009). However, subsequent biochemical characterization studies implied that the presence of the
SH2 domain is required for catalytic activity. This event stabilizes the active state of many non-
receptor tyrosine kinases. Furthermore, signaling selectivity is rapidly increased by recruiting
kinases to signaling complexes (Ardito et al. 2017). Interactions with secondary substrate
localization sites aid in selective targeting of substrates. Post-translational modifications also
help in quick propagation of signals. The SH2 domain has additional regulatory functions
including allosteric regulation of the kinase catalytic domain that is commensurate with
enhanced signaling complexities in higher order eukaryotes (Filippakopoulos, Muller, and
Knap 2009). Using a large number of biophysical and biochemical studies, the selectivity of
SH2 domains for their phosphotyrosine substrates have been determined. Approximately 50% of
the binding affinity comes from the phosphate moiety of the phosphotyrosine residue (Kaneko
et al. 2012). Directed phosphopeptide library-screening based studies indicated that residues in
positions from −2 to +4, relative to the phosphotyrosine, regulate binding specificity (Bradshaw,
Mitaxov, and Waksman 1999, Machida et al. 2007). In contrast, studies involving some co-
crystal structures suggested larger contact interfaces spanning from residues −6 to +6 (Liu et al.
2006). The overall structure of the JAK-family SH2 domain is comprised of a central β-sheet
flanked by two α-helices that resembles a canonical phosphotyrosine-binding SH2 domain
(Ferrao and Lupardus 2017). In canonical SH2 domains, two loops that go around the SH2-αB
helix form a hydrophobic groove at positions +3 and +5 relative to the pTyr opening up the
binding site for specificity determining residues (Bradshaw and Waksman 2002). In addition, the
conserved, phosphate-coordinating arginine residue located at the base of the pTyr binding
pocket is conserved in all JAKs, except TYK2, where it has been replaced with a histidine residue
(Ferrao and Lupardus 2017). In non-receptor tyrosine kinases, the conserved SH2–kinase unit is
flanked by additional domains that could include the N-terminal flanking region, a Src homol­
ogy 3 (SH3) domain, a second SH2 domain, a sequence of PH (plecstrin homology)–BTK–SH3
domains, and the FERM domain (Filippakopoulos, Muller, and Knap 2009). We discuss the
FERM domain in the next section.

2.5.4 FERM Domain

The FERM domain is conserved as a number of FERM domain-containing proteins, which are
found in present-day plants and basal eukaryotes. The FERM domain is named after
the founding protein members identified with this domain (band 4.1, ezrin, radixin, and moesin)
(Ferrao and Lupardus 2017). This domain is comprised of three subdomains: F1, F2, and F3,
JAK-STAT Structure Function 15

which are structurally similar to ubiquitin, acyl-CoA binding, and plecstrin homology­
phosphotyrosine-binding domains, respectively (Tepass 2009). The FERM domain mediates
protein–protein interactions that include adaptor and scaffolding interactions with membrane-
bound proteins. JAK-STAT signaling components also consist of several other domains that
provide accessory functions. The SOCS box is approximately 40-residue motif that mediates
interactions with proteasomal degradation pathway components, thus regulating protein half-life
(Kile et al. 2002). FERM-domain containing proteins can be divided into three broad groups:
(1) talins and kinesins; (2) ERMS, GEF, kinases, and phosphatases; and (3) myosins and KIRT
(Frame et al. 2010).
Structural model of the JAK FERM and SH2 domains and the mechanism of JAK interac­
tion with the receptors remained a challenge till five years back, owing to poor expression and
solubility of JAK protein crystallization (Lupardus et al. 2011). In 2014, co-purification with
a stabilizing receptor led to an initial X-ray crystal structure of the human TYK2 FERM and
SH2 domains, followed by crystal structures of the human JAK1 and JAK2 FERM–SH2
fragments in 2016 (Wallweber et al. 2014, Ferrao et al. 2016, McNally, Toms, and Eck 2016,
Zhang, Wlodawer, and Lubkowski 2016). These structures revealed that the JAK FERM and
SH2 domains are tightly associated to form a single receptor-binding module (Ferrao and
Lupardus 2017). Molecular modeling suggested that this domain could adopt a classical
FERM-domain structure. Two additional loops are found within the characteristic FERM
structure that are absent from other FERM domains, which are not well conserved between
JAK family members. The overall structural organization of the JAK FERM is similar to
canonical FERM domains. These subdomains pack into a canonical trilobed FERM architec­
ture (Ferrao and Lupardus 2017).
While the overall domain topology is conserved, JAK FERMs have several key differences
compared to FERM domains from the ezrin/radixin/moesin and FAK families that lead to close
contact and interaction with the SH2 domain (Ferrao and Lupardus 2017). L1, the elongated
linker joins the F1 and F2 domains to construct a major portion of the interaction surface
between the FERM and SH2 domains (Ferrao and Lupardus 2017). L1 is extended between 29
and 42 residues in JAKs that is different from the typical 13 and 15 residues in classical FERMs
(Ferrao and Lupardus 2017). Apart from the L1 linker, the SH2 domain also stacks against the
F1-α1 helix, L2 (the highly conserved F3–SH2 linker), and the L3 linker (SH2–pKD linker)
(Wallweber et al. 2014). The difference between JAK FERM and classical FERM is also evident
in the F2-subdomain structure (Ferrao and Lupardus 2017). The extension of the N-terminal
end of F2-α2 helix by one turn in JAK2 and two turns in TYK2 and JAK1 occurs due to
additional residues in the linker region between F2-α1 and F2-α2 (Ferrao and Lupardus 2017).
In JAK FERMs, the C-terminal end of the F2-α2 and the N-terminal end of F2-α3 helices also
increased by one turn. Multiple loops within the F3 subdomain display significant variability
between different JAK isoforms (Wallweber et al. 2014). The first loop contains 12 amino acids
in JAK2 between F3-β1 and F3-β2 strands that are the mostly disordered in the crystal structure
(Ferrao and Lupardus 2017). In JAK1 and TYK2, the F3-β1/β2 loop contains 22 amino acids
and 35 amino acids, respectively (Ferrao and Lupardus 2017). F3-β1/β2 loop of JAK1 forms
a β hairpin that packs against F3-β7, extending the β-sheet by a single strand, F3-β1 (Ferrao
and Lupardus 2017). Similarly in TYK2, the loop also forms a single-strand packing against
F3-β7, with an additional visible loop. In TYK2, the C-terminal end of the linker is unstruc­
tured (Ferrao and Lupardus 2017). Another difference lies between the JAK FERM and
classical FERM in the large insertions at this position that are specific to JAK-family FERM
domains (Ferrao and Lupardus 2017). The classical FERM domains contain only a short loop
at this position. An additional disordered loop of variable length is located in between F3-β3
and F3-β4. The linker lengths of F3-β3/β4 region are 34, 12, and 44 amino acids in JAK1,
JAK2, and TYK2, respectively (Ferrao and Lupardus 2017). These insertions are also not
present in canonical FERM domains thereby differing from the JAK FERM domain. However,
the length and sequence identities of these JAK F3 insertions are tightly conserved between all
higher eukaryotic species.
16 JAK-STAT Signaling in Diseases

2.6 STAT Domains

As described previously, STAT proteins have a modular structure with six well-defined domains
(Baker, Rane, and Reddy 2007). The functions of STATs are mediated by N-terminal region since
small deletions in this region do away with the STAT phosphorylation (Baker, Rane, and Reddy
2007). The N-terminal region of STAT is well conserved among family members and cooperates
with the DNA-binding domain (Baker, Rane, and Reddy 2007). Additionally, this domain also
has major roles in nuclear import, export, and receptor binding. Coiled-coil domain of STAT
interacts with regulatory proteins, adopting α-helical conformation in doing so (Kisseleva et al.
2002). This domain functions in receptor binding. Similar to the N-terminal domain, the DNA-
binding domain is also highly conserved among STAT family members (Baker, Rane, and Reddy
2007). Apart from STAT2, all STAT homodimers differentially bind more than 10 related
γ-activated sequence elements characterized by the consensus sequence, TTNCNNNAA
(Horvath, Wen, and Darnell 1995, Xu, Sun, and Hoey 1996). The linker domain functions as
a spacer to retain true conformation between the dimerization and DNA-binding domains (Baker,
Rane, and Reddy 2007). The SH2 domain, which is also found in JAKs, is highly conserved
among the STAT molecules. In playing a key role in STAT signaling, the SH2 domain recruits
STATs to activated receptor complexes. Moreover, the SH2 domain is also meant for interaction
with JAK and Src kinases (Baker, Rane, and Reddy 2007). The same domain is also required for
dimerization (both homo- and heterodimerization) of STAT molecules. This event leads to nuclear
localization and DNA-binding activities (Baker, Rane, and Reddy 2007). The transactivation
domain unsurprisingly modulates the transcriptional activation of target genes but varies among
family members. Modulation of transcriptional activation is evident when the C-terminally
truncated isoforms of STAT3, 4, and 5 minus the portions of their transactivation domains, act
as dominant-negatives (Schindler and Strehlow 2000).
In an unstimulated cell, STATs are monomeric and remain in unphosphorylated state. Cytokine
stimulation induces phosphorylation of tyrosine residues on the receptor that serve as docking
sites for STATs via their SH2 domains. Once bound to the receptor, all members of the STAT
family become tyrosine phosphorylated in response to cytokine stimulation at a conserved
C-terminal tyrosine residue. For example, phosphorylation of Y694 in STAT5 can be mediated
by growth-factor receptors as well as by JAK and Src kinases, depending on the nature of the cell
type and the ligand/receptor interactions (Nosaka et al. 1999). This phosphorylation event induces
STAT homo- and heterodimerization via the interaction of the SH2 domain of one STAT molecule
with the phosphotyrosine residue of another. Once phosphorylated, the dimerized STATs are then
able to translocate to the nucleus. In addition to tyrosine phosphorylation, several STATs are
regulated by serine phosphorylation at a conserved PSMP motif, which is located in the transacti­
vation domain (Jatiani et al. 2010). C-terminal serine phosphorylation is stimulated by several
cytokines and is mediated by serine/threonine kinases that include extracellular responsive kinase,
p38, JNK, and protein kinase C-δ (Khwaja 2006). This phosphorylation event positively regulates
the transactivation potential of these proteins. One study has revealed that the specificity of
signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation
(Kovarik et al. 2001). This culminates in differential regulation of specific target gene expression. To
acquire complete activation of STAT1, phosphorylation needs to happen at both Y701 and S727
residues (Kovarik et al. 2001). Interestingly, S727 phosphorylation of STAT1 in IFN-γ-treated
mouse fibroblasts requires an intact SH2 domain and phosphorylation of Y701 without any
requirement for p38 MAPK, ERK-1 and -2, and JNK (Kovarik et al. 2001). In contrast,
UV-induced STAT1 phosphorylation on S727 does not need SH2 domain–phosphotyrosine interac­
tions, but require p38 MAPK (Kovarik et al. 2001). Mutation of S727 differentially affected IFN-γ
target genes, at the level of both basal and induced expression. Furthermore, S727 of STAT3 is
phosphorylated in response to stimuli that differ from those for STAT1 S727, and transfer of the
STAT3 C terminus to STAT1 changed the pathway specificity of STAT1 Ser727 phosphorylation to
that of STAT3 (Kovarik et al. 2001). This series of experiments show that STAT C terminus
JAK-STAT Structure Function 17

contributes to the specificity of cellular responses by linking individual STATs to different kinase
pathways and doing so through an intrinsically different requirement for serine phosphorylation at
different target gene promoters.

2.7 Negative Regulation of JAK-STAT Pathway

It is evident that cytokines or growth factors signal through JAK-STAT pathway. Unsurprisingly,
multiple studies over several years have shown that dysregulated cytokine receptor signaling is the
foundation of many mammalian diseases. Normal cytokine signaling is dependent on the complex
tuning of cytokine-dependent signals. There are mechanisms that work against these signals to
allow a cell to be attuned to additional extracellular signals. There are multiple mechanisms by
which cytokine signaling via JAK-STAT pathway is negatively regulated. In this section, we
deliberate upon the negative regulation of JAKs via phosphatase, inhibition of STATs by protein
inhibitors of activated STATs (PIAS) and suppressor of cytokine signaling (SOCS) proteins.

2.7.1 Effect of Protein Tyrosine Phosphatases (PTPS)

Tyrosine phosphorylation leads to the signaling that emanates ensuing cytokine–cytokine receptor
interaction. The event of JAKs phosphorylating each other on tyrosine residues in activation loops is
known as transphosphorylation. Due to the critical importance of JAK tyrosine phosphorylation for
signal transduction, dephosphorylation of tyrosine residues is a mechanism employed by cells to
attenuate kinase activity of JAKs. Several protein phosphatases act as modulators of JAK-STAT
signaling. They include the SH2 domain-containing tyrosine phosphatases (SHPs), protein tyrosine
phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TC-PTP), and protein tyrosine phospha­
tase-basophil like (PTP-BL). Among these, SHPs are well characterized. The classical PTPs are
Cys-based class I PTPs that are true tyrosine-specific PTPs (Alonso et al. 2004). Based on their cellular
localization, the classical PTPs are divided into transmembrane (TM) and non-transmembrane (non-
TM) families (Tonks 2006). Few TM PTPs have been identified in T cells. Non-TM PTPs are better
characterized than their TM counterparts (Lorenz 2009). Non-TM PTPs contain a single catalytic PTP
domain flanked by N-/C-terminal extensions required for localization (Lorenz 2009).
SHPs were the first phosphatases to be identified. The human genome project has identified
more than 100 putative PTPs (Alonso et al. 2004). The major SHPs are SHP-1 and SHP-2. SHP-1
is primarily expressed in hematopoietic cells, while SHP-2 is ubiquitously expressed (Wu et al.
2003). A spliced variant of SHP-1 has been identified as SHP-1L (Jin, Yu, and Burakoff 1999).
Both SHP-1 and SHP-2 are composed of a central catalytic domain, two N-terminal SH2
domains, and a C-terminus (Poole and Jones 2005). The central catalytic domain contains the
typical PTP signature motif VHCSAGIGRTG (Poole and Jones 2005). These phosphatases are
named due to the two SH2 domains located N-terminal to the tyrosine phosphatase domain. The
SH2 domains aid in SHP recruitment to tyrosine-phosphorylated intracellular portions of many
receptors that aid in localization and activity regulation (Pao et al. 2007). SHPs inhibit JAK­
STAT signaling in two ways: by impeding interactions with other effectors and by activating the
elimination of the phosphotyrosine motifs acting as docking sites for effectors (Seif et al. 2017).
The SHP-2 SH2 domains display overlapping binding specificities with the SOCS SH2 domain,
indicating that both negative regulators compete for binding sites on phosphorylated receptors.
The interaction of the SHP SH2 domains with their binding partners is a key step toward the
activation of the phosphatase activity. At the basal state, this activity is repressed as N-terminal
SH2-domain occupies the phosphatase active site of PTP in the intramolecular fashion (Hof et al.
1998). The release of this suppression leads to an activation of the phosphatase upon engagement
of the SH2 domains. SHP-1 is a major negative regulator of receptor signaling in hematopoietic
and epidermal cells (Lorenz 2009). Apart from the SH2 domains, the C-terminus could regulate
the PTP-1 activity of SHP-1. Upon stimulation tyrosine phosphorylation of the C-terminal Y536
18 JAK-STAT Signaling in Diseases

in SHP-1, PTP activity is enhanced (Zhang et al. 2003). Phosphorylation of Tyr564 leads to
a modest increase of SHP-1 activity (Zhang et al. 2003). The role of SHP-2 in growth factor
signaling remains less understood. Additional studies are required to completely elucidate the role
of SHP-2 in hematopoiesis. The intrinsic differences in the catalytic domain allow SHP-2 to portray
different substrate specificities. SHP-2 associates with multiple growth-factor receptors, but few of
those interactions are true SHP-2 substrates. Additionally, SHP-2 could enact as an adapter and has
several phosphotyrosine motifs that can recruit downstream effectors. Phosphorylated tyrosines
have been hypothesized to generate docking sites for SH2-domain containing proteins, which could be
the basis of either an adapter function for SHP-2 or a way for preventing auto-dephosphorylation.
SHP-1L lacks the potential Y564 phosphorylation site, but retains the Y536 site of SHP-1 (Lorenz
2009). SHP-1L, however, has a proline-rich motif in the C-terminus, which could mediate binding to
SH3-domain containing proteins (Lorenz 2009). A similar proline-rich motif is absent in SHP-1, but
can be observed in the C-terminus of SHP-2 (Lorenz 2009). Critical biological functions of SHP-1
and SHP-2 have been demonstrated using mice models.
PTP1B is a negative regulator of the insulin and leptin signaling pathways. PTP1B comprises of
a single catalytic domain with N- or C-terminal extensions (Fischer, Charbonneau, and Tonks
1991). Similar to SHP-1L, PTP1B has a proline-rich region adjacent to the C-terminal and
a hydrophobic region (Liu, Hill, and Chernoff 1996). Substrates are recruited by the interaction of
the proline-rich region with the SH3 domains. The hydrophobic sequence is inserted into the
endoplasmic reticulum membrane and ties up PTP1B to the cytoplasmic side of the ER (Frangioni
et al. 1992). The substrate selectivity of PTP1B is controlled partially by the modular protein
interaction and the spatial restriction imposed by subcellular compartmentalization (Tonks 2003).
PTP1B also acts as a negative regulator of brown fat adipogenesis (Matsuo et al. 2011).
The phosphatases, PTP1B and TC-PTP, share 74% identity between their catalytic domains
(Tonks, Diltz, and Fischer 1988). TC-PTP was characterized almost three decades ago. cDNA of
PTPN2 codes for TC-PTP. TC-PTP, ubiquitously expressed, shows its highest expression in
hematopoietic tissues (Ibarra-Sanchez et al. 2001). TC-PTP could be induced by either a mitogen
(Con A) or an anti-inflammatory cytokine (IL-10) (Rajendrakumar, Radha, and Swarup 1993,
Williams et al. 2004). Expression of TC-PTP results in two isoforms due to alternative splicing. The
two forms share the conservative catalytic PTP domain of 272 amino acids, but differ in the
C-terminal (Kamatkar et al. 1996). The well-characterized form of TC-PTP has 387 amino acids
and has MW of 45-kDa (TC45), while the less-abundant form consists of 415 amino acids with a MW
of 48-kDa and expressed in the ER and the nuclear membrane (Iversen et al. 2002, Bourdeau, Dube,
and Tremblay 2005). The C-terminal of TC45 aids in nuclear localization and also binds to DNA.
However, in general, the C-terminal of TC-PTP negatively regulates the enzyme function. TC-PTP
and PTP1B have complementary specificities for dephosphorylation of JAKs and STATs. Minor
differences between JAK1/JAK3 and JAK2/TYK2 sequences determine the substrate specificity of
PTP1B and TC-PTP. JAK1 and JAK3 activation loops contain either a Thr or Val residue C-terminal
to the tandem phosphotyrosines. In contrast, JAK2 and TYK2 comprise of (E/D)-pY-pY-(R/K)
motif in their activation loops (Myers et al. 2001). This specific motif has been revealed to be the
preferred substrate of PTP1B. Both PTP1B and TC-PTP participate in STAT dephosphorylation
(Bohmer and Friedrich 2014). Biological roles of PTP1B and TC-PTP have been characterized using
mouse knockout studies. Mice deficient in TC-PTP gene exhibit multiple defects in the lymphoid
population (You-Ten et al. 1997). TC-PTP-deficient mice die neonatally from a systemic inflammatory
disease characterized by mononuclear cell infiltration. PTP1B-deficient mice demonstrate hypersensi­
tivity to various growth factors (You-Ten et al. 1997, Hendriks et al. 2008). The other phosphatase, PTP­
BL, dephosphorylates STAT1, STAT3, STAT4, STAT5, and STAT6 both in vitro and in vivo.

2.7.2 Effect of Protein Inhibitor of Activated STATs

PIAS proteins are activation-suppressing proteins for STATs incorporating transcriptional regula­
tion of genes. The PIAS family of proteins consists of five proteins in mammals: PIAS1, PIASxα,
JAK-STAT Structure Function 19

PIASxβ, PIAS3, and PIASy (Chung et al. 1997). PIAS1 has been named as it has been shown to
inhibit STAT1, while PIAS3 interacts with STAT3 (Chung et al. 1997, Liu et al. 1998). Interest­
ingly, PIAS proteins neither act only as inhibitors nor show any specificity for STATs. PIAS can
regulate transcription either positively or negatively. The domain structure of PIAS include an
N-terminal SAP domain (SAF-A/B, Acinus, and PIAS), apoptotic chromatin-condensation
inducer in the nucleus, the PINIT (Pro-Ile-Asn-Ile-Thr) motif, a C3HC4-motif type RING finger-
like zinc-binding domain, an acidic amino acid domain (AD), SUMO-interacting motif, and
a variable C-terminal Ser/Thr-rich (S/T) domain (Aravind and Koonin 2000, Jackson 2001,
Jimenez-Lara, Heine, and Gronemeyer 2002, Duval et al. 2003). The SAP domain, the PINIT
motif, and the RING domain contribute to the nuclear localization of PIAS proteins (Duval et al.
2003). The SAP domain is important for sequence/structure-specific DNA binding (Aravind and
Koonin 2000). PIAS proteins could modulate JAK-STAT signaling by various means including
SUMOylation that attenuate transcription factor activity (Niu et al. 2018). The RING domain
might interact with other proteins, which is important for SUMO-E3-ligase function (Sachdev
et al. 2001). The function of AD and S/T domain remain less explored. SUMO ligase-independent
modulation of transcription factor activity of PIAS proteins exists (Kotaja et al. 2002). One such
mechanism is blocking the DNA-binding of a transcription factor. PIAS1 has been shown to
inhibit DNA-binding activities of STAT1 (Liu et al. 1998). Similarly, PIAS3 interaction with
STAT3 inhibited the DNA-binding activity of STAT3 (Chung et al. 1997). It has been hypothe­
sized that DNA-binding activity is inhibited by PIAS, obstructing the DNA-binding site of the
transcription factor within the PIAS: transcription factor complex. This could result in
a conformational change or dissociation of a transcription complex due to PIAS engagement of
transcription factor. Co-regulators, which can get recruited to a protein complex, are important
players for PIAS-mediated regulation of transcription. PIAS proteins interact with HDAC
molecules. Knockout mice studies have indicated functional redundancy between PIAS family
members. It is evident in PIASy-deficient mice that reveal minor defects in Wnt and IFN signaling
(Roth et al. 2004). Understanding the complex in vivo functions of PIAS proteins will depend on
multiple experimental approaches.

2.7.3 Effect of Suppressor of Cytokine Signaling

JAK-STAT pathway is also negatively regulated by suppressor of cytokine signaling proteins or
SOCS proteins. These proteins are not constitutively expressed. SOCS proteins bind to JAKs,
cytokine receptors, and signaling molecules. SOCS genes encode a family of eight proteins
(SOCS1-7 and cytokine-inducible SH2 protein (CIS)) (Endo et al. 1997, Naka et al. 1997, Starr
et al. 1997). These proteins contain an N-terminal of variable length, a central SH2 domain
that determines binding specificity, and a C-terminal termed as SOCS box (40 amino acid
module) that is implicated in proteasomal degradation (Endo et al. 1997, Naka et al. 1997,
Starr et al. 1997). CIS, SOCS1, 2, and 3 are well characterized, while the biological roles of
SOCS4–SOCS7 remain poorly understood (Hilton et al. 1998). CIS/SOCS family proteins act
as E3 ubiquitin ligase, mediating protein degradation of protein families associated with them.
A 24-amino acid kinase inhibitory region (KIR) is present in the N-terminal of SOCS1 and
SOCS3 (Nicholson et al. 1999). A series of studies on SOCS proteins have revealed that a wide
range of cytokines that utilize JAK-STAT signaling are inhibited by SOCS. The SH2 domain of
SOCS1 inhibits JAK2 by binding to Tyr1007 through insertion of its KIR into the activation
loop of the kinase domain (Yasukawa et al. 1999). This prevents substrates from getting into the
catalytic pocket by acting as pseudosubstrate. SOCS1 has also shown to abrogate tyrosine phosphor­
ylation of STAT1 via direct binding to phosphorylated type I IFN receptors (Fenner et al. 2006).
A model of SH2 domain: receptor interaction and KIR-mediated inhibition have been proposed.
However, the SH2 domain of SOCS3 does not possess a strong affinity toward activation loops of
JAKs (Yoshimura and Yasukawa 2012). The absence of SOCS1 expression due to gene methylation
results in the development of a range of primary cancers (Sutherland et al. 2004). SOCS3 first
20 JAK-STAT Signaling in Diseases

binds to the receptor, followed by inhibition of JAK activity via a KIR-mediated JAK inhibition
(Sasaki et al. 1999). SOCS3 also inhibits the signaling initiated by various stimuli that induce its
expression. This inhibition is predominantly achieved by SOCS3 engagement of the activated
receptors of these cytokines. SOCS3 has been demonstrated to specifically bind to phosphory­
lated tyrosines of cytokine/growth-factor receptors. A number of these receptor-docking sites also
act as binding sites for another signaling regulator, SHP2. The relative contribution of SOCS and
SHP2 to signal modulation and pathway crosstalk is still not completely understood. Intriguingly,
SOCS3 is not phosphorylated by JAKs, but relies on other kinases, such as Src kinases and
receptor-tyrosine kinases, for activation (Sommer et al. 2005). Absence of SOCS3 has been
implicated to enhance susceptibility to chronic inflammatory diseases. A lack of SOCS3 correlates
with the development and progression of malignancies (Riehle et al. 2008). The inhibitory
mechanisms of CIS and SOCS2 differ from that of SOCS1 and SOCS3 (Krebs and Hilton 2001).
Neither CIS nor SOCS2 acts on JAKs (Krebs and Hilton 2001). The biological role of CIS is not
completely understood. There might be functional redundancy of CIS. Role of SOCS2 has been
evaluated following the development of SOCS2-deficient mice that displays gigantism phenotype
rising from dysfunction of growth hormone axis (Metcalf et al. 2000). SOCS2 partially inhibits
recruitment of STAT5 by binding pTyr487 and pTyr595 on the growth hormone receptor
(Greenhalgh et al. 2005). A number of studies have linked SOCS2 to cancer, including colon and
prostate cancer.
SOCS are basically ubiquitin ligases that promote the degradation of their partners. The SOCS
box interacts with Elongin B and Elongin C, Cullin-5, and RING-box-2 to form an E3 ligase
complex termed as ECS (Elongin B/C-Cullin-SOCS box protein) (Bullock et al. 2006). The ECS
binds to a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), allowing
the ECS complex to recognize and bind target proteins for polyubiquitination and degradation
via the 26S proteasome (Kamura et al. 2004). Proteasomal degradation has emerged as a key
mechanism of signal attenuation. The nuclear protein, SLIM, has been characterized as
a ubiquitin E3 ligase that targets STATs for degradation via the proteasome (Tanaka, Soriano,
and Grusby 2005). The interaction of the SOCS box with the ECS complex provides a clear link
between SOCS proteins and proteasomal degradation that is central to attenuating the levels of
SOCS proteins within the cell (Zhang et al. 1999). It can be argued if SOCS proteins can act as
adaptors to bridge their binding partners to the proteasome and induce their degradation.
Various overexpression studies including SOCS proteins have supported a cross-talk between
different members of the SOCS family. Physiological evidence for the importance of the SOCS
box has been garnered from in vivo studies.

2.7.4 STAT Inhibitors as Therapeutic to Treat Inflammatory Disorders

JAK-STAT pathway is critical to the pathogenesis of inflammatory disorders mediated by the
immune system. Back in the 1990s, the potential of JAK inhibitors to treat autoimmune and
inflammatory disorders had been conceived (Banerjee et al. 2017). The role of JAK-STAT
signaling in various inflammatory disorders has been extensively mentioned in this book.
Extensive efforts have been generated to develop second-class JAK inhibitors that are more
selective. Dysregulated expression of STAT molecules has been observed in various cancers.
Hence, the discovery of STAT inhibitors has mainly targeted tumor cells. In the last section of
the chapter, we will discuss the development of STAT inhibitors to treat inflammatory disorders.
We will discuss the advent of small molecule inhibitors of STAT molecules for therapeutic
purposes.

[Link] STAT1 Inhibitor

Studies have demonstrated the development of very few STAT1 inhibitors. BRD0476, a novel
suppressor of pancreatic β-cell apoptosis, was developed. BRD0476 inhibited IFN-γ-induced
JAK-STAT Structure Function 21

JAK2 and STAT1 signaling to augment β-cell survival (Chou et al. 2015). BRD0476 inhibits
JAK-STAT signaling without suppressing the kinase activity of any JAK. An analog of BRD0476
was developed by adding quinolone moieties in place of naphthyl that led to more than 1000-fold
solubility as well inhibited IFN-γ/STAT1 signal transduction (Scully et al. 2013). Pravastatin is
another STAT1 inhibitor that attenuates the IFN-γ levels and prevents STAT1 phosphorylation
by inhibiting HMG-CoA reductase (Zhou, Gao, and Sun 2009). Pravastatin treatment on
Apolipoprotein E-deficient mice fed on a cholesterol-rich diet suppressed IFN-γ levels in both
serum and atherosclerotic lesions, associated with reduced STAT1 activation, attenuated IRF1
expression, and induced activity of SOCS1 in aorta tissue (Zhou, Gao, and Sun 2009). ISS-840,
a phosphopeptide mimetic of STAT1, disrupted STAT1 dimerization leading to moderately
potent inhibition of STAT1 signaling (Gunning et al. 2007). Fludarabine, a compound used to
treat hematologic malignancies, is another STAT1 activation inhibitor causing a specific depletion
of STAT1 protein, but not of other STAT molecules (Feng et al. 2017). Fludarabine exerts
apoptosis through increasing Bax and decreasing Bid, XIAP, and survivin expression (Meng
et al. 2007).

[Link] STAT3 Inhibitor

Among the STAT-specific inhibitors, STAT3 inhibitors are most widely studied. S3I-201, a novel
STAT3 inhibitor identified from NCI chemical library, displayed potent inhibition of STAT3 DNA-
binding activity with IC50 of 86 μM in cell-free assays (Siddiquee et al. 2007a). S3I-201 inhibited
STAT3 transcriptional activities, and induced growth inhibition and apoptosis of tumor cells
expressing constitutively active STAT3 (Grandis et al. 2000). In liver fibrosis, angiogenesis and
fibrogenesis were attenuated by S3I-201 (Wang et al. 2018). Derivatives of S3I-201, S3I-201.1066,
S3I-1757, and BP-1-102, displayed increased potencies of STAT3 inhibition (Zhang et al. 2010, 2012,
2013). S3I-201 administration resulted in appreciable decrease in IFN-γ, T-bet, IL-17A, RORγt,
Stat3, IL-21, and IL-22 levels, and increase in Foxp3 and Helios production CD4+ T cells in BTBR
mice, suggesting that S3I-201 could be used to treat autism (Ahmad et al. 2018). S3I-M2001 is an
oxazole-based peptidomimetic of the STAT3 SH2 domain-binding phosphotyrosine peptide that
selectively disrupts active STAT3:STAT3 dimers in various cancers where STAT3 expression is
aberrant (Siddiquee et al. 2007b). Stattic, a nonpeptidic small molecule identified after screening of
chemical libraries, has been displayed to selectively inhibit the function of the STAT3 SH2 domain at
10 μM, independent of the STAT3 activation state in vitro (Schust et al. 2006). STAT3 activation,
dimerization, and nuclear translocation are selectively inhibited by Stattic (Schust et al. 2006).
Fragments of STX-0119 (another STAT3 SH2 domain antagonist) and Stattic were chemically fused
to generate HJC0123, which suppressed STAT3 phosphorylation and transcriptional activity in
breast cancer cells and induced anti-tumor cell effects against breast and pancreatic cancer cells
in vitro at IC50 values of 0.1–1.25 μM (Ashizawa et al. 2011, Chen et al. 2013b). By inhibiting STAT3,
Stattic reduces the growth and increases the apoptosis of NPC and sensitizes NPC to cisplatin and IR
(Pan et al. 2013). STA-21 is another small molecule inhibitor of STAT3 with a potency of 20 μM.
STA-21 inhibits STAT3 dimerization and binding to DNA resulting in a transcriptional blockade of
STAT3-dependent genes (Song et al. 2005). STA-21 has been shown to be useful in several
inflammatory disease conditions (Park et al. 2014). STA-21 suppresses joint inflammation in
a mouse CIA model through regulation of the transcription factors, Foxp3 and RORγt, in CD8+
T cells and inhibits the development and progression of rheumatoid arthritis (Ahmad et al. 2017).
Studies suggest that LLL12, another small-molecule inhibitor of STAT3 signaling, might suppress
STAT3 activation by blocking its recruitment to the receptor, preventing phosphorylation by tyrosine
kinases, and by interfering with dimerization (Bid et al. 2012). LLL12 suppressed cell viability,
induced apoptosis, and repressed colony formation and migration in vitro in studies of glioblastoma,
osteosarcoma, and breast cancer cells (Lin et al. 2010, Onimoe et al. 2012). In a drug development
program involving virtual ligand screening, 2-D similarity screening, 3-D pharmacophore analysis,
and SAR-based medicinal chemistry, C188-9, a potent small-molecule that targets the Src-homology
22 JAK-STAT Signaling in Diseases

SH2 domain of STAT3 was identified (Jung et al. 2017). C188-9 inhibited growth and survival of
several cancer cell lines in vitro, including breast cancer, acute myeloid leukemia, head and neck
squamous cell carcinoma, and non–small cell lung cancer (Jung et al. 2017). C188-9 impedes nuclear
translocation of STAT3, prevents STAT3 binding to its target gene promoters, and inhibits STAT3­
mediated regulation of gene expression (Bharadwaj et al. 2016). Another small molecule, non­
phosphorylated STAT3 inhibitor, SH-4-54 strongly binds to STAT3 protein (KD = 300 nM) (166).
SH-4-54 potently kills glioblastoma brain cancer stem cells and suppresses STAT3 phosphorylation
and its downstream transcriptional targets at low concentrations in the nM level (Haftchenary et al.
2013). Moreover, in vivo, the inhibitor exhibited blood–brain barrier permeability, potently controlled
glioma tumor growth, and inhibited pSTAT3 in vivo (Haftchenary et al. 2013). HJC0152, an
O-alkylamino-tethered derivative of niclosamide is a potent STAT3 inhibitor with remarkably
improved aqueous solubility (Chen et al. 2013a). HJC0152 exerted a significant anticancer effect on
HNSCC tumor growth and invasion (Wang et al. 2017). HJC0152 also inhibits STAT3 activation in
GC cells, and retards their growth in vitro and in vivo (Jiang et al. 2018). BBI608 is a small molecule
STAT3 inhibitor known to suppress cancer relapse, progression, and metastasis (MacDonagh et al.
2018). BBI608 can inhibit stemness gene expression, deplete CSCs, and overcome cisplatin resistance
in NSCLC (MacDonagh et al. 2018). BBI608 was initially introduced and investigated as a stemness
inhibitor in the context of tumor relapse in a xenograft model of pancreatic cancer. BBI608 was later
investigated in the context of prostate cancer progression. BBI608 inhibited cell proliferation, colony
formation, and migration, while increasing the sensitivity of prostate cancer cells to the cytotoxic
effects of docetaxel (Zhang et al. 2016). HO-3867 is an analog of curcumin that selectively suppresses
STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other
active STATs (Rath et al. 2014). This analog has been shown to induce apoptosis in BRCA-mutated
ovarian cancer cells with minimal toxicity to normal cells (Tierney et al. 2012). When combined with
cisplatin, HO-3867 has demonstrated synergistic inhibition of chemotherapy-resistant ovarian xeno­
graft tumors (174). HO-3867/cisplatin combination treatment significantly inhibited cisplatin­
resistant cell proliferation in a concentration-dependent manner (Selvendiran et al. 2011). HO-3867
induces cell apoptosis by reactive oxygen species-dependent endoplasmic reticulum stress in human
pancreatic cancer cells (Hu et al. 2017). APTSTAT3-9R is an example of specific STAT3-binding
peptide with addition of a cell-penetrating motif (Kim et al. 2014). STAT3-specific aptide APTSTAT3
has been developed for therapeutic efficacy in cancer by inhibiting STAT3 signaling (Kim et al. 2014).
The treatment of APTSTAT3-9R in various types of cancer cells blocks STAT3 phosphorylation and
reduces expression of STAT targets (Kim et al. 2014). Peptide-based STAT3 inhibitors have also been
reported. These inhibitory phospho-peptides (PYLKTK, YLPQTV), based on a shortened gp130
sequence, bind to the SH2 domain of STAT3 and block dimerization (Zhang et al. 2010). In an in vivo
setting, however, the relatively low potency and limited cell permeability of such phospho-peptides
have hampered their further use. Peptide libraries have been screened for interaction with the DNA-
binding domain (DBD) of STAT3, and the identified peptides have been shown to inhibit STAT3
DNA-binding activity (Kim et al. 2014). ISS-610 inhibited malignant cell growth and induced
apoptosis in vitro. Peptidomimetic ISS-610 also exhibited good selectivity against STAT3 and its
functions (Turkson et al. 2004). ISS-610 with 4-cyanobenzoate substitution inhibits constitutive
STAT3 activity in Src-transformed mouse fibroblasts and human breast and lung cancer cells
(Turkson et al. 2004). CJ-1383 is another class of small molecule inhibitors that targets STAT3-SH2
domain. CJ-1383 binds to STAT3 with a Ki value of 0.95 μM and inhibits cell growth with IC50 values
of 3−11 μM in two breast cancer cell lines having increased phospho-STAT3 expression (Chen et al.
2010). In a time-and dose-dependent fashion, CJ-1383 is effective in inhibition of STAT3 activity and
induction of apoptosis in the MDA-MB-468 cancer cell line (Chen et al. 2010). PM-73G is one of the
first phosphopeptides targeted to an SH2 domain that inhibits its target in vivo following systemic
administration (Auzenne et al. 2012). PM-73G possesses the non-hydrolyzable difluoromethylpho­
sphonate group, and the esterase-labile POM groups block the negative charges thus allowing passive
diffusion across cell membranes (Auzenne et al. 2012). Intratumoral and intraperitoneal administra­
tion of PM-73G to mice-bearing MDA-MB-468 tumor xenografts demonstrate significant inhibition
of tumor growth in vivo, despite having no discernible in vitro effect (Auzenne et al. 2012). Zhao et al.
JAK-STAT Structure Function 23

designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed fragment-
based drug design (FBDD) (Zhao et al. 2016). The inhibitory effect on STAT3 phosphorylation, cell
viability, migration, and colony-forming ability by LY5 were examined in human liver and colon
cancer cells (Zhao et al. 2016). LY5-inhibited constitutive IL-6-induced STAT3 phosphorylation,
STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression, and induced
apoptosis in liver and colon cancer cells (Zhao et al. 2016). LY5 had little effect on STAT1
phosphorylation mediated by IFN-γ (Zhao et al. 2016).

[Link] STAT4 Inhibitor

Most STAT4 inhibitors are natural products. STAT4 protein levels decrease in response to
berbamine treatment (Ren et al. 2008). Studies also showed that the oral administration of water
extract of cinnamon bark decreased IFN-y expression and inhibited STAT4 activation in
activated murine T cells (Lee et al. 2011). Tofacitinib has been shown to inhibit STAT4 activation
in cultured anti-CD3-stimulated T cells (Migita et al. 2011).

[Link] STAT5 Inhibitor

SH-4-54 that potently inhibited STAT3 also inhibits STAT5 with KD value of 464 nM (Haftchenary
et al. 2013). The non-peptide small molecule pimozide, a neuroleptic drug, inhibits the constitutive
STAT5 Tyr694 phosphorylation (5–10 µM for 3 h) and transcription activity (5 µM for 18 h) in Bcr-
Abl+ K562 and KU812 cultures (Nelson et al. 2011). Pimozide inhibits the activating tyrosine
phosphorylation of STAT5 (Nelson et al. 2011). However, several lines of evidence suggest that
pimozide does not function as a kinase inhibitor. Pimozide neither attenuates the autophosphoryla­
tion of BCR/ABL, nor does it decrease the tyrosine phosphorylation of other cellular proteins
mediated by BCR/ABL (Nelson et al. 2011). In addition, pimozide does not decrease the activation
of a distinct pathway downstream of BCR/ABL, MAP kinase (Nelson et al. 2011). When Berg et al.
screened large small-molecule libraries in search of compounds that can modulate SH2 domain of
STAT5, chromone nicotinyl hydrazone was discovered (Muller et al. 2008). The discovery of
chromone aldehydes as inhibitors of STAT family proteins indicates toward a model by which,
under the assay conditions, acyl hydrazones are cleaved to the respective aldehydes representing the
active species. It shows reduced potency toward the SH2 domains of STAT1, STAT3, or LCK but
suppresses SH2 domain of STAT5 at IC50 of 47 μM (Muller et al. 2008). STAT5 is activated by FLT3­
ITD, which is a constitutively active TK driving the pathogenesis of AML (Wingelhofer et al. 2018).
Wingelhofer et al. developed a novel, potent STAT5 SH2 domain inhibitor, AC-4–130, which can
efficiently block pathological levels of STAT5 activity in AML (Wingelhofer et al. 2018). AC-4–130
directly binds to STAT5, thereby disrupting STAT5 activation, dimerization, nuclear translocation,
and STAT5-dependent gene transcription. AC-4-130 shows high selectivity for STAT5 over STAT1
and STAT3 (Cumaraswamy et al. 2014, Wingelhofer et al. 2018).

[Link] STAT6 Inhibitor

There are small molecule inhibitors of STAT6. AS1517499 is a novel selective STAT6 inhibitor
with an IC50 value of 21 nM (Nagashima et al. 2007). AS1517499 is a potent STAT6 inhibitor
synthesized based on the structure of a reported STAT6 inhibitor, TMC-264, discovered from
the fungus Phoma (Sakurai et al. 2003). AS1517499 also inhibited the IL-4–induced Th2 cell
differentiation of mouse spleen T cells with an IC50 value of 2.3 nM without influencing the
IL-12–induced Th1 cell differentiation (Sakurai et al. 2003). In vivo treatment with AS1517499
ameliorates the antigen-induced bronchial smooth muscle (BSM) hyperresponsiveness by
inhibiting the RhoA up-regulation in BSMs and, at least in part, by reducing the IL-13
production in the airways in mice (Chiba et al. 2009). TMC-264 is known to inhibit both
tyrosine phosphorylation of STAT6, with an IC50 value of 1.6 mM, and the complex
24 JAK-STAT Signaling in Diseases

formation of phosphorylated STAT6 with its recognition DNA sequence (Sakurai et al. 2003).
A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by
airway fungal challenge, reversed established allergic airway disease in mice, and blocked
salmeterol-dependent enhanced allergic airway disease (Knight et al. 2015). In vitro structure–
activity relationship studies led to the development of the lead compound, PM-43I (Knight
et al. 2018). Conducting initial dose range, toxicity, and pharmacokinetic experiments with
PM-43I potently inhibits both STAT5- and STAT6-dependent allergic airway diseases in mice
(Knight et al. 2018). When cellular activity screen was performed for evaluating the binding
capacity to SH2 domains of STAT molecules, PM-86I showed the highest specificity for STAT6
and no cross-reactivity to any of the additional targets at the highest dose tested (Knight et al.
2018). PM-86I can specifically inhibit STAT6-dependent adaptive Th2 immune responses when
administered systemically (Knight et al. 2018). Other notable inhibitors of STAT6 signaling
include AS1810722, a fused bicyclic pyrimidine derivative (IC50value of 2.4 nM) (Nagashima
et al. 2009). AS1810722 is potent, orally active STAT6 inhibitors that could be developed for the
treatment of STAT6-dependent allergic diseases, such as asthma. AS1810722 showed potent
STAT6 inhibition and a good CYP3A4 inhibition profile (Nagashima et al. 2009).

2.8 Concluding Remarks

JAK-STAT is one of the well-characterized signaling pathways involved in maintaining home­
ostasis. Aberrant regulation of this pathway affects the downstream signaling molecules leading to
deleterious consequences including onset and pathophysiology of auto-immune disorders. This
chapter has elaborated on the structure of JAK and STAT domains that can be targeted for
therapeutic benefits. Subsequent chapters of the book have discussed the implication of JAK­
STAT signaling in various inflammatory disorders.

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3
MicroRNA-Mediated Regulation of JAK-STAT
Signaling in Non-Cancerous Human Diseases

Chandra S. Boosani and Devendra K. Agrawal

Department of Translational Research
College of Osteopathic Medicine of the Pacific
Western University of Health Sciences
Pomona, CA

Wanlin Jiang
Cardiovascular Research Center
Massachusetts General Hospital
Boston MA

Taylor Burke
Department of Clinical and Translational Science
Creighton University School of Medicine
Omaha, NE

3.1 Brief Background

Targeting a signaling pathway that is pathologically active and specific to a given disease could be more
enticing to pursue development of drug targets for disease prevention. Even more is when the same
pathway participates in multiple diseases and plays an important role in progression of the disease.
Certainly, of that kind would be the Janus kinase (JAK)-signal transducers and activators of transcrip­
tion (STAT) signaling pathway that has been identified as an important activator of intra-cellular
signaling molecules, which are associated with many pathological conditions. Notably, this signaling
mechanism promoting key cellular activities was identified to be directly associated with growth,
proliferation, and migration in different cell types. Its role in proliferative disorders, cancer, and
immune regulation was seen to be under tight control by STAT, which is activated by JAK. As
a result of STAT-induced transcriptional activity, expression of several key inflammatory molecules was
reported, which promote the pathological progression of the disease. Both direct and indirect activation
of STAT by JAK has been reported, which denotes their robust involvement and regulation of multiple
cross-connecting pathways, which underscores their participation in diverse cellular mechanisms.
Adding to the already-existing complexity in gene-regulatory mechanisms, the discovery of micro-
RNAs and their proven role in controlling gene expression, layers another dimension of multiple gene
regulations that are driven by a single miRNA (microRNA). At the same time, identification of
miRNA targets also facilitated the predictions of possible cross talks between different pathways. The
miRNAs are short oligomers with full amenability to modify and synthesize. In addition, the ease of
their delivery into the cells and in live animals make them a more favorable choice to use in clinical
research. Further, miRNAs as such have become a tool to drive or prevent their target pathways,
which highlights the potential of miRNAs as another therapeutic avenue to treat human and animal

35
36 JAK-STAT Signaling in Diseases

diseases. Recent advancements using bioinformatic tools have enabled identification of specific loci for
different miRNAs on the chromosomes, which furthered our understanding on the origin and
putative source of miRNAs. The increasing number of miRNAs being identified as potential
therapeutic candidates and their proven success in pre-clinical trials and animal trials show the
promise and safety of miRNAs for disease treatment.
Ample literature evidence suggest that several miRNAs regulate JAK-STAT pathways in cancer,
and many previous publications have previously discussed about this topic. In this chapter, we
specifically tried to exclude discussing about the role of miRNAs that regulate JAK-STAT pathways
in cancer due to the vast abundance and volume of literature available on this topic. At the same
time, very little literature exists that show the role of miRNAs in regulating JAK-STAT pathway in
other diseases (Table 3.1). Therefore, in this chapter, we remain focused on those miRNAs that were
validated to be regulating JAK-STAT pathway in non-cancerous diseases. Although efforts were
made to provide comprehensive information on this topic, the readers are encouraged to refer to the
bibliography below and the current literature for more details.

TABLE 3.1
List of miRNAs and their Target Transcripts that Affect JAK-STAT Signaling in Non-Cancerous Human
Diseases
Anticipated effect
Target of JAK-STAT
miRNA transcript pathway Test model Disease model Reference

miR-19a SOCS1, Inducing effect HuH-7 Liver diseases (Collins et al. 2013, e69090)
SOCS3, immortal cells
SOCS5
miR-155 STAT1 Inhibitory effect Live mice Liver injury (Lv et al. 2015, 6013–6018)
miR-1264 DNMT1 Inhibitory effect Smooth Coronary artery (Boosani et al. 2015,
muscle cells disease 1365–1376)
miR-181a To be char- Inducing effect Dendritic cells Heart disease (Zhu et al. 2017, 2884–2895)
acterized
miR-150 To be char- Inhibitory effect Dendritic cells Heart disease (Zhu et al. 2017, 2884–2895)
acterized
miR-146b STAT5A Inhibitory effect Heart tissue Cardiac (Feng et al. 2014, 361–368)
hypertrophy
miR-499 STAT2 Inhibitory effect Heart tissue Cardiac (Feng et al. 2014, 361–368)
hypertrophy
miR-19a TNF-α, Inducing effect Human Arthritis (Li et al. 2016 2531–2536)
STAT3, PBMCs
SOCS3
miR-21 TNF-α, Inducing effect Human Arthritis (Li et al. 2016 2531–2536)
STAT3, PBMCs
SOCS3
miR-1246 HNFγ Inducing effect Chondrocytes Arthritis (Wu et al. 2017, 2010–2021)
miR-1246 PRKAR1A, Inducing effect Epithelial cells Inflammation and (Bott et al. 2017,
PPP2CB cancer 43897–43914)
miR-203 MCL-1 Inhibitory effect Human carti- Osteoarthritis (Zhao et al. 2017 171–178)
lage cells
miR-29b To be char- Inducing effect Osteoblasts Rheumatoid (Figueiredo Neto and
acterized arthritis Figueiredo 2017, 6365857)
miR-21 To be char- Inducing effect Osteoblasts Rheumatoid (Figueiredo Neto and
acterized arthritis Figueiredo 2017, 6365857)
miR-155 SOCS1 Inducing effect CD4+ T-cells Immune disorders (Yao et al. 2012, e46082)

(Continued )
JAK-STAT Pathway Regulation 37

TABLE 3.1 (Cont.)

Anticipated effect
Target of JAK-STAT
miRNA transcript pathway Test model Disease model Reference

miR-1225-3p GAB3 Inhibitory effect Huh7 cells Influenza A virus (Cheng et al. 2018,
5975–5986)
miR-203 DR1 Promotes JAK­ A549 Influenza A virus (Zhang et al. 2018, 6797­
STAT signaling 018-25073-9)
miR-324-5p CUEDC2 Negative regulator A549 H5N1 (Kumar et al. 2018, 10.1128/
JVI.01057–18. Print 2018
Oct 1)
miR-130a To be char- Inducing effect J6/JFH-1 Hepatitis C viral (Li et al. 2014, 121–128;
acterized immortal cells infection Murayama et al. 2007,
8030–8040)
miR-122 To be char- To be Liver cells Hepatitis C viral (Jopling et al. 2005,
acterized characterized infection 1577–1581)
miR-373 JAK1, IRF9 Inhibitory effect Human liver Hepatitis C viral (Mukherjee et al. 2015,
biopsies infection 3356–3365)
miR-155 SOCS1 Inducing effect HepG2 HBV infection (Lu et al. 2008,
cells, and 10436–10443)
B lymphocytes
miR-103 CDK5R1 Inhibitory effect Stem cells Spinal cord injury (Li et al. 2018, 292–300)
miR-210 Presumably Inhibitory effect In vivo in mice Spinal cord injury (Dai et al. 2018b,
JAK2 6609–6615)
miR-125b JAK1 Inhibitory effect In vivo in mice Axon regeneration/ (Dai et al. 2018a, 582–589)
STAT1 spinal cord injury
miR-155 To be char- Inducing effect Retinal pig- Eye diseases (Kutty et al. 2010, 390–395)
acterized ment epithe­
lial cells
miR-155 To be char- Inducing effect Lymphatic Angiogenesis (Yee et al. 2017,
acterized endothelial 20,683–20,693)
cells
miR-9 SOCS5 Inducing effect Endothelial Angiogenesis (Zhuang et al. 2012,
cells 3513–3523)
miR-155 SOCS1 Inducing effect Microglial Neurodegenerative (Cardoso et al. 2012, 73–88)
cells diseases
miR-146a IL-6 Inducing effect Microglial Neurodegenerative (Saba et al. 2012, e30832)
cells diseases
miR-216a JAK2 Inhibitory effects Neuronal cells Neuroprotection (Tian et al. 2018, 977–988)

3.2 JAK-STAT Targeting miRNAs in Liver Diseases

A group of tumor-suppressor proteins, which together were classified as the suppressor of cytokine
signaling proteins (SOCS), have been well characterized with defined functions toward inhibition of
JAK kinase and STAT transcription factor in the canonical JAK-STAT signaling pathway. The
inhibition of both JAK and STAT by SOCS proteins was found to be mediated through protein
interactions and through epigenetic mechanisms, which are regulated by specific miRNAs. The miRNA
miR-19a targets most of the SOCS transcripts, which include SOCS1, SOCS3, and SOCS5, in addition
to Cullin (Collins et al. 2013, e69090). As JAK-STAT regulation is influenced by SOCS proteins and
since miR-19a inhibits SOCS3, the functional role of miR-19a would be to curtail the inhibitory effects
of SOCS3 on JAK and STAT activities. This was reported recently in immortal “HuH-7 cells”
where IL-6 and IFN-α enhanced activation of pSTAT3 in presence of miR-19a. These results suggest
38 JAK-STAT Signaling in Diseases

that miR-19a indirectly promotes JAK-STAT-mediated expression of pro-inflammatory mediators

(Collins et al. 2013, e69090). It would be more interesting to see if this microRNA miR-19a had any
impact on activation of JAK proteins, since SOCS3 binds to the receptor of JAK in its transmembrane
region and stereotypically prevents its interactions with JAK.
In a significantly large animal study using BALB/c mice with n = 40 in each group, an in vivo study
was performed to evaluate the role of specific miRNAs associated with lipopolysaccharides-(LPS-)
induced sepsis as liver injury model (Lv et al. 2015, 6013–6018). While LPS was reported to have
effectively induced the anticipated liver injury in mice, there was a concomitant increase in the
expression of the miRNA miR-155 in the group that was treated with LPS alone. In the mice group
that were treated with LPS and with miR-155 specific inhibitor, SOCS1 protein was reported to be
significantly upregulated. Further, in presence of the miR-155 specific inhibitor, the expression levels of
STAT1 were shown to be greatly reduced. Also, the levels of tumor necrosis factor alpha (TNF-α) and
interleukin-10 (IL-10) cytokines were reported to be significantly decreased in the mice group that was
treated with LPS and miR-155 specific inhibitor. Since STAT1 drives the expression of the miRNA
miR-155, and miR-155 targets SOCS1 and prevents its expression, the above study provides a clear
mechanistic regulation between the microRNA miR-155 and SOCS1, where miR-155 inhibits SOCS1
to promote JAK-STAT signaling in LPS-induced liver damage (Kutty et al. 2010, 390–395).
Compared to the normal liver tissues and the para-tumor tissues in patients with hepatocellular
carcinoma (HCC), the levels of microRNA miR-409 were reported to be drastically reduced in the
tumor tissues. This downregulation of miR-409 was presumed as a possible cause associated with the
progression of the HCC (Zhang et al. 2019, 146–154). The authors also reported the existence of an
inverse correlation between the expression of miR-409 and the protein levels of JAK2 and STAT3.
Further, in cell-culture system, when HCC cells were transfected with both the microRNA mimic and
inhibitor separately, reduced cell viability and increased apoptosis was observed in the miR-409
transfected cells. As can be anticipated, the opposite results were found in cells that were transfected
with the inhibitor, which blocks the functions of miR-409. Both the cell culture data and the histological
observations indicate that miR-409 targets JAK-STAT pathway to prevent the progression of HCC.
The reports, which showed E74-like factor 2 as a direct target of miR-409 and c-Met as a target for
miR-409-3p, suggest that miR-409 may not be directly targeting JAK or STAT transcripts.
However, all the current evidence point to the same common observation, which indicate that miR­
409 regulates JAK-STAT pathway, and which is more likely to be an indirect cause. In another study,
HCC development was found to be directly correlated with the expression of two miRNAs belonging to
the same class—miR-196a and miR-196b—which regulate JAK-STAT pathway (Ren et al. 2019, 333­
019-1530-4). These two miRNAs were reported to increase cell proliferation, migration, invasion, and
apoptosis induction. However, each of these was found to target two different transcription factors. The
microRNA, miR-196a, primary target was identified as FOXO1, while the target for miR-196b was
FOXP2. Interestingly, both these miRNAs were also found to prevent SOCS2 expression, affecting the
JAK2–STAT5 pathway in HCC. The above reports suggests that in normal hepatic cells these miRNAs
establish a tight control of JAK-STAT pathway as a means to prevent oncogenic transformation.

3.3 Specific MicroRNAs that Regulate JAK-STAT Pathways in Heart Diseases

An indirect epigenetic mechanism, which we recently reported, was the direct targeting of the
main DNA methylase (DNMT1, DNA methyltransferase 1) by miRNA miR-1264 (Boosani et al.
2015, 1365–1376). DNMT1 promotes methylation of CpG residues in the genome DNA, and this
non-specific methylation of DNA would affect all genes, which have CpG islands. Since SOCS3
genomic sequence harbors a well-defined CpG island in its promoter region, it is highly subjected
to methylation by DNMT1. This we recently reported in vascular smooth muscle cells wherein the
miRNA miR-1264, which has strong affinity to bind to the DNMT1 transcript and inhibit its
expression, and thus in absence of DNMT1, SOCS3 expression remain unaffected (Boosani et al.
2015, 1365–1376). Subsequently, SOCS3 was able to prevent the activation of JAK. The miRNA
JAK-STAT Pathway Regulation 39

miR-1264 also prevented the DNMT1 activity induced by TNF-α and insulin-like growth factor 1
(IGF-1) when treated together in smooth muscle cells. These observations establish the existence of
an epigenetic mechanism, which involves both miRNA and a DNA methylase. Also, inhibition of
SOCS3 was identified to be an important cause for the progression of intimal hyperplasia and
restenosis (Gupta et al. 2011, 346–352). Cumulatively, these studies clearly indicate that multiple
mechanisms are involved in preventing SOCS3 expression during restenosis and the miRNA miR­
1264 is a key mediator that can help restore SOCS3 expression to prevent neointima formation in
the arteries.
The role of miRNAs specific to JAK-STAT pathway and its implications in immune regulations
associated with myocardial infarction were reported recently using bone-marrow–derived dendritic
cells from mice (Zhu et al. 2017, 2884–2895). In an interesting approach, the authors used the
cultured supernatant from the necrotic cardiomyocytes as a means to mimic myocardial infarction
environment on dendritic cells. The authors subsequently identified specific miRNAs that can
potentially regulate inflammatory responses mediated by the dendritic cells. The supernatant from
the necrotic cells were reported to upregulate maturation markers in the dendritic cells, such as CD40,
CD83, and CD86, along with increased levels of proinflammatory cytokines. Concurrent increase in
the expression of the miRNA, miR-181a, and simultaneous downregulation of miR-150 was also
reported. Treatment with the necrotic supernatant was shown to activate the JAK-STAT pathway and
also facilitated nuclear translocation of nuclear factor kappa B (NF-κB) and c-Fos. These reports
highlight the significance of the miRNAs—miR-181a and miR-150—in regulating JAK-STAT
signalling, which controls the hypoxic cell death in cardiomyocytes.
In a rat model to study the role of different miRNAs in cardiac hypertrophy and heart failure,
transverse aortic constriction (TAC) was performed in 20 rats (Feng et al. 2014, 361–368). RNA isolated
from the hearts of these rats showed differential expression of multiple miRNAs that regulated different
cellular pathways. The miRNAs, which directly or indirectly targeted JAK-STAT pathway in the above
study, were discussed here. Expression of the microRNAs let-7c, let-7f, miR-1, miR-300-5p, miR-347,
and miR-874 were found downregulated. These miRNAs indirectly act in regulating JAK-STAT
pathway by targeting different interleukins. At the same time, the expression of miRNAs let-7i, miR­
22, miR-29, miR-30a, miR-101, miR-125b-5p, miR-146b, miR-214, and miR-499 were found to be
increased. Notably, only miR-146b and miR-499 were found to act directly on STAT5 and STAT2,
respectively. Cumulatively, this comprehensive miRNA profiling in the cardiac hypertrophy model
resulting from pressure overload due to TAC, identified signature miRNA species. Although a significant
role of JAK-STAT signaling was evident in this study, several other effective target transcripts and other
potent miRNAs regulating different cellular pathways exist which were well characterized.

3.4 MicroRNAs Targeting JAK-STAT Pathways in Arthritis

In efforts to identify specific miRNAs that regulate JAK-STAT signaling in patients with arthritis,
a recent human clinical study was conducted using peripheral blood monocytes (PBMCs) collected
from 20 normal individuals and 20 patients who were suffering from symptomatic juvenile idiopathic
arthritis (Li et al. 2016, 2531–2536). Although the study showed minimal analysis to identify specific
miRNAs, it was interesting to note that two selected miRNAs, miR-19a and miR-21, were reported to
be expressing at a very low level in the patients. Importantly, the authors conclude that TNF-α,
STAT3, and SOCS3 transcripts are the targets for these two miRNAs, miR-19a and miR-21. The
increased expression of TNF-α and STAT3 in the arthritic patients agrees with the other reports in the
literature. However, the expression of SOCS3 can be debatable but it should be noted that SOCS3 has
also been shown to encourage inflammatory responses in a few other diseases, such as in asthma
(Zafra et al. 2014, e91996). Also, the study includes juvenile patients, and presumably is focused on
the local ethnic group, which greatly limits extension of the findings to other population groups unless
evaluated. Additional analysis in this study could have been more beneficial, such as conducting
a microarray-based analysis of all the samples and evaluating the samples to identify what specific
40 JAK-STAT Signaling in Diseases

JAK or STAT molecules are activated through phosphorylation. Another notable limitation in this
study was using real-time PCR as means for quantifying the gene expression. The procedure as such
depends on the primer choice, and thus the estimated gene expression based on the available RNA
needs to support through additional means of evaluation, such as using a protein array or ELISA or
western blotting.
LPS is widely known to initiate many proinflammatory mechanisms in different cell types. Several
volumes of information in the literature shows that inflammatory mediators, which include interleukins,
cytokines, and growth factors, can be upregulated by LPS, both in vitro and in animal models. TNF-α is
certainly one of them, which has the potential to mediate multiple signaling mechanisms and the
evidence is accumulating, which supports the role of TNF-α in inducing epigenetic mechanisms that
promote disease progression. We have earlier identified a clear role of TNF-α in exacerbating
neointimal hyperplasia in the coronary arteries (Gupta et al. 2011, 346–352). Recently, we have also
shown that TNF-α regulates different intracellular pathways including the JAK-STAT signaling (Dhar
et al. 2013, H776-85; Boosani et al. 2015, 1365–1376). LPS-induced expression of IL-1b, IL-6, IL-8, and
TNF-α was identified to promote inflammation in the chondrogenic cells “ATDC5.” LPS-induced
expression of these inflammatory mediators were found to regulate cell viability and apoptotic
pathways that are mediated by JAK1, STAT1, STAT3, Akt, and PI3K (Wu et al. 2017, 2010–2021).
Notably, the regulation of JAK-STAT pathway and its cross-talk with PI3K-Akt pathway by LPS was
found to be mediated through the miRNA, miR-1246. The hepatic nuclear factor gamma “HNFγ”
was reported as a specific target of miR-1246 in the putative chondrocytes. The study highlights
multiple functions of HNFγ, and its inhibition by the miRNA miR-1246 in presence of LPS as
a potential mechanism by which JAK-STAT promotes intracellular inflammation.
In mesenchymal stem cells, the miRNA miR-1246 was reported to be expressed in higher levels
and interestingly, this miRNA expression was independent of the inflammatory signal induced by
TNF-α. However, the main functions of miR-1246 reported was to target and prevent the
expression of two tumor-suppressor genes—cAMP-dependent protein kinase type I-alpha
“PRKAR1A” and the protein phosphates 2C-beta “PPP2CB” (Bott et al. 2017, 43897–43914).
An interesting aspect was that the conditioned medium from the miR-1246 transfected cells was
found to induce JAK-STAT signaling in the normal and tumor epithelial cells.
To understand the role of JAK-STAT pathway and miRNAs that affect JAK-STAT signaling in
osteoarthritis, LPS was used as means to induce inflammation and cellular injury in cultured human
cartilage cells C28/I2 (Zhao et al. 2017, 171–178). The authors report that when chondrocytes were
treated with LPS, expression of a specific miRNA miR-203 was significantly upregulated with
concomitant decrease in cell viability and increase in cellular apoptosis. Further, the authors also
report that overexpression of miR-203 in the chondrocytes exaggerates LPS-induced inflammatory
responses in the transfected cells. Since the direct target of the miRNA, miR-203, is the myeloid
cell leukemia-1 transcript (MCL-1), it was speculated that MCL-1 would play a critical role in
LPS-mediated inflammation during osteoarthritis. Further, as the effector pathways for MCL-1
are Wnt/β-catenin and JAK-STAT, it was speculated that the inflammatory responses initiated
during the progression of osteoarthritis are mediated through upregulation of the miRNA
miR-203, which indirectly affects JAK-STAT pathway through the expression of MCL-1.
In treating rheumatoid arthritis patients, it is not just sufficient to address inflammation since bone
erosion occurs which is also a complication seen in these patients. In this direction, IL-27 has been
shown to promote mineralization in the osteoblasts, which can prevent bone erosion. In attempts to
identify the factors that enhance the effects of IL-27, a microarray-based study revealed that two
specific miRNAs—miR-29b and miR-21—augment the expression of IL-27 in osteoblasts (Figueiredo
Neto and Figueiredo 2017, 6365857). The study showed that treatment with IL-27 not only promoted
osteoblast differentiation, but it also inhibited the proteins that prevent osteoblast formation. Two main
signaling pathways were identified to be associated with osteoblast differentiation, which include JAK­
STAT and TGF-β/BMP/SMAD signaling. In another study using human bone marrow stromal cells
which were induced for osteogenic differentiation, specific miRNAs were identified that were character­
ized as osteogenic specific miRNAs, and these were reported to be targeting JAK-STAT pathway
(Vimalraj and Selvamurugan 2014, 194–202).
JAK-STAT Pathway Regulation 41

3.5 JAK-STAT Specific miRNAs Regulating Immune Responses

CD4+ T-cells are an important class of immune cells with multiple functions and are primarily
involved in suppressing immune reaction. IL-6 and STAT3 were shown to be essentially required
for the differentiation of immune cells, such as T-regulatory and Th17, cells from uncommitted
CD4+ T-cells. In a recent report, inhibition of SOCS1 by the miRNAs miR-155 was shown to
induce differentiation of T-regulatory cells and Th17 cells (Yao et al. 2012, e46082). This cell
differentiation was further supported by the observations that when CD4+ T-cells were trans­
fected with the miRNAs miR-155, increased expression of activated STAT3 and STAT5 were
observed, which correlated with decrease in the expression of SOCS1 in the same group. These
reports clarify a clear role of JAK-STAT signaling in immune cell differentiation, and SOCS1
prevents activation of JAK kinases, which is required to induce STAT3 and STAT5 activation. In
another study to identify the significance and role of miRNAs in regulating proliferation and
death of T-cells, a comprehensive microarray analysis was carried out which was based on the
observations that a drastic decrease in JAK1 expression was noted in IL-2 depleted cells (Ranji
et al. 2015, 169–183). The study identified several miRNAs that can specifically target important
pathway mediators of JAK-STAT signaling to regulate immune responses.

3.6 miRNAs Regulating Viral Diseases Induced by JAK-STAT Pathways

Chronic hepatitis resulting from hepatitis C viral infections can be a major cause that can lead to
hepatocellular carcinoma and other complications in humans. A recent report showed a strong
correlation between the expression of the miRNA miR-130a and the replication of hepatitis C virus
(Li et al. 2014, 121–128). Specifically, the expression of miRNA miR-130a was reported to
significantly inhibit the virus replication in chimeric J6/JFH-1 cells. It is to be noted that the J6/
JFH-1 cell line, which has the chimeric genomic regions that encode J6 structural proteins and
JFH-1 nonstructural proteins, enables the J6/JFH-1 cells to replicate autonomously and produce
infectious HCV particles (Murayama et al. 2007, 8030–8040). The finding that overexpression of
miR-130a in these cells upregulates the expression of type I interferons and other essential proteins
which initiate innate responses clearly indicates the role of interferon in stimulating genes that have
been earlier reported to activate JAK-STAT pathway (Martensen and Justesen 2004, 1–19).
Together, the above reports would provide strong inference, which indicates the potential role of
miR-130a in inhibiting replication of hepatitis C virus in humans. Another important observation
reported in the above studies is the indirect effects of the miRNA miR-130a that inhibits the
expression of another miRNA miR-122. The miRNA miR-122 has been previously reported to
promote proliferation of hepatitis C virus (Jopling et al. 2005, 1577–1581).
Since SOCS3 is a competitive inhibitor of JAK kinases, miRNAs that inhibit SOCS3 would
have JAK–promoting effects. Conversely, those miRNAs that promote SOCS3 expression would
confer indirect inhibitory effects on JAK activation. Such an indirect role of miRNA miR-122
was recently reported to affect interferon treatment, which is the current standard of care to treat
hepatitis C viral infections. While overexpression of the miRNA miR-122 suppressed the
interferon-stimulated response element-mediated gene expression, silencing of miR-122 was
reported to enhanced interferon-induced functions, and as a consequence, the expression of
SOCS3 was reportedly affected (Yoshikawa et al. 2012, 637).
In another study, elevated levels of the miRNA miR-373 was reported in hepatitis C virus
infected human primary hepatocytes, and also in the liver biopsies obtained from human patients
(Mukherjee et al. 2015, 3356–3365). A direct target of miR-373 indicated in the above article is the
JAK1 transcript and the interferon regulating factor-9 and therefore, activation of STAT1 was found
to be decreased. Subsequently, knockdown of miR-373 in the hepatocytes was found to prevent the
expression of JAK1 and IRF9, and, therefore, the authors speculate that the hepatitis C virus induces
the expression of miR-373 in hepatocytes and this is required for its replication inside the cell.
42 JAK-STAT Signaling in Diseases

The Epstein–Barr virus infection of B lymphocytes has been recently shown to elevate the expression
levels of the miRNA miR-155, which contributes to immortalization (Lu et al. 2008, 10436–10443).
Subsequently, in HepG2 human hepatocellular carcinoma cell lines, it was reported that expression of
miR-155 upregulates the interferon inducible anti-viral response. However, overexpression of miR-155
was also found to inhibit SOCS1 expression and, as a consequence, STAT1 and STAT3 activation was
induced (Su et al. 2011, 354-422X-8-354). SOCS1 being a negative regulator of JAK-STAT pathway, the
above reports provide a clear understanding that the miRNA miR-155 enhances antiviral immune
response by activating JAK-STAT pathway, and this is required to prevent HBV infection in humans.
Diagnostic biomarkers, if detected early in the disease, can be very helpful in strategizing an effective
treatment plan to cure the disease. In this line, using the blood samples from 50 patients diagnosed with
HBV infections and hepatic fibrosis, circulating miRNAs were screened using miRNA microarray
(Zhang et al. 2015, 5647–5654). The authors reported that 12 miRNAs were differentially expressed, of
which 10 were overexpressed and 2 were downregulated. Among the target genes identified, 31 of them
were specific for JAK-STAT pathway. Although the study identified putative biomarkers for early
diagnosis of HBV-associated hepatic fibrosis, further validation studies are required.
As identified earlier in this manuscript, the role of miR-203 is evident in regulating different cellular
pathways in many disease conditions. Apart from the above-discussed studies, the role of miR-203 in
regulating viral infections was also reported. Within the 2500bp promoter region of miR-203, the
binding sites for two transcription factors, NFκB and ISGF3, were identified which are known to
regulate the JAK-STAT signaling pathway. ISGF3 is primarily involved in IFN-mediated regulation
of JAK-STAT pathway with STAT1, STAT2, and IRF9 as key mediators. Interestingly, in the IFN-
deficient cells that were infected with influenza A virus, increased expression of miR-203 was
observed. These reports suggest a direct correlation between the increased expression of miR-203
with influenza A virus infection; however, the same study also identifies indirect role of JAK-STAT
signaling during influenza A viral infection (Zhang et al. 2018, 6797-018-25073-9). IFNs are
commonly administered to treat viral infections. In both cancerous and non-cancerous liver cells,
treatment with IFN was reported to downregulate the levels of miR-1225-3p. Since viral infection
induces the production of endogenous IFN levels, Huh7 and 293T cells when infected with RNA
viruses, such as Hepatitis C Virus, Sendai Virus, and New castle Disease Virus, was also reported to
lower the levels of miR-1225-3p (Cheng et al. 2018, 5975–5986). Since IFN directly regulates JAK­
STAT pathway through its cell surface receptors, the above reports indicate IFN-mediated regulation
of JAK-STAT signaling by miR-1225-3p during viral infections.
The highly pathogenic influenza A virus, H5N1, showed more than 50% mortality rate in infected
people. In mice infected with H5N1, the expression of miR-324-5p was reported to be down-
regulated by Poly(IC), which is a viral pathogen-associated molecular pattern protein. By targeting
the RNA polymerase subunits PB1 and PB2, the miR-324-5p prevents H5N1 viral replication in the
host (Kumar et al. 2018). Further transcriptome analysis of the miR-324-5p identified one of its
direct target transcripts CUEDC2, which is a negative regulator of JAK-STAT signaling. It was also
shown that ectopic expression of miR-324-5p induces the expression of antiviral gene expression,
notably the IFNs. Taken together these reports show an essential role of different miRNAs in
regulating viral infections, primarily through IFN-mediated JAK-STAT signaling pathway compris­
ing different isoforms of JAK and STAT proteins.

3.7 Involvement of miRNAs in Other Diseases Regulated by

JAK-STAT Pathways
3.7.1 Spinal Cord Injury
The transcription factor SOX2 is critically required for tissue development and homeostasis in the
cells. In addition, for stem cells, SOX2 expression is required to maintain their self-renewability
and pluripotency. Just recently, the miRNA miR-103 was identified to prevent two important
JAK-STAT Pathway Regulation 43

cellular functions, autophagy and apoptosis, by regulating SOX2 expression. The miRNA miR-103
was shown to indirectly inhibit both MAPK-ERK and JAK-STAT pathways by upregulating SOX2
in PC12 cancer cells (Li et al. 2018, 292–300). Further in the same article, the authors showed that
in rats with spinal cord injury, delivery of the miRNA miR-103 agomir, increased the expression of
SOX2. The treatment with miR-103 agomir was also found to prevent the expression of Beclin-1,
Bax, activated caspase-3 and caspase-8, and in contrast, LC3-1, p62, and BcL-2 were reported to be
upregulated. Although whether miR-103 can directly regulate SOX2 expression remained unclear, it
was evident that SOX2 plays a critical role in tissue repair. Further, since CDK5R1 is a known
direct target of miR-103, it remains to be elucidated whether SOX2 is at a pivotal point between
CDK5R1 and JAK-STAT pathways, and their possible cross talk.
A mouse model of spinal cord injury due to contusion was reported as a feasible study to evaluate
axon growth and regeneration (Dai et al. 2018a, 582–589). In this mouse model, significantly reduced
expression of the miRNA miR-125b was noted due to spinal cord contusion. Both JAK1 and
STAT1 transcripts were identified as the direct targets of this miRNA miR-125b, suggesting
the direct role of JAK-STAT signaling in regulating spinal cord injury and axon regeneration.
The authors further extended the mice results to a rat model and reported that miR-125b reduces
protein levels of caspase3, which is a known activator of cellular apoptosis. The results presented
in the above study clearly indicate that miR-125b promotes axon growth and regeneration by
inhibiting the JAK-STAT signaling pathway. The same group also evaluated the role of another
miRNA miR-210 in the mice with spinal cord injury (Dai et al. 2018b, 6609–6615). A key
observation made in this study was the late recovery to grasping strength in mice with spinal cord
injury, compared to sham control group. The late recovery was found to correlate with the
reduced levels of the miRNA miR-210. Correlating with this observation, the mice group that was
administered with miR-210 mimic was found to show higher grip strength. In assessing the target
gene transcripts of miR-210, increased expression of JAK2 and STAT3 proteins was observed in
the control group. Their observations were further supported from the results seen in the mice
with miR-210 mimic group, which showed reduced JAK2 and STAT3 levels. The above two
studies point to the possibility of targeting JAK-STAT proteins to promote spinal cord injury. In
these lines, JAK or STAT inhibitors can be tested in animal models to show their effects in
promoting axon regeneration and recovery from spinal cord injury.

3.7.2 Eye Diseases

Eye transplant is vital to restoring vision in suitable patients. In efforts to address the inflammatory
responses in the retinal pigment epithelial cells (RPE) from donor eyes, the role of several cytokines
was recently evaluated (Kutty et al. 2010, 390–395). Three important cytokines, TNF-α, IL-1β, and
IFN-γ, were tested in RPE cells. The authors reported that all three were effective in inducing the
inflammatory responses in the RPE cells. While the effects of each cytokine were noticed in RPE cells
when treated individually, the cellular inflammation was reported to be exaggerated when RPE cells
were treated with all three cytokines at the same time. Importantly, the combined treatment was
shown to enhance the expression of miRNA miR-155, and the RPE cells when treated with the three
cytokines along with JAK1 inhibitor, the expression of miR-155 was drastically reduced. In addition,
the authors also showed that STAT1 activation was elevated in presence of the three cytokines and
the same was inhibited when the cells were treated with JAK1 inhibitor. These reports suggest that
miR-155 promotes JAK-STAT signaling to induce inflammation in the cells. However, additional
studies are required to precisely elucidate specific transcripts that are targeted by miR-155 and the
molecular intermediates that promote proinflammatory signaling in these cells.

3.7.3 Endothelial Cell Migration and Angiogenesis

Within this last decade, several studies have identified that targeting the programmed death ligand
1 “PD-L1” could be an efficient strategy to prevent progression of certain cancer types, especially
44 JAK-STAT Signaling in Diseases

with its initial success in treating melanoma patients (Wolchok et al. 2013a, 1–13, 2013b,
122–133). However, in many non-cancerous diseases, upregulation of cytokines is also a
characteristic feature such as in atherosclerosis where there is an increased expression of TNF­
α. Whether the increased expression of cytokines and growth factors in non-cancerous diseases
would have any impact on normal cells and tissues would enable strategizing a better treatment
plan. In these directions, using normal human lymphatic endothelial cells, the effects of the
cytokines TNF-α and IFN-γ were recently reported where the combined presence of TNF-α and
IFN-γ significantly elevated the expression of PD-L1 expression (Yee et al. 2017, 20683–20693).
A key finding in the above study was that TNF-α induced the expression of miRNA miR-155
and this effect was enhanced by treatment with IFN-γ. Further, the same effects on PD-L1 and
miR-155 expression were also reported in normal dermal fibroblasts. Importantly, it was
reported that the cytokines TNF-α and mostly the IFN-γ, initiates JAK-STAT pathways and
drives the activation of STAT1 and STAT3 contributing to the induced expression of miR-155
and PD-L1.
SOCS5 is another member of SOCS family, which is a negative regulator of JAK-STAT
pathway as it prevents activation of JAK. The miRNA miR-9 was reported to be increased in
different tumor types (Ma et al. 2010, 247–256; Shigehara et al. 2011, e23584). However, this
miRNA was found to target and inhibit SOCS5 expression by binding to its 3ʹUTR sequence
and relieves the inhibition on JAK activation (Zhuang et al. 2012, 3513–3523). Transfection of
the miRNA miR-9 into human and mouse endothelial cells was shown to induce phosphoryla­
tion of JAK1, JAK2, STAT1, and STAT3, and simultaneously inhibit SOCS5 expression.
Further, miR-9 was also shown to promote endothelial cell migration and angiogenic sprouting
in HUVECs.

3.7.4 Neuroprotective Functions

Besides the above-discussed biological effects of the miRNA, miR-155, its proinflammatory
functions through inhibition of SOCS1 were also reported in immortal mouse microglial cells
(Cardoso et al. 2012, 73–88). Upon transfection of the microglial cells with miRNA miR-155, the
expression of SOCS1 was significantly downregulated. On the contrary, knockdown of the
miRNA miR-155 not only upregulated the expression of SOCS1, but it also prevented induction
of nitric oxide synthase and inflammatory cytokines. It is to be noted that SOCS1 prevents JAK
and STAT activation. The authors report that when the microRNA miR-155 was inhibited
in microglial cells and treated with LPS, the LPS induced effects on the expression of TNF-α
and IL-6 were significantly reduced. These findings indicate that under chronic inflammatory
conditions, inhibition of miR-155 could confer neuroprotective effects and may prevent neuronal
cell death, which can be an effective treatment strategy for neurodegenerative diseases.
In the prion-infected mice brain, the levels of miRNA miR-146a was reported to be elevated.
Further, TLR2 and TLR4 activation also was found to hold strong correlation with the increased
expression of miR-146a (Saba et al. 2012, e30832). Interestingly, in the murine microglial cells, the
authors report that upon transfection with the miRNA miR-146a, the expression of the proin­
flammatory cytokine IL-6 was drastically reduced. However, the expression of the master
proinflammatory transcription factor, NFκB was affected in absence of miR-146a, in addition to
the JAK-STAT pathway mediators. Although these studies demonstrate the effect of miR-146a in
modulating the cellular inflammation, the cross talk between NFκB and JAK-STAT pathway is
still warranted.
Cerebral ischemia is a major cause of permanent damage to health that can lead to disability
and even death of the individual. Inflammation initiates several cellular mechanisms, which
eventually cause neuronal damage. In the cultured primary neuronal cells from mouse brain and
also in the mouse model for ischemic stroke induced due to middle cerebral artery occlusion,
targeting JAK2 was reported to confer neuroprotection against ischemic injury (Tian et al. 2018,
977–988). The cultured neuronal cells, when subjected to oxygen-glucose deprivation and
JAK-STAT Pathway Regulation 45

FIGURE 3.1 Regulation of JAK-STAT signaling pathway by microRNAs.

reoxygenation, appear to mimic the ischemic conditions. Expression of JAK2 and the downstream
proinflammatory molecules such as iNOS, MMP9, TNF-α, and IL-1β were reported in this
in vitro cell culture model. In this study, the miRNA miR-216a was identified as a direct target
for JAK2 and inhibition of JAK2 in the neuronal cells transfected with the miR-216a prevented
the expression of the above inflammatory mediators. Based on the above published reports,
targeting JAK2-mediated signaling appears to be a beneficial approach to protect neuronal
damage resulting from the ischemic injury.
Figure 3.1 shows known miRNAs that can directly target JAK and STAT transcripts and
prevent their translation. Also, specific miRNAs that can potentially promote JAK-STAT signal­
ing pathway by indirectly targeting the expression of proteins, such as the suppressors of cytokine
signaling proteins, which inhibit JAK1 and JAK2, were highlighted. In addition, the miRNAs,
which intercept the crosstalk between the JAK-STAT and PI3K–Akt pathways were shown in the
above figure.

3.8 Summary and Future Directions

The information presented here clearly shows that JAK-STAT pathway mediators promote patho­
logical progression of many human diseases. Several important miRNAs have been identified, which
have been proven to be key regulators of JAK-STAT signaling. In Figure 3.1, we have summarized
few miRNAs, which affect JAK-STAT pathway. Also, specific miRNAs were found to activate
JAK-STAT pathway in more than one disease conditions. Since inflammation and cell proliferation
are key cellular mechanisms that are associated with many diseases, and JAK-STAT pathway
promotes both these important cellular mechanisms, it appears very promising to identify and test
46 JAK-STAT Signaling in Diseases

those miRNAs that can modulate JAK-STAT pathway. As current bioinformatic tools were proven
to be effective in target identification and characterization of different miRNAs, high throughput
screening and cell-based assays would facilitate validation of the putative miRNAs to identify their
therapeutic benefits. Since miRNAs have gained entry into human clinical trials, their initial
evaluation through pre-clinical trials in animal models is already underway in many research
laboratories. It is anticipated that there would be a significant change in the therapeutic strategies
to treat different human diseases using miRNA-based medicine.

ACKNOWLEDGEMENTS
This work was supported by the State of Nebraska LB692 grant to CSB by Creighton University
and research grants R01 HL112597, R01 HL116042, and R01 HL120659 to DK Agrawal from
the National Institutes of Health, USA. The content of this book chapter is solely the responsi­
bility of the authors and does not necessarily represent the official views of the National Institutes
of Health or the State of Nebraska.

COMPETING INTERESTS
The authors declare no other relevant affiliations or financial involvement with any organization
or entity with financial interest or financial conflict with the subject matter or materials discussed
in this chapter. No writing assistance was utilized in the production of this manuscript.

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4
JAK-STAT Signaling in Asthma and Allergic
Airway Inflammation

Amina Abdul Qayum, Tristan Hayes, and Mark H. Kaplan

Department of Pediatrics and Herman B Wells Center for Pediatric Research
Indiana University School of Medicine
Indianapolis, Indiana

4.1 Introduction
Classical asthma is an inflammatory disease mediated by TH2 and TH9 cells characterized by
increased serum IgE, eosinophilia, and responsiveness to steroids. Non-canonical asthma, which
affects less than 10% of all asthmatics, is thought to be promoted by TH17 cells and is non-atopic,
characterized by steroid-resistant neutrophilia.1 Cytokines that are well known to implement the
asthmatic phenotype include IL-4, IL-5, IL-9, IL-13, IL-17, TSLP, and IL.-312,3 These cytokines
signal through the JAK-STAT (see Table 4.1) pathway to mediate communication between cells and
their environment. In this chapter, we will discuss the current understanding of JAK-STAT signaling
in multiple cell types as it relates to asthma and allergic lung inflammation in animal models.

4.2 Janus Kinase

Janus kinase (JAK) proteins are a family of intracellular non-receptor tyrosine protein kinases
(PTKs) that aid in signal transduction of JAK-STAT pathways. There are four known JAK kinases:
JAK1, JAK2, JAK3, and TYK2. The structures of these kinases contain seven homology domains
(JH1-JH7). The kinase domain (JH1) has catalytic activity while the pseudo-kinase domain (JH2)
located at the carboxy terminus end, does not have catalytic activity. JH2 domains interact with and
regulate the JH1 domain.4,5 The kinase and pseudo-kinase domains are the defining feature of the
JAK family of proteins. This name is derived from the Roman God of duality, Janus, who is depicted
as having two heads; one looking to the past and one looking to the future. The JH3–JH4 domains
contain homology to SH2 domains and putatively play a structural role and stabilize JAK protein
conformation. The JH5–JH7 domains are located at the amino-terminus and serve as the FERM
proteins that are critical for receptor binding of JAKs.6,7
When a ligand binds to its receptor, JAKs associated with that receptor phosphorylate each
other as well as the cytokine receptor. The phosphorylated receptor serves as a docking site for
STAT proteins. When the STAT proteins bind to the receptor, they become phosphorylated by
JAKS. Activated STATs can then translocate to the nucleus where they directly bind DNA and
promote RNA transcriptoin. There is also evidence for the role of nuclear JAK proteins in cell
signaling. Some studies have shown that JAKs can modify other non-STAT transcription factors
and modify histone proteins.8,9 However, the main function defined for JAKs is in the activation
of STAT proteins.

49
50 JAK-STAT Signaling in Diseases

TABLE 4.1
Th2 Cytokine Usage of JAK-STAT Proteins
JAK/STAT Role in Signaling Mutation Phenotype References

JAK1 IL-4, IL-13, IL-5, IL-9, KO prenatally lethal 6

TSLP, IL-31
JAK2 IL-5, TSLP, IL-31 KO embryonically lethal 6,7
JAK3 IL-4, IL-13, IL-9 KO causes SCID (similar in humans) 12,8,11
Defective B and T cell maturation
Tyk2 (IFNs, IL-12) KO susceptible to infections 10
Defective STAT3 activation
STAT1 (IFNs), IL-5, IL-31, IL-9 Defective in Type I/II IFN signaling 18,21,19
STAT2 IL-5 KO phenotype not yet reported 18,21,19
STAT3 IL-5, IL-31, IL-9 KO embryonically lethal and mutation causes HIES 27
STAT4 (IL-12) Defective Th1 Development 33,34,35
Enhanced Th2 development
STAT5a/b IL-5, TSLP, IL-31, IL-9 STAT5a/b: defects in hematopoietic cell development and 33,34,35,38,40
immune cell signaling
STAT6 IL-4, IL-13 Defective Th2 and Th9 development 46
Protected from AHR 132, 178
KO, knock out; AHR, airway hyperresponsiveness; TSLP, thymic stromal lymphopoietin; SCID, severe combined
immunodeficiency; IFN, interferon. Brackets indicate canonical activators of the pathway that are not Th2 cytokines.

4.2.1 JAKs and Asthma

The importance of JAK proteins in human diseases is evident by the presentation of autoimmune
and inflammatory disorders in humans with JAK mutations. Somatic missense mutations in JAKs
are linked to the development of malignancies. Genome-wide association studies (GWAS) have
not yet identified small nucleotide polymorphisms (SNPs) in JAK genes linked to asthma.8
In mice, the loss of JAK1 or JAK2 is lethal, respectively, at perinatal or embryonic stages of
development. Based on this evidence, loss of function mutations in JAK1 and JAK2 are predicted
to have high level of intolerance in humans.
Tyk2−/− animals show increased susceptibility to viral and intracellular infections.10 The deficiency
in Tyk2 affects Type II interferon activity and IL-12 signaling.11 Only a few patients have been
identified with Tyk2 mutations and all of them show high susceptibility to infections.11 One patient
with a homozygous recessive Tyk2 mutation was diagnosed with hyper-IgE syndrome (HIES), which
is an immune deficiency characterized by high levels of IgE, eczema, and recurrent lung infections.11
HIES patient cells are also defective in several cytokine signaling pathways (IL-6 and IL-12).10,11 Of
note is that Tyk2−/− mice do not develop HIES despite defects in cytokine signaling (IL-12).10 This
case serves as a good example of differences in JAK signaling between human and animal models,
though it could also indicate differences between loss of the entire gene and hypomorphic mutations
in patients, highlighting the complexity behind developing and testing treatments.
JAK3 is unique in that it is not expressed in all cell types like the other three JAKs. JAK3
expression is predominantly present in hematopoietic cell lineages and it only associates with IL-2
common gamma chain receptor. JAK3−/− animals have defective T and B cell maturation.
Mutations in JAK3 cause autosomal recessive severe combined immunodeficiency syndrome
(SCID). Human mutations of JAK3 are limited to defects in the immune system, which has
made targeted inhibitors of JAK3 promising immunosuppressants.8,9,12

4.2.2 JAKinibs in Asthma

The criticality of JAKs in cytokine signaling has led to the development of numerous JAK
inhibitors; both specific to certain JAKs and broad tyrosine kinase inhibitors. Some of these
Asthma and Allergic Airway Inflammation 51

TABLE 4.2
Use of JAK-STAT Protein Inhibitors in Mouse Models of Asthma
Inhibitor Targeted Protein Mouse Asthma Model References

TyrA1 All JAKs ↓STAT3 activation 15

↓Eosinophilia
WHI-P97 JAK3 ↓Eosinophilia 14
↓AHR
VR588 All JAKs ↓STAT3 activation 16
↓AHR
↓Immune cell infiltrate into lung
Decoy ODN STAT1 ↓BAL infiltrate, AHR, IL-5 56
C1889 STAT3 ↓Th2, Th17 cell development 57
R256 JAK1, JAK3 ↓Eosinophilia 62
↓AHR
↓Mucus production
AS1517499 STAT6 ↓RhoA Expression 196
Leflunomide STAT6 ↓RhoA Expression 197
AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage.

inhibitors are FDA approved and are being prescribed for conditions like rheumatoid arthritis
(Ruxolitinib, Tofacitinib, Oclacitinib), where they show considerable efficacy.13 These inhibitors
are in clinical trials for their potential in the treatment of other immune-related diseases as well.13
While JAK inhibitors have not yet been tested in patients with asthma, mouse model studies of
asthma have employed JAK inhibitors (see Table 4.2). One study shows that inhibiting JAK3
specifically (WHI-P97) reduces leukotriene synthesis in mast cells and in vivo administration can
prevent eosinophilia and airway hyperresponsiveness (AHR).14
A pan-JAK inhibitor (TyrA1) has been used which resulted in reduced STAT3 activation and
eosinophilia during a mouse model of allergic asthma.15 Another pan JAK inhibitor (VR588) also
resulted in reduced STAT3 activation, AHR, and immune cell infiltration in mice during allergic
asthma.16 These studies provide clear evidence that JAKs are important in orchestrating asthma
and can be targeted for inhibition. However, it is still not clear how they will affect asthmatic
patients.

4.3 STATs and Asthma

STATs are latent cytoplasmic proteins classified as transcription factors. There are seven known
mammalian STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) (see
Table 4.1). The structure of STAT proteins allows them to serve a dual role of both signal
transduction and DNA binding. Sequentially from the amino terminus end to the carboxy
terminus, these proteins have a STAT dimerization domain, coiled-coil domain, DNA binding
domain, SH2 domain, and transactivation domain. STAT proteins use their SH2 domains to bind
phosphorylated cytokine receptors. STATs are then phosphorylated by JAK kinases and released
from the receptor. This allows STATs to form homo or hetero dimers, which are poised for
translocation into the nucleus and transcriptional activation.17

4.3.1 STAT1 and STAT2

The overall importance of STAT1 and STAT2 for asthma is unclear. Type I, Type II, and Type III
interferons signal through STAT1 and STAT2. While some evidence exists that interferons may
52 JAK-STAT Signaling in Diseases

perpetuate asthma in general, Interferons are thought to be protective in asthma development

because they skew the immune system to Th1-like responses.18–20
Through the use of knockout animals and administration of recombinant IFNs during allergic
asthma, studies have shown that IFN-λ can reduce IL-5-, IL-13-, and IL-17-positive T cells and
dampen allergic asthma in mice.19–21 One study found that indirect activation of STAT1 (through
interferons or IL-12) can be protective in allergic asthma.22 There is also evidence that STAT1
induces pulmonary chemokines indicating that under some conditions it may not play a protective
role in mouse models of asthma.23 Evidence for the upregulation of STAT1 activity in epithelial
cells of asthmatic patients has also been documented.24

4.3.2 STAT3
Loss of function mutations in STAT3 in humans is the underlying cause of dominant negative
hyper-IgE syndrome (HIES), while gain of function mutations in STAT3 is associated with the
development of autoimmunity and immunodeficiencies. No association has been found between
SNPs or mutations in STAT3 and occurrences of asthma.25 It has been demonstrated that STAT3
is a positive regulator of migration and function of eosinophils from asthmatic patients.26
Germ-line Stat3−/− animals are difficult to assess because they die early in embryonic develop­
ment. However, conditional mutant mice that lack STAT3 in specific tissues have been used to
address the role of STAT3 in allergic inflammation. The importance of STAT3 in house dust mite
(HDM) models of asthma has been demonstrated using STAT3 airway epithelial cell conditional
mutant animals. These animals have reduced airway eosinophilia, lung TH2, and TH2 cytokine
levels in a chronic asthma model.15
STAT3 is known to play an important role in the development of TH2, TH9, and TH17 cells.
Mice with Stat3−/− T cells fail to develop allergic asthma. STAT3 can bind to TH2 cytokine loci
and this process is critical for STAT6 induced TH2 cell development.27 STAT3 is also critical for
inhibiting stability of TH9 cell development.28 In TH17 cells, STAT3 can activate several cytokines
and this process is well documented. T cells that are Stat3−/− have reduced RORγt,
a transcription factor that is critical for TH17 cell development. Similarly, TH17 cells fail to
develop in HIES patients.27,29 However, in contrast to the mouse system, STAT3 is required for
human TH9 development.29

4.3.3 STAT4
STAT4 is expressed in lymphoid and myeloid cells.30 It is implicated in the development and
regulation of TH1, TH2, and follicular helper T cells.31–35 STAT4 is capable of exerting cytokine
regulatory effects in other cell types such as regulatory T cells, TH17 cells, mast cells, and natural
killer cells.36–39 Beyond its role in cell development, STAT4 is associated with increasing the
severity of airway diseases including chronic obstructive pulmonary disorder (COPD), allergic
rhinitis, and asthma.40–43 At present, however, the direct role that STAT4 plays on the develop­
ment of these diseases remains under-investigated. The role of STAT4 on the regulation of several
cytokines important for the development of airway diseases such as asthma, has been explored
using mouse models of asthma and airway hypersensitivity.
STAT4 is a transcription factor that was initially described as being a master controller of the
development of TH1 cells. STAT4, which can be activated by IL-12, is capable of inducing effects
ranging from increased IFN-γ production, lymphocyte proliferation, and enhanced natural killer
cell cytotoxicity.33,35 During viral infections, STAT4 binds to the IFN-γ gene suggesting that
STAT4 alone is capable of regulating the IFN-γ pathway.44
As asthma is an airway disease with a complex interplay between TH1, TH2, and TH17 cells, the
cytokine milieu can have conflicting effects on TH cell development. IL-12 promotes the differ­
entiation of naïve T cells into TH1 cells while suppressing the generation of TH2 cytokines such as
IL-4 and IL-5. However, TH2 cytokines such as IL-4 inhibit the development of TH1 cells. Much
Asthma and Allergic Airway Inflammation 53

of this interplay can be attributed to transcriptionally activated T-bet which induces IL-12
receptor beta 2 subunit (IL-12Rβ2) expression and subsequently IL-12-dependent STAT4 activa­
tion. T-bet conversely represses the expression of several transcription factors including GATA-3.
In an environment with IL-4, IL-5, and IL-13, IFN-γ production and IL-12Rβ2 gene expression
are suppressed.45–48 TH17 cells are additional players in some asthma models and IL-12-induced
STAT4 activation and the induction of T-bet can drive them to an IFN-γ producing phenotype
further suppressing TH17 cell development.49–54

[Link] The IL-12/STAT4/IFN-γ Pathway in Mouse Models of Asthma

In mouse models of allergen-induced asthma, direct treatment with IL-12 can inhibit the
development of asthma-like symptoms, including airway hyperresponsiveness and eosinophilic
infiltration of the airways.55–57 Several studies elucidate the mechanism that drives the control
of STAT4 and IL-12 on airway hyperresponsiveness in allergen-induced asthma.58 Mice
deficient in STAT4, on both the C57BL/6 and BALB/C background, develop airway hyperre­
sponsiveness and airway eosinophilia.59,60 When these mice were given an IL-12 blocking
antibody during allergen re-challenge, airway hyperesponsiveness was reduced.61 In this model,
Stat4−/− mice showed decreased numbers of peribronchial eosinophils as compared to
controls. As expected, when profiling the airways for cytokines, IL-12 was lower as compared
to WT controls, perhaps suggesting that the lack of IL-12-induced STAT4 directly affects
eosinophils.61 IFN-γ was not altered in this model, suggesting an IFN-γ-STAT4-independent
process in some models of allergen-induced asthma. One such mechanism has been
described.61–63 Interestingly, in other studies using WT mice, when IL-12 is applied directly to
the airways of OVA-sensitized mice, IFN-γ levels are increased after antigen challenge. In this
model, eosinophil recruitment is still inhibited.58 Additionally, when mice are treated with TH2
cytokine reducing compounds, such as CpG Oligodeoxynucleotide, the ratio of IFN-γ and
IL-12 increases as compared to IL-5. This, in turn, delays the development of asthma-like
symptoms.64 Thus in these studies, the role that IL-12/STAT4 plays in asthma could be
beneficial.
In other mouse models of allergen-induced asthma, STAT4 contributes to airway hyperrespon­
siveness. However, its contribution compared to IFN-γ appears dependent upon concentration of
allergen used. For example, in one study, low dosage of LPS (0.1 µg) induced type 2 asthma
(airway hyperresponsiveness and eosinophilic infiltration) while high dosages (10 µg) induced type
1 asthma (airway hyperresponsiveness and non-eosinophilic infiltration). In the high dose model,
the asthma was not able to develop when using IFN-γ-deficient mice. Levels of lung inflammation,
IgG2a production, and airway infiltration by IFN-producing cells (CD8+ helper T cells) were
decreased. Interestingly, when using Stat4−/− mice, high dosages of LPS did not induce pulmon­
ary inflammation.65 A separate study utilizing cockroach antigen-treated control and Stat4−/−
mice showed that pulmonary levels of chemokines CCL5, CCL6, CCL11, and CCL17 were
reduced when compared to WT mice. Additionally, and unlike the previous model, TH2 cytokines
including IL-4 and IL-13 were not reduced.61 Allergen-induced airway hyperresponsiveness was
found to be impaired by STAT4-deficiency in a methacholine-challenge airway hyperresponsive­
ness model. Though IL-12 levels remained the same between WT and Stat4−/− groups, lung
infiltration by IFN-γ producing cells was only enhanced in WT mice.66 Thus, most data suggest
that IL-12-signaling and STAT4-dependent IFN-γ production are critical to controlling airway
hyperresponsiveness, but in some models they can be dispensable or even required for develop­
ment of disease.

[Link] STAT4 in Patient Asthma

Several population-based studies have found that IL-12 levels are reduced in asthmatics and that
polymorphisms in the IL-12 gene can serve as a risk factor for the development of asthma.67,68
54 JAK-STAT Signaling in Diseases

Following inhaled corticosteroid treatments, IL-12 levels are increased in these patients.69 How­
ever, when using high dose-inhaled corticosteroids on populations with defects in the STAT4 gene,
asthma symptoms do not improve.43
Additionally, human airway disease studies have mixed conclusions in correlating IFN-γ and
disease severity.70–76 For example, sputum IFN-γ expression is higher in severe asthmatics as
compared to mild to moderate asthmatics.65 In a study examining discordant twins, hypermeth­
lyation of IFN-γ was associated with asthma severity.77 Another study found hypermethlyation of
the IFN-γ promoter to be associated with the increased risk of the development of occupational-
related asthma.78 These suggestions indicate that the role of IFN-γ may be more subtle and
context-dependent.
In the human population, polymorphisms of STATs, including STAT4 and STAT6, are
associated with airway diseases including asthma and COPD.79,80 In studies using a Japanese
population of asthmatics, patients homozygous for the STAT4 SNP (rs925847) display reduced
lung function and increased bronchial airway remodeling.43 In a study examining an asthmatic
Korean population, that same SNP was associated with an increased risk of the development of
allergic asthma.81 In studies of children, STAT4 SNPs (rs16833215 and rs4853546) were asso­
ciated with childhood wheezing and increased asthmatic exacerbations.82 Finally, in studies
incorporating IFN-γ, STAT4, and STAT6 polymorphisms, SNPs in any of these genes were
correlated with the development of asthma.83 Taken together, these studies suggest that defects
in the STAT4-IFN-γ pathway could lead to the development of asthma and increased severity of
symptoms.

4.3.4 STAT5
There are two genes encoding STAT5 proteins: Stat5a and Stat5b which are 96% similar in
sequence but have some distinct biological functions. Stat5a/b−/− animals have prenatal
lethality and severe immunodeficiency similar to JAK3−/−.84 In humans, STAT5b deficiency
causes autoimmune disease in part due to dysfunctional T regulatory cells.85 Patients with severe
refractory asthma (SRA) were found to have lower levels of STAT5a in their peripheral blood
mononuclear cells.86
STAT5 proteins can be activated via JAK and Src kinases. It has been well documented that
STAT5 plays an important role in the development of cells that drive airway disease such as TH2,
TH9, ILC2s, mast cells, and eosinophils.87–89 STAT5 is critical in mast cell development and
IgE-mediated mast cell function.87 In TH9 cells, IL-9 gene transcription is in part dependent on
STAT588,90,91 STAT5 activation, through IL-2 signaling, has been shown to induce T cell
proliferation in asthma models.92

[Link] STAT5, IL-5, and IL-2

Eosinophilic lung inflammation and high peripheral blood eosinophil numbers have been asso­
ciated with severity of asthma in humans. Eosinophils are also implicated in airway remodeling
because they are the source of remodeling molecules such as tumor growth factors (TGF), VEGF,
and metalloproteinases.89 IL-5 is undoubtedly an important mediator of asthma because it is
important for the maturation and migration of eosinophils. In the asthmatic lung milieu, IL-5 is
primarily produced by T cells, eosinophils, mast cells, and innate lymphoid cells. In humans, high
levels of IL-5 protein can be found in the mucosa of asthmatic airways.93 Studies of anti-IL-5
treatment (Mepolizumab) have shown reduced disease exacerbation in severe asthmatics with
eosinophilia. Anti-IL-5 treatment in these patients reduced peripheral blood and sputum eosino­
phil counts.94–96
Ovalbumin-challenged Il5−/− mice show decreased numbers of circulating eosinophils and
airway eosinophilia.97,98 Conversely, inhalation of IL-5 has been shown to increase eosinophils
and airway hyperresponsiveness. IL-5 primarily signals through the JAK2/STAT5 pathway. It has
Asthma and Allergic Airway Inflammation 55

been demonstrated that Stat5a−/− and Stat5b−/− animals have diminished antigen-induced
eosinophil recruitment in the airways. Interestingly, this effect is due to both diminished IL-5
responsiveness of eosinophils and defects in antigen-specific TH2 cell proliferation.99,100 TH2 cell
differentiation is thought to be orchestrated by IL-2 signaling, which primarily signals through
STAT5. It has been shown that constitutively active STAT5a, independent of STAT6, can increase
accessibility of the IL-4 gene leading to TH2 cell differentiation.101 Consistent with this concept,
mice that are Stat5a−/− Stat6−/− have severely decreased allergic airway inflammation with low
eosinophilia and TH2 cell differentiation compared to Stat6−/− alone.102

[Link] STAT5 and TSLP

TSLP is a type I cytokine that signals through JAK2/JAK1 to activate STAT5 (and other STATs
to a lesser degree).103 In the lung, TSLP is primarily expressed in epithelia and stromal cells and
some nonstructural cells such as DCs.104 TSLP supports maturation of B and T cells in addition
to modulating many immune cells that play a role in development of lung inflammation like DCs,
mast cells, and eosinophils. TSLPR-deficient animals have been used to demonstrate that without
TSLP signaling, mice do not develop allergic asthma. Inflammation can be restored in these
animals when wild type T cells are reconstituted, emphasizing the importance of TSLP signaling
for T cells.105,106 It has also been demonstrated that TSLP inhibits the development of tolerance
to an allergen in the lung. TSLP can suppress development of antigen-specific human and mouse
T regulatory cells.107,108

4.3.5 STAT6
In mice and humans, IL-4 induces the tyrosine phosphorylation and DNA binding activity of
STAT6109,110 IL-13 activates STAT6 in cells that express the receptor including macrophages.46,111,112
Some evidence suggests that IL-15 is additionally capable of activating STAT6; however, more
investigations are warranted.113–115 Activated STAT6 has roles in the regulation of several genes
including the TH2 transcription factor GATA3, IL-4Rα receptor, eotaxin-3, CCL17, and
FIZZ.146,115–124 STAT6 also inhibits the expression of CD40 and E-selectins.125,126
IL-4 and IL-13 are cytokines that induce the activation of STAT6. In asthma, these cytokines
play numerous roles. For example, activated TH2, NK, and mast cells secrete IL-4 and IL-13 to
drive the activation of B-cells. Activated B-cells initiate an IgE-producing phenotype, which allows
mast cell priming.127–130 In asthmatics, both cytokines are increased in airway smooth muscle
cells.131 These cytokines promote goblet cell metaplasia which enhances mucus secretion and airway
hyperresponsiveness.127,130,132,133 Therefore, it is of interest to identify the link between IL-4 and IL-13
with STAT6 in asthma. Indeed, asthma patients treated with the IL-4/IL-13 signaling inhibitor,
dupilimab, experience decreased asthma exacerbations and improved lung functions.134
STAT6 plays several roles in helper T-cell development. While it mainly skews the differentia­
tion of naïve T-cells towards a TH2 producing phenotype, it can regulate both TH9 and TH17 cell
development.35,46,135–137 In airway diseases, STAT6 affects numerous cell types. For example, in an
Alternaria-allergen challenge model of asthma, STAT6 controlled eosinophilia.138 Another study
linked the control of eosinophilia by STAT6 to PARP1, a poly (ADP-ribose) polymerase.139
In asthmatics, STAT6 positively correlates with increased serum IgE levels.140 Furthermore,
several studies indicate that increased STAT6 levels also correlate with more severe forms of
allergic rhinitis and asthma.141–145 In patients who undergo glucocorticosteroid treatments,
STAT6 controls cytokine secretion in cell types such as innate lymphoid cells (ILC2s).146

[Link] STAT6 and IL-4 in Patient Asthma

Observations of induced sputum in asthmatics reveal that STAT6 and the IL-4 receptor are
expressed more highly in these patients than in control subjects.147 Mutations in the IL-4 receptor
56 JAK-STAT Signaling in Diseases

are wide ranging and can result in increased serum IgE. Several mutations are linked with the
increased incidence of atopic asthma and defective STAT6 DNA binding (Q576R and
S503P).148–153 For example, a mutation in an extracellular region of IL-4R (V50) leads to prolonged
STAT6 phosphorylation.153 Another IL-4R mutation (Ala57Thr) is associated with decreased atopy
among patients in Greenland.150
Several STAT6 variants are associated with asthma risk.154 The variant rs324015 has a
protective effect on atopic asthma. Variant rs7180246 is associated with an increased risk of
asthma in all but an Indian population.143,154 In Caucasians, the variant rs324011 is associated
with asthma risk.154 Variant rs324011 though not associated with asthma risk, is associated with
recurrent wheezing in early childhood.155 A recently characterized STAT6 variant, rs167769, is
associated with asthma risk in a multi-ancestry study.156

[Link] Pharmaceutical and Biological Inhibition of STAT6

Researchers have used various inhibitors to block the IL-4/STAT6 pathway. Fasudil, an
approved clinical drug that can block genes such as RhoA/Rho kinase from enhancing
hypermucus secretion in asthmatics, was used in mouse studies to further clarify the role of
STAT6 in asthma. In an OVA-challenge mouse model, secreted IL-4 was decreased in Fasudil­
treated animals compared to untreated wild types. Importantly, this correlated with a decrease
in both phosphorylated STAT6 and STAT6 protein levels as compared to OVA-challenged
untreated controls.157
Another drug, heparin, limits the amount of secreted cytokines from asthmatic cells and prevents the
development of asthma in mice, sheep, and pigs.158–161 In humans, it limits bronchoconstriction.162 In
an OVA-sensitized mouse model of asthma using sulfated non-anticoagulant low molecular weight
heparin (S-NACH), a low molecular weight form of heparin, S-NACH acted as an inhibitor of STAT6.
This led to reduced secreted IL-4. Transcription factor protein amounts, such as GATA3, were also
reduced.163
Reductions in protein expression levels of IL-4 in mouse lungs occur when STAT6 siRNA is
used in asthmatic models.164 Similarly, in models of allergic rhinitis, sneezing and nasal rubbing in
STAT6 siRNA-treated mice are decreased as compared to untreated controls. When submandib­
ular lymph node cells were stimulated with OVA in vitro, secreted IL-4 amounts were undetected
in cells originating from the STAT6 siRNA-treated mice.165 These results corroborate with those
from similar studies. In chronic fungal asthma mouse models using Stat6−/− mice, IL-4 levels
increased much slower than in WT mice.166 The lack of complete abolishment of IL-4 suggests
that STAT6 does not prevent all features of asthma from developing.167 Chemokines such as
MCP-1, RANTES, and eotaxin were altered compared to WT animals. This confirmed the results
of several Stat6−/− in vitro studies.166,168,169 Studies using STAT6-deficient mice and acute and
chronic challenge with OVA, also show decreased amounts of IL-4 in BAL.167
Several chemical compounds provide insights into the role of STAT6 and IL-4 in allergic airway
disease. In rat allergic airway models using YM-341619 hydrochloride, a compound that exclu­
sively suppresses the differentiation of T cells to TH2 cells, STAT6 expression was suppressed.
When the compound was used on cultured T-cells, IL-4 production was decreased. Airway
hyperresponsiveness was similarly decreased when the compound is administered orally in a DNP-
Ascaris asthmatic-mouse model.170 This finding mimics the role of Prednisolone, an orally
available corticosteroid, in altering IL-4 levels in asthma. In an OVA sensitization and methacho­
line-challenge model using BALB/c mice, the use of Boswellic acid led to decreased IL-4 secretion,
phopho-STAT6, and GATA3. Overall, airway inflammation was attenuated.171 In a study using
tetrahydrocurcumin (THC), a major metabolite of Curcumin, a pigment from the Indian spice
turmeric, OVA-induced asthmatic mice displayed decreased asthmatic symptoms, such as nasal
rubbing, tissue eosinophilia, macrophage infiltration, and mucus production. Cytokine levels in
BAL fluid such as IL-4, IL-5, and IL-13 were reduced compared to OVA-challenge THC-
untreated mice. In examining protein secretion and phosphorylation levels, the data suggested
Asthma and Allergic Airway Inflammation 57

that these decreases were due to decreased expression of IL4 receptor, STAT6, and GATA3,
further suggesting a role for these proteins in asthma.172
Natural anti-inflammatory compounds such as ursolic acid have been used in lung inflamma­
tion treatments. In one study, ursolic acid prevented the development of airway eosinophilia in
OVA-challenged mice. Levels of several cytokines such as IL-13 and IL-17 were also decreased as
compared to wild type controls. Interestingly, when examining the levels of transcription factor
expression, both STAT6 and GATA3 were decreased compared to controls.173

[Link] STAT6 and IL-13

Eotaxin is of great interest in studies of asthma and atopy. Elevated levels of eotaxin are routinely
present in asthma patients.174 Several cell types secrete eotaxin in response to IL-13 treatment.
For example, airway epithelial cells induced by IL-13 secrete eotaxin.175 In human skin cells, when
treated with STAT6 siRNA and stimulated with IL-13, eotaxin levels are decreased.164 When
human bronchial epithelial cells are treated with IL-13, STAT6 is activated, suggesting a link
between IL-13, STAT6, and eotaxin, and potentially explaining the mechanism for increased
levels of eosinophils in some forms of asthma.176 In in vitro studies using human airway cell lines,
such as A549, eotaxin family levels (CCL11, CCL24, CCL26) are upregulated in response to IL-4
and IL-13. In this same study, STAT6 siRNA blocked the upregulation of these eotaxins. When
secreted protein levels were examined, only CCL26 was detectable while the others were not. In
this same study, eotaxin gene expression was decreased when primary human bronchial smooth
muscle cells and primary epithelial cells were treated with IL-13 and STAT6 siRNA.177
In a study where transgenic mice that expressed STAT6 under control of a CC10 promoter,
a protein that is specific to airway epithelial cells, were crossed to STAT6 deficient mice, they
selectively expressed STAT6 only in lung epithelium cells. These mice were crossed further with
transgenic IL-13 mice and examined for the requirement of STAT6 in IL-13 induced asthma. While
researchers found that STAT6-deficient mice were protected from airway hyperresponsiveness, they
saw that mice that only expressed STAT6 in the airways still developed airway hyperresponsiveness.178

[Link] STAT6 and IL-13/IL-17

Like IL-4 and IL-13, IL-17A is increased in asthmatics. IL-17A induces bronchial cells to secrete
cytokines such as IL.-6179 IL-17 can induce the secretion of IL.-13180 In studies that transferred
TH17 cells into both WT and STAT6-deficient mice, IL-13 protein expression was assessed after
OVA challenge. Harvested lungs from both groups showed higher IL-13 protein expression when
compared to PBS controls. When comparing both the OVA-treated groups, the STAT6-deficient
mice displayed higher levels of IL-13 protein expression. Overall, STAT6 negatively regulated
IL-13 protein expression.181
The role of IL-17 on receptor-mediated signaling has been explored. The IL-4Rα chain can link
with the IL-13Rα2 receptor to form the type II IL-4/IL-13 receptor. When this happens, IL-17
can induce IL-13 secretion. Thus, studies have attempted to identify the role of IL-17 on the IL-13
receptor complex.182 Recent studies focusing on IL-13rα2, which has traditionally not been linked to
the development of asthma, have shown that it makes a modest contribution to the development of
airway hyperresponsiveness.183 When IL-13rα2-deficient mice were given IL-17, IL-13 driven airway
hyperresponsiveness was enhanced.180 While IL-13rα1 clearly plays a role in STAT6-mediated
signaling, it appears that IL-13rα2 does not. When non-transgenic mice are compared to IL-13rα2
deficient mice, and STAT6 levels are examined, differences in STAT6 activation are not observed.183

[Link] STAT6 and Mucus Gene Overproduction

Since one of the hallmark features of asthma is mucus overproduction, studies have defined ways
that STAT6 may, directly or indirectly, play a role in the regulation of mucus genes.127,132,184
58 JAK-STAT Signaling in Diseases

Twelve mucin-related genes are expressed in humans and several of these, such as MUC2 and
MUC4, are sometimes increased in asthmatics when compared to healthy controls.185,186 One of
the more heavily studied genes, MUC5AC, is increased up to 60% higher than normal in
asthmatic patients.185
Intratracheal IL-13 induces MUC5AC in wild type but not STAT6-deficient mice. Consistent
with this, when cells were isolated and then treated with IL-13 for 24 hours, MUC5AC
expression was only slightly increased in STAT6 deficient animals compared to untreated cells,
and to wild type controls.132 Moreover, another gene involved in goblet cell metaplasia, Gob-5,
was unresponsive in cells deficient in STAT6 as compared to wild type controls, suggesting
a role for STAT6 in controlling both genes.132 In a study using STAT6 siRNA to knockdown
STAT6 levels, Muc5ac promoter activation was strongly decreased.187 In another study using
STAT6 siRNA, repression of STAT6 in the presence of IL-13 led to decreased MUC5AC
expression as well as SAM domain-containing prostate-derived Ets factor (SPDEF), which is
a transcription factor that plays a role in goblet cell hyperplasia and mucus secretion.188
Reductions in IL-13-mediated expression of the transmembrane protein 16A, a calcium-
activated chloride channel that is a key regulator of mucus overproduction in airway epithelial
cells, were shown to be a result of STAT6 inhibition. This reduction corresponded to decreased
MUC5AC expression, further highlighting the role of STAT6 in mucin expression.189 In a study
exploring the effects of using a Chinese herb extract, Glycyrrhiza uralensis, on asthma, the
flavonoid 7′4′-dihydroxyflavone was shown to possess anti-inflammatory properties. When this
compound was used on human cell cultures stimulated with PMA, Muc5AC secretion was
decreased compared to untreated controls. This decrease was positively correlated with declining
phosphorylated STAT6.190
The role of STAT6 in controlling mucin genes has additionally been explored in Lyn kinase­
deficient mice. Lyn kinase negatively regulates the progression of asthma.191 In an in vitro study
using human bronchial epithelial cells treated with either IL-4 or IL-13, when Lyn siRNA was
used, STAT6 expression and phosphorylation increased in both conditions, as compared with
untreated cells. When Lyn was overexpressed in their system, STAT6 expression was decreased.
Taken together, the results suggest that Lyn regulates MUC5AC expression via the STAT6
pathway.187

4.3.6 STAT Inhibitors

Theoretically, like JAKinibs, inhibitors of STAT proteins could be promising in immune-related
disorders (see Table 4.2). Efforts in studying STAT inhibitors have been challenged by a lack of
bioavailability and low efficacy and specificity. Some groups have tried to circumvent issues of
specificity by employing oligonucleotide-based STAT inhibitors. One example is the STAT1 decoy
oligonucleotide which, in mouse models, has been shown to reduce lung infiltration and antigen-
specific AHR.192
A STAT3 inhibitor that targets the SH2 domain (C1889) has been used to treat animals
intranasally during allergic asthma model. This treatment significantly reduced TH2 and TH17
development and allergic asthma.193 Currently, some small molecule inhibitors of STAT3 are in
clinical trials to be tested for indications other than asthma.194,195
In mouse models of allergic bronchial asthma, IL-13 administered intranasally leads to induced
phosphorylation of STAT6 as well as the upregulation of the RhoA protein, a protein involved in
smooth muscle contraction. In this same study, when human bronchial smooth muscle cells were
used and treated with a STAT6 inhibitor, leflunomide, STAT6 phosphorylation and the RhoA
upregulation induced by IL13 were abolished.196 In another study, when the STAT6 inhibitor,
AS15174999, was used concurrent with a 1-hour exposure of human bronchial smooth muscle
cells to IL-13, STAT6 phosphorylation was inhibited in a dose-dependent manner. Similarly,
RhoA expression was decreased in these cells. In the same study, when STAT6 siRNA was used,
RhoA expression was partially inhibited.197
Asthma and Allergic Airway Inflammation 59

Another approach taken to find inhibitors of STAT proteins is the use of clinically available
drugs that, through unknown mechanisms, reduce STAT activation. For example, the psycho-
trophic drug pimozide has been shown to reduce the phosphorylation of STAT5198 This drug was
recently used in an ex-vivo study to show that the steroid resistance seen in the ILC2 cells of
asthmatic humans was dependent on TSLP and STAT5 activation.199 Additionally, JAK inhibi­
tors are used to assess the role of STAT proteins in vivo. In particular, the JAK1/3 inhibitor
(R256) can prevent STAT5 activation and TH2 development in culture. Treatment of animals with
R256 can reduce AHR, eosinophilia, and mucus production.200

4.4 Conclusion
Despite some of the challenges, JAK/STAT pathway constituents remain attractive targets for
pharmaceutical intervention. Several intrinsic properties of JAK/STAT signaling highlight the
complexity of this pathway. STAT proteins can be expressed in a cell- and tissue-specific manner.
One cytokine can activate multiple JAK and STAT proteins to varying degrees. STAT proteins
can create homodimers and heterodimers which allow for differential DNA binding.7,201 STAT
proteins may also be able to compete with or compensate for one another and some evidence does
exist supporting this claim.7,202 It has therefore been a challenge to study the effects of inhibiting
STAT proteins in multicellular and multi-organ systems and existing studies that tackle this
question should be interpreted cautiously.
The JAK/STAT pathway is clearly central in the development of asthma. STAT3, STAT5,
and STAT6 are required in multiple cell types for myriad cytokine responses. While the role of
other STAT proteins is less clear, there are likely some situations where they are required in the
spectrum of asthma endotypes that span disease pathologies from purely TH2 to more mixed
TH cell phenotypes. As the use of JAKinibs and more specific STAT inhibitors move more
broadly into the clinic, it will be interesting to see how they affect various patients with asthma
and how personalized medicine can help direct the use of drugs to inhibit these critical
pathways.

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193. Gavino, A. C., Nahmod, K., Bharadwaj, U., Makedonas, G., & Tweardy, D. J. STAT3 inhibition
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asthma model. Allergy. 71, 1684–1692 (2016).
194. OPB-51602 in locally advanced nasopharyngeal carcinoma prior to definitive chemoradiotherapy ­
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68 JAK-STAT Signaling in Diseases

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197. Goto, K. et al. The proximal STAT6 and NF-kappaB sites are responsible for IL-13- and
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198. Nelson, E. A. et al. The STAT5 inhibitor pimozide displays efficacy in models of acute myelogenous
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asthma: The role of thymic stromal lymphopoietin. J. Allergy Clin. Immunol. 141, 257–268.e6 (2018).
200. Ashino, S. et al. JAK1/3 signaling pathways are key initiators of TH2 differentiation and lung
allergic responses. J. Allergy Clin. Immunol. 133, 1162–1174.e4 (2014).
201. Delgoffe, G. M. & Vignali, D. A. A. STAT heterodimers in immunity. JAK-STAT 2, e23060 (2013).
202. Yang, X.-P. et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions
of STAT3 and STAT5. Nat. Immunol. 12, 247–254 (2011).
5
Role of JAK-STAT Signaling in Atopic Dermatitis

Radomir M. Slominski
Department of Dermatology
Indiana University School of Medicine
Indianapolis, Indiana

Matthew J. Turner
Department of Dermatology
Indiana University School of Medicine
Indianapolis, Indiana
Richard L. Roudebush VA Medical Center
Indianapolis, Indiana

5.1 Introduction
Atopic dermatitis (AD) is one of the most common inflammatory diseases known to humankind.
Severe itch, lost sleep, and secondary skin infections are major contributors to the morbidity of this
disease (Holm et al. 2006; Ong and Leung 2010). Allergic skin inflammation and associated impaired
skin barrier function are key drivers of AD pathogenesis, and Th2 (and other) cytokines are
implicated in initiation and preservation of skin inflammation in AD. Targeted therapeutics have
recently entered the market for treatment of AD, including monoclonal anti-human IL-4Rα antibody,
dupilumab, which was FDA approved in 2017 for treatment of moderate-to-severe AD (Beck et al.
2014). Several other types of targeted therapeutics are either FDA approved or under investigation;
the latter group includes oral and topical forms of Janus kinase (JAK) inhibitors. This chapter will
discuss clinical and histopathologic aspects of AD, and mechanisms of AD immunopathogenesis,
including the role of JAK/STAT signaling in AD, and conclude with a review of available data
regarding use of JAK inhibitors in human and canine AD.

5.2 Clinical Characteristics of AD

Based on estimates of up to 17% of children in the United States developing atopic dermatitis
(AD) and 2009 U.S. census data, more than 13 million children in the United States have AD
(Laughter et al. 2000). The worldwide prevalence of AD in adults is 1–3%, equating to millions of
additional cases (Katsarou and Armenaka 2011). There are two different phases of AD lesions,
acute and chronic (Rajka 1986). Acute lesions are red (erythematous), scaly, minimally elevated
plaques with poorly defined borders. In contrast to acute lesions, chronic lesions are more well
defined, often less scaly, and are more elevated appearing with accentuated skin lines (i.e.
lichenified). Chronic lesions develop from acute lesions, in part as a result of repetitive rubbing
and scratching of the skin (Rajka 1986). The areas of the body affected by AD (i.e. the
distribution) change with age. In infancy, the flexor aspects of the extremities and face are more
prominently involved. In contrast, the involvement of the extensor aspects of the extremities is

69
70 JAK-STAT Signaling in Diseases

more common later in childhood and adulthood. As implied above, the extent of skin affected
by AD patients can be limited to specific body regions; however, disease can be much more
extensive involving the majority of a patient’s skin. In addition to AD lesions themselves, AD
patients often have other associated clinical findings including dry skin (xerosis), ichthyosis
vulgaris (fish-like scaling of the skin caused by autosomal semidominant mutations in the
Filaggrin gene) and keratosis pilaris as well as comorbid allergic diseases such as asthma, allergic
rhinitis and food allergies (Rajka 1986; Smith et al. 2006).
As noted in the introduction, severe itch (pruritus), poor sleep and secondary skin infections are
major sources of morbidity in AD. Regardless of the extent of skin involvement, itch is one of the
defining features of AD (Rajka 1986). Patients often respond to itch by scratching the skin, which can
have several effects including: minor bleeding, inoculation of injured skin with microbial pathogens,
promotion of acute to chronic lesion conversion, and development of scarring and/or skin dyspig­
mentation. In addition, itching and scratching are important contributors to sleep disruption in AD
(Stores et al. 1998). With respect to secondary skin infections, Staphylococcus aureus, which also
frequently colonizes skin in AD patients without causing disease, is probably the most common
pathogen (Ong and Leung 2016; Totte et al. 2016). Typically, S. aureus infections manifest as
impetigo, a superficial skin infection that may resolve even without antibiotic therapy if a patient’s AD
is treated with anti-inflammatory agents (Travers et al. 2012). In contrast, herpes simplex virus
infections of AD skin lesions (eczema herpeticum) benefits from prompt treatment with systemic
antiviral therapies, particularly in the case of those that put the patient at risk of developing eye
infections, which can result in permanent vision loss (Young et al. 2010). As a result of the
aforementioned morbidities, the quality of life in AD can be dramatically impaired. For example,
children with generalized AD reported greater impairment in quality of life than patients with
asthma, diabetes, and epilepsy (Beattie and Lewis-Jones 2006). Healthcare costs for AD are also
significant (Mancini et al. 2008). In light of the above and the historical lack of widely effective
treatments for moderate-to-severe disease, patients and the medical community recognize the need to
develop more effective therapeutic agents for AD.

5.3 Histologic Aspects of AD

This section begins with a review of the histologic architecture of healthy skin and the histopathologic
changes seen in AD. These findings are summarized in Figure 5.1. Although the architecture varies
between body regions, the skin can generally be thought of as being composed of three layers, the upper
layer (epidermis), a middle layer (dermis) and the underlying subcutaneous fat. The outermost layer of
the skin, the epidermis, is also composed of multiple layers as detailed below. While this simplistic three-
layer concept of the skin will be discussed below for understanding AD pathogenesis, it is worth noting
that the skin is a highly specialized and complex organ system composed of many resident cell types and
structures including nerves, blood vessels, hair follicles, eccrine glands, apocrine glands, and others. The
role of all of these structures and cell types in AD is beyond the scope of this discussion.
The lowest (basal) layer of the epidermis is composed of stem cells and undifferentiated keratinocytes.
These undifferentiated keratinocytes leave the basal layer and transit upward through the epidermis,
undergoing progressive and eventually terminal differentiation, at which point their nucleus is lost and
they become protein-rich devitalized structures called corneocytes that are embedded in a lipid-rich
matrix. This composite of corneocytes and lipid matrix is the uppermost layer of skin called the stratum
corneum, a key barrier structure that prevents harmful irritants, allergens, and pathogens from entering
the skin and promotes water retention in the skin (Matsui and Amagai 2015).
Histopathologic changes in AD include increased thickness of the stratum corneum (ortho- and
parakeratosis), increased epidermal thickness (acanthosis) due to epidermal hyperplasia, spongio­
sis due to intercellular edema in the epidermis, and lymphocytic and histiocytic inflammatory cell
infiltration into the dermis and epidermis. In comparing acute and chronic AD lesions, acanthosis
and inflammatory infiltrates are more prominent and spongiosis is often less prominent in chronic
Role of JAK-STAT Signaling in Atopic Dermatitis 71

FIGURE 5.1 Basic histologic features of healthy human skin and atopic dermatitis lesional skin. The epidermis is
predominately composed of keratinocytes. Other cells in the epidermis include melanocytes and Langerhans cells (not
shown). The four layers of the epidermis include the stratum basale (SB), stratum spinosum (SS), stratum granulosum
(SG), and the stratum corneum (SC). The SB is the proliferative and least differentiated layer. Keratinocytes in the SG
have keratohyalin granules (purple dots), which contain filaggrin. The SC is the devitalized terminally differentiated
layer of the epidermis and is composed of corneocytes embedded in a lipid matrix. The SC and SG are major
contributors to epidermal barrier function. Spongiosis, which is caused by intercellular edema leading to keratinocyte
separation, is a common histologic feature of atopic dermatitis; however, spongiosis is not always observed in chronic
atopic dermatitis lesions. A perivascular lymphohistiocytic infiltrate is also seen in atopic dermatitis lesional skin; the
density and composition of this infiltrate varies between acute and chronic atopic dermatitis lesional skin and different
atopic dermatitis endotypes. Other histologic features that can be seen in atopic dermatitis lesional skin include
parakeratosis (thickened SC composed of corneocytes with retained nuclei), hypergranulosis (increased numbers of
keratohyalin granules in the SG), and lymphocyte exocytosis (lymphocytes in epidermis). Of note, the histologic
features of atopic dermatitis are not distinguishable from many other types of eczematous dermatitis.

lesions. Immunohistochemical and other analyses demonstrate that T cells are abundant in the
epidermal and dermal inflammatory cell infiltrates (Zachary et al. 1985).

5.4 Pathogenesis of AD
The pathogenesis of AD is mediated by a complex interplay between skin barrier dysfunction and
immune system dysregulation (Werfel et al. 2016). Atopic dermatitis is a complex genetic disease;
Filaggrin gene mutations are perhaps the best-characterized genetic defect in AD (Palmer et al.
2006; Weidinger et al. 2006). There is also a great deal of genetic, clinical, histologic, and
immunologic variation between subjects with AD. For example, most AD patients do not
harbor Filaggrin gene mutations, and there is variation in the type and frequency of Filaggrin
mutations between different populations (Akiyama 2010). The presence (extrinsic AD) or absence
(intrinsic AD) of elevated total serum IgE levels provide another was to subdivide AD (Novak
and Bieber 2003). There are also differences in clinical, histologic and immunologic characteristics
of AD in certain Asian populations who can manifest with a more psoriasiform phenotype
compared to classical AD features commonly seen in those of European descent (Noda et al.
2015). Taken together, these observations highlight the heterogeneity of AD and support the
72 JAK-STAT Signaling in Diseases

concept that there are subtypes (endotypes) of disease (Werfel et al. 2016). However, there
are characteristic clinical, histologic, cellular, and molecular aspects of disease that help
explain AD pathogenesis. As this chapter is focused on JAK-STAT signaling and JAK inhibition
as a potential treatment of AD, much of what follows in this section examines the immunologic
aspects of AD pathogenesis, some of which are summarized in Figure 5.2.

5.4.1 Epidermal Barrier Dysfunction

Development of AD involves two key and interrelated processes, epidermal barrier dysfunction
and T helper type 2 (Th2) cell-polarized immune responses (Bieber 2008; Sehra et al. 2008b; De
Benedetto et al. 2009). The stratum corneum is the outermost part of the epidermal barrier and is
composed of devitalized keratinocytes (i.e. corneocytes) embedded in a lipid matrix (Elias and
Schmuth 2009; Kypriotou et al. 2012). Cornified envelope proteins including filaggrin, loricrin and

FIGURE 5.2 Immunopathogenic mechanisms of atopic dermatitis that involve JAK-STAT signaling. In AD, a variety
of cytokines are produced by keratinocytes (TSLP, IL-33), Th2 cells (IL-4, IL-5, IL-13 and IL-31), and Th22 cells
(IL-22). With the exception of IL-33, these cytokines activate receptors (green) that employ JAK-STAT pathways. Top
panel: the upper layer of the skin (epidermis) is composed of keratinocytes that undergo progressive differentiation. As
keratinocytes differentiate and progress upward through the epidermis, they acquire the ability to produce the
epidermal barrier protein, filaggrin (FLG), which is stored in intracellular granules (purple circles). Keratinocytes
then lose their nucleus and become devitalized corneocytes embedded in a lipid matrix; this layer is called the stratum
corneum (SC) and forms a key part of the skin barrier. Stimulation of keratinocytes with IL-4 reduces keratinocyte
maturation, filaggrin expression, and proper stratum corneum formation. In contrast, IL-22 stimulation of keratino­
cytes promotes epidermal hyperplasia. Middle panel: Stimulation of Th2 cells with IL-4, IL-33, and TSLP promotes
Th2 cell differentiation and/or cytokine production. Bottom panel: Stimulation of B cells with IL-4 promotes IgE class
switching and production. Peripheral eosinophilia is stimulated by IL-5 and IL-33. Cytokines that act directly on
neurons to stimulate itch include IL-4, IL-31, and TSLP.
Role of JAK-STAT Signaling in Atopic Dermatitis 73

involucrin are key structural and functional proteins in corneocytes and are critical for proper
epidermal barrier function (Elias and Schmuth 2009; Kypriotou et al. 2012). These proteins are
encoded within the epidermal differentiation complex (EDC) gene cluster on human chromosome
1q21 (Elias and Schmuth 2009; Kypriotou et al. 2012). Barrier dysfunction in AD has been
classically attributed to inherent defects (i.e. genetic) in the keratinocyte; the most well-established
example of such a defect is the presence of mutations in the Filaggrin gene (Palmer et al. 2006;
Weidinger et al. 2006).
Filaggrin is a cornified envelope protein that normally aggregates keratin filaments in
corneocytes facilitating the ordered formation of a ‘bricks and mortar’ type structure of the
stratum corneum and an effective barrier that helps impede water loss from the skin and entry
of certain microbes and environmental agents into the skin (Elias and Schmuth 2009; Scharsch­
midt et al. 2009; Kypriotou et al. 2012). Filaggrin mutations are detected in a significant
proportion of patients with AD and result in insufficient quantities of functional protein thereby
leading to defective bundling of keratin filaments and disarray in the organization of the
stratum corneum (Manabe et al. 1991; Brown and McLean 2012). Filaggrin breakdown
products are important components of the so-called natural moisturizing factor (NMF) that
acts as a humectant helping to retain water in the skin. In patients with Filaggrin gene
mutations and AD, decreased levels of filaggrin and as a result decreased levels of NMF, are
thought to contribute to skin dryness (Rawlings and Harding 2004; Kezic et al. 2008). As noted
earlier, Filaggrin gene mutations also cause ichthyosis vulgaris, thus explaining the association
between this scaling skin condition and AD.
The preceding discussion on Filaggrin shows that intrinsic defects in keratinocytes can and
often do occur in AD, resulting in defective epidermal barrier function. These primary defects in
keratinocyte function leading to impaired skin barrier function can in turn promote cutaneous
inflammation in AD (Kim and Leung 2018). Interestingly, the majority of patients with AD do
not harbor Filaggrin gene mutations (Margolis et al. 2012). This suggests the impaired keratino­
cyte differentiation and function in AD do not necessarily require intrinsic defects in keratino­
cytes. One explanation for this observation is that in AD, the Th2 cytokine IL-4 signals through
its cognate receptor on keratinocytes leading to impaired keratinocyte differentiation and epider­
mal barrier dysfunction (Howell et al. 2007; Sehra et al. 2010). This suggests that inflammation is
sufficient to induce keratinocyte and skin barrier dysfunction in AD. Finally, environmental
insults can induce epidermal barrier disruption and resultant production of keratinocyte-derived
cytokines (e.g. TSLP, IL-33), which can in turn promote Th2-polarized inflammation in AD
(Oyoshi et al. 2010). Thus, extrinsic and intrinsic pathways can promote keratinocyte/epidermal
barrier dysfunction and immune dysregulation. Furthermore, there is a bidirectional positive
feedback loop between keratinocyte/epidermal barrier dysfunction and Th2 cytokine-mediated
immune dysregulation in AD pathogenesis. These data suggest a central role for IL-4-mediated
signaling in AD and suggest JAK inhibition could counteract the ability of IL-4 to impair
keratinocyte function in this disease.

5.4.2 T Cell Subsets and Cytokines

Histologic analyses of nonlesional skin from patients with AD reveal sparse perivascular T cell-
rich inflammatory infiltrates, while acute lesional skin reveals denser T cell-rich inflammatory
infiltrates of CD4+ and less abundant CD8+ T cells (Zachary et al. 1985). In acute lesions, Th2
cells and Th2 cytokines such as IL-4, IL-5 and IL-13 are predominant. In chronic lesions, Th2
cells and cytokines persist, but the T cell infiltrate becomes more diverse to include Th1 cells
(producing IFNγ) and other subsets (Hamid et al. 1994, 1996; Leung and Soter 2001). Clinical
investigations using atopy patch testing to mimic AD lesion formation revealed similar patterns of
T helper cell infiltrates with an early influx of Th2 cells and a later accumulation of Th1 cells
(Grewe et al. 1998). Th2 cytokines contribute to AD pathogenesis through multiple mechanisms
including promotion of Th2 cell differentiation, class switching and IgE production by B cells,
74 JAK-STAT Signaling in Diseases

eosinophil recruitment into the skin and decreased EDC gene expression by keratinocytes (Howell
et al. 2007; Kim et al. 2008; Sehra et al. 2010; Carmi-Levy et al. 2011). Th2 cells can also produce
IL-31, which can activate sensory neurons causing itch (Bilsborough et al. 2006; Sonkoly et al.
2006; Takaoka et al. 2006). As IL-4, IL-5, IL-13, and IL-31 receptors activate JAK/STAT
signaling, inhibition of JAK activity at these receptors could theoretically counteract the effects
of these cytokines thereby attenuating Th2 cell differentiation and cytokine production, IgE
production, peripheral eosinophilia, and itch while enhancing keratinocyte differentiation in
subjects with AD.
Other T cell populations are also implicated in AD pathogenesis. Subsets of CD4+ and CD8+
T cells designated Th22 and Tc22 cells, respectively, are implicated in AD pathogenesis. Inter­
leukin (IL)-22 produced by these cells is thought to promote the epidermal hyperplasia seen
in AD and other cutaneous inflammatory diseases like psoriasis (Nograles et al. 2009). An IL-9
producing T helper population (Th9) has also been identified in lesional skin of patients with AD,
though the role of these cells in disease is still under investigation (Schlapbach et al. 2014).
Regulatory T cells may also mediate AD pathogenesis as an AD-like eczematous dermatitis
develops in patients with immunodysregulation polyendocrinopathy enteropathy X-linked
(IPEX) syndrome, a disease characterized by defective Treg development and function (Halabi-
Tawil et al. 2009). However, more work is required to understand if and how skin-derived
regulatory T cells are involved in AD pathogenesis. In conclusion, involvement of T cells in AD
is well established, but much work remains to understand the effector mechanisms by which these
cells interact with one another and keratinocytes to regulate pathogenesis.

5.4.3 Epithelial Cytokines

Epithelial cytokines linked to AD pathogenesis include thymic stromal lymphopoietin (TSLP) and
IL-33 (Schmitz et al. 2005; Saenz et al. 2008; Meephansan et al. 2012; Milovanovic et al. 2012;
Savinko et al. 2012; Kim et al. 2013). Production of TSLP and IL-33 by keratinocytes is increased
in lesional skin of AD (Soumelis et al. 2002; Briot et al. 2009). Epidermal barrier disruption, toll-
like receptor 2 activation by membrane fragments of S. aureus, and inflammatory cytokines can
promote TSLP production by keratinocytes (Angelova-Fischer et al. 2010; Vu et al. 2010; Ziegler
2012). Stimulation of dendritic cells with TSLP also polarizes naïve T cells differentiation towards
Th2 cells and promotes production of the chemokines CCL17 and CCL22, which can act as
chemoattractants for Th2 cells (Soumelis et al. 2002). Moreover, TSLP stimulation of Th2, ILC2,
basophils, and mast cells promotes Th2 cytokine production (Hammad and Lambrecht 2015).
Recently, TSLP was shown to directly stimulate sensory neurons in the skin leading to pruritus
(itch) in mice injected with recombinant TSLP (Wilson et al. 2013; Turner and Zhou 2014). These
findings indicate that TSLP can directly promote allergic inflammation (via actions on leukocytes)
and itch (via actions on neurons). Similar to TSLP, IL-33 can activate Th2 and other cell types to
promote allergic inflammation. While contribution of IL-33 to AD pathogenesis is less clear,
transgenic overexpression of IL-33 in basal layer keratinocytes is sufficient to cause an AD-like
phenotype in mice (Imai et al. 2013). Although IL-33 receptor signaling does not utilize JAK/
STAT pathways, TSLP does; thus TSLP-mediated effects on promoting allergic inflammation and
itch could be therapeutic targets for JAK inhibition.

5.4.4 JAK/STAT Signaling in Preclinical Models of AD

With the exceptions of IL-25 and IL-33, the cytokines described above that are involved in AD
pathogenesis utilize JAK/STAT signaling. As noted earlier, the Th2 cytokine IL-4 is an important
mediator of AD pathogenesis. Activation of the IL-4 receptor promotes STAT6 phosphorylation
and downstream inflammatory cascades. To study the impact of STAT6 function in T cells, a T
cell-specific transgenic mouse expressing a constitutively active mutant human STAT6 protein,
STAT6VT, was generated; subsequent analyses determined these (Stat6VT) mice develop allergic
Role of JAK-STAT Signaling in Atopic Dermatitis 75

skin inflammation referred to hereafter as an AD-like phenotype (Sehra et al. 2010). Scaly
erythematous papules and plaques that can develop on the head, neck, trunk, extremities, and
tail characterize the AD-like phenotype in these mice. Two phases of disease, early and late
lesions, mirror the clinical, histologic, and cytokine profiles of human AD (DaSilva-Arnold et al.
2018). Histologic features of AD-like lesions in these mice include acanthosis and spongiosis and
a moderately dense perivascular and interstitial lymphocyte- and eosinophil-rich infiltrate (Bruns
et al. 2003; Sehra et al. 2008a, 2010; Turner et al. 2014). Interleukin 4 is a key mediator of disease,
as the AD-like phenotype in Stat6VT mice does not develop on an IL-4 deficient background
(Sehra et al. 2008a). Notably, Stat6VT mice develop an AD-like phenotype despite having
‘normal’ keratinocytes, providing a model to study the function of ‘normal’ keratinocytes in the
context of inflammatory cascades initiated by Th2 cells. The fact that these mice develop
an AD-like skin phenotype in the absence of any other genetic lesions or environmental interven­
tions demonstrates the importance of JAK/STAT signaling in this mouse model of AD. In addition
to STAT6 signaling, endogenous STAT3 activity was also required for this disease phenotype
(Stritesky et al. 2011).
Mouse models of AD have also been used to study the effects of JAK inhibitors on allergic skin
inflammation. For example, Amano and co-workers studied the effects of topical JTE-052 (0.5%
[wt/vol] in acetone with 1% [vol/vol] DMSO) on the NC/Nga mouse model of AD. Compared to
vehicle-treated controls, JTE-052-treated NC/Nga mice exhibited less severe clinical scores and
reduced transepidermal water loss as well as increased filaggrin levels and natural moisturizing
factor (NMF) concentrations in the epidermis. Conversely, phosphorylation of STAT3 and
STAT6 was reduced in the epidermis of JTE-052-treated NC/Nga mice. This study also demon­
strated topical JTE-052 increased filaggrin levels and NMF concentrations in healthy human skin
xenografted onto athymic nude mice. In addition, JTE-052 treatment of human skin equivalents
and human keratinocytes promoted keratinocyte differentiation even in the presence of IL-4 and
IL-13 (Amano et al. 2015). Tanimoto and co-workers also studied JTE-052 in a mouse model
of AD that is induced by intradermal injection of ear skin with TSLP; systemic administration of
JET-052 also reduced AD severity (i.e. ear thickness) in a dose-dependent manner in this model
(Tanimoto et al. 2018).
In summary, there is a significant body of evidence that JAK/STAT signaling contributes
to AD-like skin inflammation. Studies with the Stat6VT mouse model demonstrate the
sufficiency of constitutive STAT6 activity to cause AD-like inflammation. Conversely, inhibi­
tion of Janus kinase activity with JTE-052 in mouse models of AD reduced phosphorylation
of STAT6 and STAT3, reduced disease severity, and improved several markers of skin barrier
function. Interestingly, JAK inhibition even enhanced skin barrier function in healthy skin.
This latter observation may reflect an ability of JTE-052 to inhibit the basal IL-4 stimulation
of keratinocytes that occurs in mouse epidermis under homeostatic conditions (Sehra et al.
2010). These preclinical studies provide mechanistic insights into how JAK inhibition could
impact AD pathogenesis.

5.5 Clinical Experience with JAK Inhibitors for AD

As discussed above, multiple cytokine signaling pathways and cell types implicated in AD
pathogenesis utilize JAK/STAT signaling. This coupled with studies of JAK/STAT signaling in
preclinical models of AD provide a strong rationale for the potential utility of JAK inhibition
for the treatment of AD. Currently, no JAK inhibitors are FDA-approved for treatment
of AD in humans. In contrast, the JAK inhibitor, oclacitinib, was FDA-approved in 2013 for
treatment of canine AD. The first half of this section will discuss data on use of JAK
inhibitors for treatment of AD in humans. The second half will discuss canine AD and use
of oclacitinib for treatment of this condition. Table 5.1 summarizes some of the data presented
in the text below.
76 JAK-STAT Signaling in Diseases

TABLE 5.1
Summary of JAK Inhibitors Under Investigation for Use in Atopic Dermatitis
Target
Drug Route Dosing population Study type Efficacy

Tofacitinib Oral 5 mg QD-BID Human Case SCORAD ↓66%; Itch ↓ 76.3%;

series Sleeplessness ↓ 100% (at 8–29 weeks)
Topical 2% oint Human IIa EASI ↓ 81.7% vs. 29.9% for placebo (at 4 weeks)
Baricitinib Oral 2–4 mg QD Human II EASI ↓ 64-65% vs. 46% for placebo (at 16 weeks)
SCORAD ↓ 41-47% vs. 21% for placebo
JTE-052 Topical 0.25, 0.5, 1, Human II Modified EASI ↓ 41.7–72.9% vs. 12.2% for
3% ointment BID placebo
and 62% for tacrolimus ointment (at 4 weeks)
Ruxolitinib Topical 1.5% ointment Human IIb Not currently available in peer-reviewed
BID publication
Upadacitinib Oral 7.5, 15, 30mg QD Human IIb Not currently available in peer-reviewed
publication
Oclacitinib Oral 0.4–0.6 mg/kg Dog RCT CADESI-02 ↓ 48.4 vs. 3.6% for placebo (at
BID →QD 4 weeks)
Itch ↓ 47.4 vs. 10.4% for placebo
Dog Open Quality of life ↑ 91%
label

5.5.1 JAK Inhibition for Treatment of AD in Humans

There are currently no JAK inhibitors approved for any dermatologic indications in humans (Shreberk-
Hassidim et al. 2017). Oral and/or topical formulations of tofacitinib, baricitinib, JTE-052,
ruxolitinib, and upadacitinib have been and/or are under investigation for treatment of AD. As
data for use of ruxolitinib and upadacitinib in AD have not been published, the section will focus
on studies of tofacitinib, baricitinib, and JTE-052. Prior to discussing the safety and efficacy
data related to these agents, the next paragraph will review the metrics used to evaluate AD in
clinical trials.
There are multiple scoring systems used to evaluate AD in clinical research. The two most
common disease scoring instruments are the eczema area and severity index (EASI) and scoring
atopic dermatitis (SCORAD) system. The EASI score calculation requires estimation of the
surface area affected by AD in a given body region (i.e. head/neck, trunk, upper limbs, lower
limbs) and the severity of several clinical parameters of AD (i.e. redness, induction, excoriation,
and lichenification) in each of those body regions (Hanifin et al. 2001). Improvement in the EASI
score is often written as EASI-x where ‘x’ represents a percent reduction in disease severity from
baseline. For example, EASI-50 means a patient or group of patients (or study subjects) had
a 50% or greater improvement in their EASI score compared to their first EASI score. If 65% of
patients achieved EASI-50, that means 65% of patients had a 50% or greater improvement from
their initial EASI score. Similar to the EASI, the SCORAD calculation includes a determination
of the body surface involved by AD but differs from the EASI in that the SCORAD only
evaluates severity (intensity) of a ‘representative’ area of AD and also factors in the symptoms of
itch and sleeplessness (Kunz et al. 1997). Other clinical scoring systems focus on specific
symptoms like itch and quality of life, but will not be discussed further in this section.
Tofacitinib is the first JAK inhibitor reported for treatment of AD (Levy et al. 2015). Levy et al.
reported off-label use of oral tofacitinib to treat six consecutive patients with recalcitrant moderate-
to-severe AD. This medication was used due to the recalcitrant nature of these cases of AD. Patients
in this study were treated with tofacitinib 5 mg by mouth daily (one patient) and twice daily (five
patients), and their disease was assessed with the SCORAD index. Patients were allowed to continue
Role of JAK-STAT Signaling in Atopic Dermatitis 77

use of topical corticosteroids and topical calcineurin inhibitors as needed; one patient also received
low-dose oral corticosteroids (prednisone 5 mg by mouth twice daily) at the beginning of the study.
The average SCORAD measurement decreased by 54.5% (SCORAD 36.5 to 16.5) at the initial
follow-up visit (4–14 weeks after starting tofacitinib) and by 66% at the second follow-up visit
(8–29 weeks after starting tofacitinib). In addition, the severity of itch (pruritus score) decreased by
69.9% and 76.3% at the first and second follow-up visits, respectively; sleeplessness was reduced by
71.2% and 100% at those same time points. No adverse events were reported in this cohort of six
patients. Limitations of the study included the small sample size and lack of a placebo control arm.
There are no randomized placebo-controlled (RCT) trials of oral tofacitinib for AD.
Topical formulations of tofacitinib have been studied in AD. Bissonnette and co-workers
reported results from a phase IIa RCT of 69 adult AD patients treated twice daily with tofacitinib
2% ointment or vehicle control for 4 weeks (Bissonnette et al. 2016). Patients had mild-to­
moderate AD. Disease severity was evaluated using EASI score. After 4 weeks of treatment,
EASI scores improved by 81.7% in the tofacitinib-treated group versus 29.9% improvement in the
vehicle-treated group. Significant reductions in itch were also reported in the tofacitinib treatment
group. Treatment emergent adverse events occurred in 31.4% of tofacitinib-treated subjects and
55.9% of vehicle-treated subjects. In the tofacitinib treatment group, gastroenteritis occurred in
one subject, a second developed bronchitis, and a third developed a furuncle; two cases of
nasopharyngitis and one case of upper respiratory tract infection developed in each treatment
group. No severe or serious adverse events or deaths occurred in either treatment group.
Baricitinib was studied in a 16-week phase II randomized double-blinded placebo controlled trial of
124 adult subjects from the United States and Japan with moderate-to-severe AD (Guttman-Yassky
et al. 2018). Prior to randomization, all patients were treated for 4 weeks with topical corticosteroid
(triamcinolone 0.1% cream). Subjects were then randomized to a placebo group or one of two
baricitinib treatment groups, but triamcinolone use was permitted throughout the study. The first
baricitinib treatment group received 2 mg by mouth once daily; the second group received 4 mg by
mouth once daily. All three groups were treated with placebo or baricitinib for 16 weeks. Response to
therapy was evaluated with EASI and SCORAD instruments. At week 16, the mean reduction in
EASI scores from baseline was 64% and 65% in groups that were treated with 2 and 4 mg baricitinib,
respectively, and 46% in the placebo-treated group. The mean reduction in SCORAD scores was 41%
and 47% in groups that were treated with 2 and 4 mg baricitinib, respectively, and 21% in the placebo-
treated group. Treatment emergent adverse events occurred in 46% and 71% in 2 and 4 mg baricitinib
groups, respectively, and 49% in the placebo group. During the treatment phase of the study, one
serious event was reported (i.e. a benign polyp of the large intestine was detected in one patient in the
4 mg baricitinib group). Headache and elevated blood creatine phosphokinase were reported in
3–13% of subjects in the baricitinib-treated groups but not in the placebo-treated group. There was no
difference in frequency of infections between the groups that were treated with placebo and baricitinib.
No severe events or deaths occurred in any group.
Topical therapy with the JAK inhibitor JTE-052 was also studied in a Phase II study of 327 Japanese
subjects (16–65 years of age at enrolment) with moderate-to-severe AD (Nakagawa et al. 2018). Study
subjects were randomly assigned to one of the following six study groups: 0.25%, 0.5%, 1% and 3% JTE­
052 ointment, vehicle ointment or tacrolimus 0.1% ointment twice daily for four weeks. Therapeutic
response was evaluated using a modified EASI score (excludes evaluation of disease on head/neck
region). At week 4, the mean reduction in the modified EASI score from baseline for the JTE-052
treatment groups was 41.7% (0.25% ointment), 57.1% (0.5% ointment), 54.9% (1% ointment), and
72.9% (3% ointment). These improvements were all significantly greater than the 12.2% mean reduction
in the modified EASI score seen in the vehicle-treated group. The mean reduction in the tacrolimus
treated group was 62%, similar to that seen with the higher concentrations of JTE-052. Itch was also
evaluated using the pruritus numerical rating scale (NRS). Based on the NRS, daytime and night-time
itch were both significantly improved at 1 week in the groups treated with 0.5%, 1%, and 3% JTE-052
compared to vehicle-treated groups, with improvements in pruritus beginning on day 1. The percentage
of total adverse events in the groups treated with JTE-052 was 16% compared to 19.2% and 43% in the
vehicle- and tacrolimus-treated groups, respectively. All adverse events were mild or moderate severity
78 JAK-STAT Signaling in Diseases

with no serious or severe events, including death, occurring during the study. The most common adverse
event noted in the JTE-052 treatment group was nasopharyngitis (3.4%).

5.5.2 JAK Inhibition for Canine AD

While no JAK inhibitors have been FDA approved for treatment of AD in humans, oclacitinib,
marketed as Apoquel, was FDA approved in 2013 for treatment of AD in dogs (canine AD).
Canine AD is a common dermatologic disease; one study demonstrated 8.7% of dogs were
reported to have AD (Lund et al. 1999). Canine AD shares similarities to human AD with
respect to some clinical, histologic, and microbiologic features and treatment modalities used
(Marsella and Girolomoni 2009; Cosgrove et al. 2013; Gedon and Mueller 2018). Environmental
and food allergens also appear to have a more prominent role in contributing to skin inflamma­
tion in canine AD (Marsella and Girolomoni 2009). Although the pathogenic mechanisms of
canine AD differ in some respects to human disease, Th2 cytokines are important drivers of
allergic skin inflammation in both human and canine AD (Gedon and Mueller 2018).
Cosgrove et al. performed a randomized double-blinded placebo-controlled trial of oral oclacitinib
for canine AD (Cosgrove et al. 2013). The study used 299 dogs (12 months of age or older) with AD
and was divided into placebo, 0.4 mg/kg and 0.6 mg/kg oclacitinib treatment arms. Dogs first
received placebo or oclacitinib by mouth twice daily for 14 days, followed by once daily dosing
thereafter for 98 additional days (total treatment duration 112 days). Response to therapy was
determined periodically using the canine AD extent and severity index (CADESI-02) determined by
the clinician and the dog owner’s assessment of their dog’s itching (owner pruritus visual analogue
scale). Disease severity and itch were both significantly reduced in oclacitinib-treated dogs compared
to placebo-treated controls. For example, a 48.4% reduction in CADESI-02 score was noted on days
14 and 28 in oclacitinib-treated dogs compared to 1.7% and 3.6% reductions in placebo-treated dogs
on days 14 and 28. With respect to itching, pruritus scores on days 1, 2, 7, 14 and 28 improved by
29.5–66.7% in oclacitinib-treated dogs compared to 3.9–10.4% improvement in the placebo group.
Data analysis after day 28 was limited by the fact that most (>86%) placebo-treated dogs experienced
worsening itch and/or AD and were therefore transitioned to an open-label arm in which they
received oclacitinib. Cosgrove et al. also performed a long-term study of oclacitinib therapy in
canine AD in which dogs were followed for up to 683 days of treatment with oclacitinib; in this
study, dog owners reported 91% of dogs exhibited an improved quality of life (Cosgrove et al. 2015).
In the 2013 study by Cosgrove et al., the majority of placebo-treated dogs were switched to oclacitinib
by day 16 due to worsening AD. This switch limited the ability of the investigators to compare adverse
events between oclacitinib and placebo-treated groups beyond day 16. Adverse events between days 0
and 16 in the placebo and oclacitinib groups included diarrhea, vomiting, anorexia and lethargy; these
events affected less than 5% of dogs with no clear difference between study groups and resolved in the
majority of dogs despite continuation of treatment. Of the 299 dogs used in this study, 283 received at
least one dose of oclacitinib. Adverse events affecting at least 5% of oclacitinib-treated dogs included
pyoderma (12%), nonspecified dermal nodules (12%), otitis, (9.9%), vomiting (9.2%) and diarrhea (6%).
Leukopenia due to neutropenia occurred in some dogs but the percentage of dogs affected was not
reported. Although some variance in hematologic parameters was seen, cell counts remained in the
normal range, as did serum chemistry values and urinalysis parameters. In a long-term investigation of
oclacitinib therapy for canine AD, Cosgrove et al. studied dogs for up to 683 days of treatment with
oclacitinib (Cosgrove et al. 2015). Adverse events and laboratory values in oclacitinib-treated dogs were
similar to those reported in the author’s earlier short-term study.

5.6 Conclusions
After decades of studying atopic dermatitis, some key aspects of pathogenesis are clear. Skin
inflammation, skin barrier disruption, and itch are key manifestations and targets for treatment
Role of JAK-STAT Signaling in Atopic Dermatitis 79

of AD. Cellular mediators of these manifestations include epithelial cells (keratinocytes), Th2 and
other immune cells and neurons. Each of these cell types utilize JAK/STAT signaling to respond
to one or more cytokines implicated in AD. In light of this, there is a strong rationale for the
realized and potential utility of JAK inhibitors for treatment of this disease. In canine AD,
systemic therapy with the FDA-approved JAK inhibitor, oclacitinib, has helped revolutionize
treatment of AD in dogs. As clinical studies progress, it will be interesting to see how JAK
inhibitors impact treatment of AD in humans.

ACKNOWLEDGMENTS
MJT was supported by awards from the Dermatology Foundation (Physician Scientist CDA), the
Veterans Administration (VA CDA2; IK2CX001019) and the Showalter Trust as well as Indiana
University School of Medicine. MJT also thanks Dr. Stephen Shideler for his kind support. RMS
was supported by the Indiana Cutaneous Biology Training Program NIH T32 fellowship award
(T32AR062495).

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6
JAK-STAT Signaling Pathway and Gliosis in
Neuroinflammatory Diseases

Han-Chung Lee
Laboratory Centre
Xiamen University Malaysia
Sepang, Malaysia

Kai-Leng Tan
Institute of Biomedical and Pharmaceutical Sciences
Guangdong University of Technology
Guangzhou, China

Pike-See Cheah
Department of Human Anatomy, Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
Seri Kembangan, Malaysia

King-Hwa Ling
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
Seri Kembangan, Malaysia
Department of Genetics
Harvard Medical School
Boston, Massachusetts United States

6.1 Introduction
It is commonly perceived that the brain composes of glia and neuron cells (GNR) in the ratio of
10:1. However, a recent review suggested that the GNR could be only about 1:1 (von Bartheld,
Bahney, and Herculano-Houzel 2016). In the human cerebral cortex, there are about 10–20 billion
neuronal cells, while the number of glial cells could be about 15–30 billion. There are three types
of glial cells in the human cerebral cortex with 45–75% of them are oligodendrocytes, 19–40% are
astrocytes, and 10% or less are microglia (von Bartheld, Bahney, and Herculano-Houzel 2016).
Each type of glial cell has a different role; for example, oligodendrocytes are the myelinating
cells that function to produce the insulating sheath of axons. On the other hand, microglia act as
immune cells in the central nervous system. In contrast, astrocyte has broader functions, which
include the maintenance of water and ion homeostasis, the blood–brain barrier, and modulation
of synaptic activity (Jakel and Dimou 2017; Dossi, Vasile, and Rouach 2018). However, reactive
microglia and astrocytes could have a detrimental effect on neuropathological conditions, for

83
84 JAK-STAT Signaling in Diseases

example, when they are stimulated by factors that are toxic to neurons such as β-amyloid and
α-synuclein, which are the critical molecule in the pathogenesis of Alzheimer’s disease (Hardy and
Higgins 1992) and Parkinson’s disease (Zhang et al. 2005), respectively.
Neuroinflammation is pathological inflammatory responses of the central nervous system
(CNS), which can lead to further damage/insult to the brain and spinal cord. The pathological
conditions in the brain include neurodegenerative diseases such as Alzheimer’s disease, Parkin­
son’s disease, and autoimmune diseases. These conditions induce both astrocytes and microglia,
which are the most significant and smallest neuroglial cells, respectively, to become reactive in
concert. Shreds of evidence now support that the reactive state of astrocytes and microglia are
deleterious and neurotoxic to the brain cells.
The Janus kinase–signal transducers and activators of transcription (JAK-STAT) signaling
pathway is an intracellular signaling pathway that involves the activation of two families of
proteins: The Janus kinases (JAK) and the signal transducer and activator of transcriptions
(STAT). JAK is a class of four cytoplasmic protein tyrosine kinases that includes JAK1, JAK2,
JAK3, and TYK2. The STAT family contains seven transcription factors: STAT1, STAT2,
STAT3, STAT4, STAT5A, STAT5B, and STAT6 (Kisseleva et al. 2002). JAK-STAT signaling
pathway is essential for the initiation of innate immunity, subsequently coordinating the adaptive
immune mechanism and ultimately constraining inflammatory responses (O’Shea and Plenge
2012). Specifically, the JAK-STAT signaling pathway is critical for T-cell proliferation, myeloid
and lymphoid cell differentiation, B cells, and macrophage activation. Under normal condition,
the protein expression level of JAK-STAT signaling pathway candidates such as JAK2, STAT1,
and STAT3 in the brain is low, which probably is due to the low cytokine secretion as the number
of immune cell present is small, and the glial cells are not in a reactive state (Na et al. 2007; Yan
et al. 2018).
Neurons, astrocytes, microglia, and immune cells are the essential sources for the cytokine
production in the brain (Galic, Riazi, and Pittman 2012). In general, cytokines are secreted in
response to infection or injury, and whether they are pro-inflammatory or anti-inflammatory
cytokines, the outcome will be different. Pro-inflammatory cytokines have been shown elevated
in many neurodegenerative diseases, which have a detrimental effect—such as damage to CNS
tissue—and are toxic to neurons and glial cells (Wang et al. 2015). Various cytokines have been
reported to activate the JAK-STAT signaling pathway, and different cytokines have the
propensity to activate a specific JAK and STAT. These include the interferons family such as
IFN-α, IFN-β, IFN-γ, IL-10, IL-19, IL-20, and IL-22; the gp130 family including IL-6, IL-11,
oncostatin M, leukaemia inhibitory factor, cardiotrophin-1, granulocyte colony-stimulating
factor, IL-12, leptin, ciliary neurotrophic factor, and cytokines under γ-chain family, such as
IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (Rawlings, Rosler, and Harrison 2004; Schindler, Levy,
and Decker 2007).
Aberrant activation of the JAK-STAT pathway is becoming evident in contributing to neuroin­
flammatory diseases, with the involvement of activated microglia and astrocytes. Importantly, the
downregulation of JAK2–STAT3 signaling pathway was reported to cause memory impairment in
Alzheimer’s disease (Chiba et al. 2009), and potently promote the neurogenic-to-gliogenic shift in
Down syndrome brain (Lee et al. 2016). In this review, we aim to review the role of JAK-STAT
signaling pathway in neuroinflammation such as neurodegenerative disorders (e.g., Alzheimer’s
disease, Parkinson’s disease, Huntington’s disease), autoimmune disease (e.g., multiple sclerosis),
and developmental disorder due to chromosomal abnormalities (e.g., Down syndrome).

6.1.1 JAK-STAT and Reactive Astrogliosis and Microgliosis

Reactive gliosis is a pathological term referred to as the accumulation of astrocytes and microglia
at the injury site in the CNS (Streit, Mrak, and Griffin 2004). Advances in the neurobiology field
have pinpointed that glial cells, particularly astrocytes and microglia, are the essential players in
neuroinflammation (Heneka et al. 2015). Microglia are the resident phagocytes within the CNS
JAK-STAT Pathway and Neuroinflammation 85

(Heneka et al. 2015) and make up about 5–12% of the glial cell population (Gomez-Nicola and
Perry 2015). Generally, microglia exist in two distinct states, which are surveillance/resting state
versus activated state. Microglia in the resting state usually is elongated in shape with long
processes to facilitate its surveillance function. They are highly mobile, actively scouting their
niche by extending and retracting ramified processes while remaining stationary (Hristovska and
Pascual 2016). Upon detection of immunogen or pathogenic debris, microgliosis occurs and
initiates an immune response (Heneka et al. 2015).
When activated, they alter the cell morphology (Streit and Graeber 1993) and transform into
amoeboid shape with extensive cell processes toward the lesion site. It is the first-line defense
that protects the CNS after traumatic injury or infection (Gaudet et al. 2018). However, this
supposed beneficial mechanism when it is prolonged, becomes detrimental to neurons, with
or without the chronic persistence of the injury or immunogen (Cherry, Olschowka, and
Kerry O’Banion 2014). Activated microglia are further categorized into two different states:
M1 and M2 states. In neuroinflammation, the dynamic between M1 and M2 polarization is
crucial to determine the possible beneficial or detrimental outcomes (Cherry, Olschowka, and
Kerry O’Banion 2014). M1 microglia are responsive to harmful stimuli and secrete pro-
inflammatory cytokines such as TNF-α, IL-6, IL-1β, IFN-γ, and IL-23 that are known to
kick-start the inflammation cascades. In contrast, M2 microglia express inflammation inhibitory
cytokines, clear cellular debris through phagocytosis, and are capable of restoring homeostasis
(Hu et al. 2015).
JAK-STAT signaling pathway is the canonical pathway in immune and inflammatory responses.
It plays a vital role in microglial M1/M2 polarization (Hu et al. 2015). Extracellular signals such
as IFN-γ, IL-2, IL-6, and TNF-α trigger M1 polarization in microglia, while IL-4, IL-10, and
Galectin-1 stimulate M2 phenotype polarization. Members of the STAT family, including STAT1,
STAT3, and STAT6, are the intracellular switches in microglia M1/M2 phenotype interchange.
IFN-γ is found to induce M1 polarization via STAT1, while IL-13 and IL-4 stimulation polarize
microglia to M2 phenotype via STAT6 (Wang et al. 2015). Upon IFN-γ binds to its receptor,
JAK1/2 was activated, and STAT1 was phosphorylated, which subsequently trigger the expression
of inflammatory genes such as iNOS and IL-12. In contrast, JAK1/JAK3 or JAK1/Tyk2 was
activated by IL-4 or IL-13, respectively, which leads to phosphorylation of STAT6 and the release
of anti-inflammatory cytokine including IL-10 and IL-1RA (Tugal, Liao, and Jain 2013; Orihuela,
McPherson, and Harry 2016).
These microglial-secreted factors are acting through autocrine and paracrine manner to neurons
and astrocyte. The interplay between these cells can either further damage or repair the injury site.
Furthermore, cytokines (such as IFN-β and TNF-α) released from activated microglia are known
to stimulate astrocytes to secrete more inflammatory mediators including CCL2, CXCL1,
CXCL2, CXCL10, GM-CSF, and IL-6, as a secondary immune response (Pugazhenthi et al.
2013; Yan et al. 2018).
In response to pathological brain conditions, astrocytes exhibit cardinal features by becoming
hypertrophied and expressed a high level of GFAP, vimentin, and S100β (Zamanian et al. 2012).
This reactive state of astrocyte can be detrimental or beneficial to neuronal function, which
depends on the reactive state of the astrocyte, A1 or A2 type. Neuroinflammation-induced
astrocyte into A1 type led to downregulated genes such as Gpc6, Sparcl1, Mertk, and Megf10
that are critical for promoting synapse formation and phagocytic capacity (Liddelow et al. 2017).
A2 reactive astrocyte induced by ischemia upregulated many neurotrophic factors such as
CLCF1, LIF, IL6, and thrombospondins, which promote survival and growth of neurons, and
synapse repair (Zamanian et al. 2012; Liddelow et al. 2017).
Interestingly, activation of the JAK-STAT3 pathway has been reported as the central player in
the induction of reactive astrocytes, suggesting the pathway as a common mediator of astrocyte
reactivity was found highly conserved between disease states, species, and brain regions (Ben
Haim and Ceyzeriat 2015). Mounting evidence suggests that the JAK2–STAT3 pathway serves as
the critical regulator for astrocyte reactivity. See Figure 6.1 for a schematic illustration of
microglia and astrocyte activation in response to pathological brain conditions.
 86

FIGURE 6.1 Microglia and astrocyte activation in response to pathological brain conditions. (A) Neuroinflammation process involving the activation of both resting microglia
and astrocytes into M1/M2 and A1/A2 states, respectively. (B) Multistep cellular activation of a JAK-STAT signaling pathway in microglia, astrocytes, or neurons upon the
activation by pro-inflammatory mediators. The activation of the JAK-STAT signaling pathway leads to the upregulation or downregulation of the transcription of targeted genes.
TSS: transcription start site.
JAK-STAT Signaling in Diseases
JAK-STAT Pathway and Neuroinflammation 87

6.1.2 JAK-STAT Signaling Pathway in Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, which has been associated
with the extracellular deposition of neurotoxic β-amyloid (Aβ) plaques and intracellular neurofi­
brillary tangles (composed of hyperphosphorylated tau protein). There is ample evidence to show
that the deposition of Aβ neuritic plaques activates both the astrocytes and microglia to secrete
pro-inflammatory cytokines that are capable of inducing neuroinflammation (Lyman et al. 2014).
Pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ, TNF-α, and TNF-β have been reported
increased in human AD brain (Su, Bai, and Zhang 2016) and transgenic mouse models of AD
(Benzing et al. 1999; Apelt and Schliebs 2001). These cytokines exert an inflammatory effect
through activation of the JAK-STAT signaling pathway (Pugazhenthi et al. 2013; Rothaug,
Becker-Pauly, and Rose-John 2016), particularly the JAK2–STAT3 pathway. It was reported
that the activation of the JAK2–STAT3 pathway increased astrocyte reactivity, which in turn
increased β-amyloid deposition (Ceyzeriat et al. 2018). The mechanism is still largely unknown.
STAT3, however, has been associated with the β site APP cleaving enzyme 1 (BACE1), which is
responsible for Aβ generation (Wen et al. 2008). Besides, the upregulation of STAT3 promotes
inflammation genes expression such as iNOS and COX-2 and thus promotes neuroinflammation
(Lee et al. 2017).
Cytokines of the interleukins family activate the JAK-STAT signaling pathway. Upon
cytokine binding, the kinase JAK is activated and leads to STAT3 phosphorylation, which
dimerizes, translocated to the nucleus, and, subsequently, regulates the transcription of its
target genes. STAT3 activation in neurons is triggered in response to the increased release of
TNF-α from surrounding activated glial cells (Wan et al. 2010; Carret-Rebillat et al. 2015). It
is noteworthy that the upregulation of Tyk2 increases the phosphorylation of STAT3 and
contributes to neuronal apoptosis (Wan et al. 2010). The JAK-STAT signaling pathway was
found activated in microglial even before the first plaque deposition (Boza-Serrano et al.
2018). When amyloidogenesis continues and elevates Aβ plaque deposition, microglia
and astrocyte can be activated via toll-like receptor (TLR) and receptor for advanced
glycoxidation end products (RAGE)-dependent pathways. Subsequently, the activation of
caspases and signal-dependent transcription factors, such as NF-kB and activator protein 1
(AP-1) in microglia and astrocytes, leading to the release of pro-inflammatory cytokines
(Glass et al. 2010).
Furthermore, a pro-inflammatory cytokine, such as TNF-α, induced phosphorylation of STAT3,
which potently bound to 14-3-3 epsilon gene promoter and increased the expression that could lead
to more microglia activation (Xu et al. 2007; Tanabe et al. 2010; Zhang et al. 2012; Eufemi et al.
2015). Activated microglia also secrete several factors, IL-1α, TNFα, and complement component 1,
q subcomponent (C1q), which can induce more astrocytes into the reactive state (Figure 6.2)
(Liddelow et al. 2017). Indeed, activation of STAT3 promotes more microglia and astrocyte into
a reactive state. Reactive astrocytes are observed close to amyloid depositions in both patients
with AD (Probst, Ulrich and Heitz 1982) and mouse models of AD (Itagaki et al. 1989). Similar to
microglia, upon activated, reactive astrocytes reorganized arborization with increased number and
length of GFAP-positive processes (Wilhelmsson et al. 2006), polarize toward the site of amyloid
plaques aggregation (Nagele et al. 2003). It is unknown whether inhibition of STAT3 reduces β­
amyloid (Aβ) plaques aggregates. However, a study showed that suppression of STAT3 reduced the
expression of Aβ-induced gene transcription, such as iNOS, which markedly attenuates neuronal
cell death (Wan et al. 2010).

6.1.3 JAK-STAT Signaling Pathway in Parkinson’s Disease

Parkinson’s disease (PD) is an age-related and ranked as second most frequent chronic neurode­
generative disease worldwide (Darnell, Kerr, and Stark 1994; Troncoso-Escudero et al. 2018),
typically presented with decreased motor activity. Loss of dopaminergic neurons (DA) in the
substantia nigra pars compacta (SNc) and their projecting fibers in the striatum are observable
88 JAK-STAT Signaling in Diseases

FIGURE 6.2 Neuroinflammation in Alzheimer’s Disease.

Aβ neuritic plaques activate both the astrocytes and microglia to secrete pro-inflammatory cytokines such as IL-1β, IL-6,

IFN-γ, TNF-α, and TNF-β. The pro-inflammatory cytokines activate the JAK-STAT3 pathway, which, in turn, increases

the expression of 14-3-3 epsilon protein that leads to more microglia activation. Activated microglia secrete several

factors, IL-1α, TNFα, and complement component 1, q subcomponent (C1q), which induces more astrocytes into

a reactive state.

at the cellular level (Olanow and Tatton 1999). Reactive astrogliosis is observed upon neuroin­
flammation on various PD animal models, for example, familial PD associated-Nurr1 mutant
mice (Saijo et al. 2009), human alpha-synuclein (α-syn) stimulated mouse astrocytes (Fellner et al.
2013), and PD-related A53T mutant α-syn mice (Darnell, Kerr, and Stark 1994; Xing-Long et al.
2010). Overexpression of astrocytic A53T mutant α-syn caused reactive astrogliosis and microglial
activation accompanied by significant degeneration of DA and motor neurons in mice (Xing-Long
et al. 2010; Laurence et al. 2012). This neuroinflammation has long been considered a downstream
response to the death of dopaminergic neurons. However, mounting evidence suggests that astro­
cytes have an initiating role in PD pathophysiology.
The pathological hallmark of PD is the cytoplasmic inclusions of Lewy bodies that resulted from
misfolded and aggregated protein α-syn in presynaptic cells (Schulz-Schaeffer 2010). They are
proven to be toxic to DA (Petrucelli et al. 2002; Maries et al. 2003). Upon exposure to endogenous
stimuli, such as α-syn, resting microglia are activated and acquired the neurotoxic pro-inflammatory
M1 phenotype, which is deleterious to the DA neurons (Subramaniam and Federoff 2017). In PD
model, both wild type and mutant α-syn are found to potentiate microglia-driven neuroinflamma­
tion and further induce chronic progressive dopaminergic neurodegeneration (Gao et al. 2011,
Hoenen et al. 2016). JAK-STAT activation that promotes inflammatory M1 phenotype is proven
with the overexpression of STATs (STAT1, STAT2, STAT3) in α-syn stimulated microglia (Hoenen
et al. 2016, Qin et al. 2016) and α-syn overexpressed PD rat model (Qin et al. 2016). The pro-
inflammatory cascade activated in microglia and chronic PD progression is inhibited by the
inhibitors of inducible nitric oxide synthase (iNOS) and NADPH oxidase (Gao et al. 2011), which
JAK-STAT Pathway and Neuroinflammation 89

FIGURE 6.3 Summary of JAK-STAT expression in microglia M1/M2 phenotype polarization and released factors at

the presence of alpha-synuclein (α-syn) in PD models.

The presence of α-syn induced M1 phenotype in microglia via overexpression of STATs (STAT1, STAT2, and STAT3)

and release pro-inflammatory cytokines (IFN-γ, IL-6, IL-1β, TNF-α, and NO). Exposure of IL-4 induced M2

phenotype in microglia via the upregulation of STAT6 expression and facilitate phagocytosis clearance on α-syn.

are the two major free-radical synthesis enzymes in microglia. M2 microglia phenotype polarisation
is modulated by STAT6 activation via IL-4 stimulation (Park et al. 2016). This M2 polarised
microglia showed enhanced extracellular α-syn phagocytotic clearance that facilitate neuronal
survival (Park et al. 2016) (Figure 6.3).
Ample evidence indicates that the JAK-STAT pathway in neuroinflammation has a direct impact
on PD pathogenesis. Microglia release pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α,
IFN-γ, and NO (Kawanokuchi et al. 2006; Nakagawa and Chiba 2015). IFN-γ and IL-6, two of the
most potent activators of the JAK-STAT pathway, are elevated in PD (Zhao et al. 2005; Chen et al.
2008; Gough et al. 2008; Yoda et al. 2010; Sherer 2011) and involved in the activation of the M1
phenotype. IFN-γ contributes to the degeneration of DA neurons through a mechanism involving
microglia. IFN-γ-deficient mice are protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) neurotoxicity with reduced loss of DA, striatal tyrosine hydroxylase, and dopamine
transporter fiber density (Zhao et al. 2005; Mount et al. 2007). In the presence of microglia,
exogenous IFN-γ ligand alone is enough to cause DA cell loss under neurotoxin treatment.
Whereas, IFN-γ receptor deficient-neurons are unable to resist cell death induced by a neurotoxin
(Zhao et al. 2005; Mount et al. 2007). These observations indicate the direct participation of IFN-γ­
induced microglial JAK-STAT on modulating DA loss in MPTP-induced PD models, despite the
existence of IFN-γ receptor on DA neurons. The consequence of the pro-inflammatory M1
microglia activation could be the key incident in neuroinflammation that leads to signature DA
loss observed in PD.
Downstream signaling pathways of M1 microglia could serve as important targets to inhibit the
pro-inflammatory damage. Human α-syn upregulates the expression of classical STAT-inducible
genes (iNOS, IL-6, TNF-α, MHC Class II, CIITA, and IRF-1) in primary mouse microglia (Qin
et al. 2016). AZD1480, a JAK1/JAK2 inhibitor, has been proven effective in inhibiting the JAK­
STAT signaling pathway and reducing STAT1 and STAT3 activation in α-syn-induced primary
mouse microglia (Nateghi Rostami et al. 2012; Qin et al. 2016). AZD1480 strongly inhibits the
subsequent overexpression of MHC Class II protein. In the same study, flow cytometry analysis
showed that AZD1480 suppressed microglial activation in the SNc of α-syn overexpressed PD rat
90 JAK-STAT Signaling in Diseases

model, compared to the baseline level in naïve rats. Most importantly, the stereological analysis of
tyrosine hydroxylase stained DA showed that AZD1480 attenuated the ~50% DA degeneration in
the SNc of AAV2-α-syn-transduced rat model after 3 months (Qin et al. 2016). RNAseq analysis
from the same sample also highlighted IFN-inducible genes, particularly IFNAR, STAT1, IFNB1,
IRF7, and TRIM24 that are regulated by the JAK-STAT signaling pathway in α-syn transduced
group. This proposes that repressing microglia activation via the JAK-STAT signaling pathway
might be beneficial to PD. It has been shown that in vivo LPS-induced microglia activation led to
PD-like symptoms, including the aggregation of α-syn and it was IL-1 dependent (Sakai et al.
1995; Tanaka et al. 2013). Collectively, the JAK-STAT signaling pathway in neuroinflammation is
a possible target to halt PD progression.

6.1.4 JAK-STAT Signaling Pathway in Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disorder. It is characterized by progressive
motor dysfunction, psychiatric disorders, and cognitive deficits (Ross and Tabrizi 2011). The
disease is caused by a mutation in the huntingtin (HTT) gene (Träger et al. 2013; Crotti and Glass
2015). The CAG repeat expansion in the HTT gene encodes an expanded polyglutamine tract in
the huntingtin protein (HTT), resulting in a mutant protein. Mutant HTT protein with 40 or
more CAG repeats causes HD with full penetrance (MacDonald et al. 1993). While HD brain
quintessentially shows selective loss of neurons in the striatum and cortex, neuroinflammation has
been one of the leading clues to understand the pathogenesis of HD neurodegeneration. Notably,
the expression of mutant HTT increased microglial activation (Kraft et al. 2012). Immunostaining
of microglial marker, ionized calcium-binding adaptor molecule 1 (IBA1) in HD transgenic mice
striatum showed an increase of reactive microglia compared to wild-type mice (Simmons et al.
2007). Furthermore, mutant HTT expressing microglia promotes not only cell-autonomous
pro-inflammatory gene expression such as IL-6, but also triggers neighboring cells to secrete
pro-inflammatory cytokine (Crotti et al. 2014). The upregulation of several pro-inflammatory
cytokines, such as IL-6 and IL-8, was observed in the cerebrospinal fluid of HD patients (Bjorkqvist
et al. 2008). In addition, IL-6 upregulation was also reported in the cortex, cerebellum, and striatum
of HD patients (Silvestroni et al. 2009).
Increasing pro-inflammatory cytokine IL-6 potentially activates JAK-STAT3 signaling, which
in turn promotes astrocytic activation (Ben Haim and Ceyzeriat 2015; Luo and Zheng 2016).
The reactive astrocyte was observed in the striatum and cortex of patients with HD and HD­
transgenic mouse models. Besides, the increase of mutant HTT expression also contributes to
astrogliosis. A study showed that astrocytes were more hypertrophic and had increased STAT3
expression when the mutant HTT was delivered into the brain cells derived from mice and
monkeys (Ben Haim and Ceyzeriat 2015). Bioinformatics analysis identified STAT1 and STAT2
as potentially involved in the progression of HD. Protein–protein interaction network analysis
based on the differentially expressed genes (DEG) in the mutant huntingtin mouse cell line,
STHdh111/111, has identified 18 hub genes, including STAT1 and STAT2, due to the high
connectivity to the network; however, the study did not further investigate the role of these
genes in HD (Dong and Cong 2018). Besides, the analysis also found a novel regulatory
pathway (miR-124-STAT3-RNASE4) that could be a possible target for HD treatment (Dong
and Cong 2018). RNASE4 has been reported to have a neuroprotective effect, and it was
significantly upregulated in HD. MiRNA and transcription factor prediction showed that
RNASE4 is regulated by miR-124-STAT3 complex (Li et al. 2013; Dong and Cong 2018).
Taken together, the data showed that JAK-STAT signaling pathway is associated with the
pathogenesis of HD; however, to what extent that the pathway involves in HD neuroinflamma­
tion remains unknown.
Immunostaining analysis on post-mortem brain tissue from patients with HD showed that
many astrocytes in the brain were complement component 3 (C3)-positive, which is the specific
marker for A1 astrocytes (Liddelow et al. 2017). A1-reactive astrocytes lost the normal function
JAK-STAT Pathway and Neuroinflammation 91

and secreted many cytokines and chemokines, such as IL-6, chemokine CXCL-1, and IL-8 (Liu
et al. 2018). In consequence, this may attract circulating leukocytes to pass through the blood–
brain barrier and contribute to the chronic inflammatory progression in the brain (Liebner et al.
2018). On the other hand, the elevation of pSTAT5 level in HD monocytes has been reported
previously, which enhances the DNA-binding activity of NFκB leading to IL-6 production
(Kawashima et al. 2001; Träger et al. 2013). STAT5 does not directly activate the IL-6; it binds
to NFκB promoter and transactivates the promoter of IL-6, which has the NFκB-binding site
(Kawashima et al. 2001). Notably, the increased level of plasma IL-6 has been detected in HD
patients (Chang et al. 2015). Moreover, gene set enrichment analysis (GSEA) also found
differential expression of the JAK-STAT signaling pathway in HD monocyte (Miller et al. 2016).
Taken together, it is postulated that the elevation of circulating pro-inflammatory cytokine
released by HD monocyte infiltration into the brain and leading to chronic inflammation. In
brief, the existence of mutant HTT in the glial cells potentially induce neuroinflammation via
JAK-STAT signaling pathway in HD brains, both locally and systemically.

6.1.5 JAK-STAT Signaling Pathway in Multiple Sclerosis and Experimental

Autoimmune Encephalomyelitis
Multiple sclerosis (MS) is a demyelinating disease of the CNS with chronic neuroinflammation
and neurodegeneration (Constantinescu et al. 2011). MS exhibits four pathological features,
which are inflammation, demyelination, axonal loss, and gliosis. The pathological hallmark of
MS is demyelination, which is the destruction of the myelin sheath or the oligodendrocyte cell
body by the inflammatory process (Dendrou, Fugger, and Friese 2015). The etiology of MS
remains unknown, but it is widely accepted that neuroinflammation, specifically auto-reactive
lymphocytes, CD4+ T cells target CNS components and initiate the disease (Goverman 2009).
T helper type 1 (Th1) and T helper type 17 (Th17) lymphocytes produce IFN-γ and IL-17,
respectively (Constantinescu et al. 2011). In MS patients, T cells are peripherally primed by the
antigens, which possess molecular similarity with some CNS antigen (Sospedra and Martin 2005).
Later they differentiate into Th1 and Th17 cells that are capable of crossing the blood–brain
barrier (BBB) and causing a series of myelin-targeted autoimmune neuroinflammation. Other
immune cell types CD8+ T cells, B cells, and myeloid cells are also involved in promoting lesion
formation and neuronal damage (Yan et al. 2018).
Experimental autoimmune encephalomyelitis (EAE) is the animal model of MS. Auto-antigens,
such as myelin basic protein, proteolipid protein, and preferably myelin oligodendrocyte glyco­
protein (MOG), has been used to induce EAE in mice. Infiltrating macrophages and activated
microglia are the common populations found on the lesion site (Yan et al. 2018), and the EAE
model showed autoimmune response and MS-like clinical symptoms (Robinson et al. 2014). The
M1/M2 polarization of microglia and infiltrating macrophage is determined by the environmental
cytokine produced by Th1 cells and Th17 cells in EAE. The summary of how JAK-STAT
interplays between auto-reactive T cells and microglia polarization is shown in Figure 6.4. On
the infiltrating macrophage, IFN-γ secreted by Th1 cells promotes M1 polarization by activating
STAT1 in the JAK-STAT signaling pathway, while GM-CSF produced by Th17 cells promotes
M1 polarization via JAK2–STAT5 activation (Hamilton 2008). M1 macrophage polarization is
a disastrous event for MS and EAE progression. This has been proven by a study that showed
mice with GM-CSF receptor-deficient-CCR2+ monocytes are completely unresponsive from
EAE induction (Croxford et al. 2015). Subsequently, M1 macrophages produce IL-12 that
polarize T cells into Th1 cells via JAK2–TYK2 and STAT4 activation (Natarajan and Bright
2002; Krausgruber et al. 2011). Besides, IL-23 and IL-6 from M1 macrophages facilitate Th17
polarization by activating JAK1/2 and STAT3 in T cells (Samoilova et al. 1998; Cua et al.
2003). Thus, M1 macrophages are the kick starter of the vicious cycle in MS and EAE by
activating JAK-STAT signaling pathway within. From a different perspective, it is also shown
that macrophage and microglia are the most feasible candidates to halt the neuroinflammation
in MS or EAE. Studies showed that external stimuli, such as IL-4 (Francos-Quijorna et al.
92 JAK-STAT Signaling in Diseases

FIGURE 6.4 Summary of JAK-STAT expression in macrophage or microglia M1/M2 phenotype polarization and

released factors in MS and EAE models.

T lymphocytes transformed into autoreactive T helper type I (Th1) and Th17 lymphocytes in MS. Th1 and Th17

release IFN-γ and GM-CSF, respectively. These two factors induced M1 phenotype via STAT1 and JAK2–STAT5

activation. Pro-inflammatory cytokines, such as IL-12, IL6, and IL-23, further activate T cells into Th1 and Th17 cells.

IL-17 is Th17 autocrine factor. IL-4, IL-13, and IL-10 drive M2 phenotype and secrete IL-10 that are capable of

inactivating Th1 and Th17 auto-activation.

2016; Liu et al. 2016), IL-13 (Rutschman et al. 2001; Wynn, Chawla, and Pollard 2013), and
IL-10 (Lang et al. 2002; Gordon 2003), could drive them into M2 polarization. M2 macro­
phages and microglia exhibit strong anti-inflammatory reaction via an elevated level of IL-10
production (Moore et al. 2001; Lobo-Silva et al. 2016) reduced production of IL-23 (Correa
et al. 2011) and IL-6 (Sanchez-Ventura et al. 2019). Furthermore, M2 macrophage-secreted
IL-10 demonstrated a strong suppression on Th1 and Th17 phenotypes via STAT3 activation,
which then alleviate EAE symptoms (Qin et al. 2012; Liu et al. 2013; Li et al. 2018; Lotfi
et al. 2019). These showed that M2 macrophage and microglia are the pivots in relieving
clinical symptoms of MS and EAE. In brief, the JAK-STAT signaling pathway and the
responded cytokines or interleukins are utilized by both auto-reactive T cells and macro­
phages. It can be either deleterious or beneficial to the MS and EAE neuroinflammation, by
tweaking the M1/M2 phenotype polarization and the environmental cytokines via the JAK­
STAT signaling pathway.
Astrocytes are also responsive to environmental cytokines. They respond to IFN-β and secrete
suppressors of cytokine signaling (SOCS) via activation of the JAK-STAT signaling pathway to
negatively regulate immune cell infiltration, such as monocytes and CD4+ T cells, into the CNS (Qin
et al. 2008). IFN-β is a successful MS treatment as it reduces exacerbation rates and delays MS
progression (Weinstock-Guttman et al. 1995; Dhib-Jalbut 1997; Hohlfeld and Wekerle 2004). IFN­
β induced SOCS protein expression via STATs activation in primary murine astrocyte (Qin et al.
2008). The induction of SOCS-1 and SOCS-3 expression are dependent on STAT1α and STAT3,
respectively. In the same study, SOCS-1 and SOCS-3 silenced-astrocytes show greater chemoattrac­
tion to bone marrow-derived primary macrophages and CD4+ T cells under IFN-β stimulation
(Qin et al. 2008). This strengthens the importance of the JAK-STAT signaling pathway in
ameliorating neuroinflammation in MS. The depletion of IFN-γ receptors on astrocytes has
been shown to reduce the severity of EAE, which suggest that by ignoring IFN-γ detected in the
niche may lower the chance to trigger neuroinflammation (Ding et al. 2015). Similar to M1
macrophages, the secretion of IL-6 by astrocytes in response to endoplasmic reticulum stress
JAK-STAT Pathway and Neuroinflammation 93

(Meares et al. 2014) has been reported to be a determining factor of EAE neuroinflammation
(Quintana et al. 2009; Giralt et al. 2013). IL-6 deficient mouse is completely resistant to EAE
induced by MOG (Giralt et al. 2013). In the same study, the mouse with the only astrocyte
produced IL-6 demonstrated demyelination, leukocytes infiltration, and astrogliosis, restricted
within the cerebellar region, upon MOG stimulation. Like Th17 cells, it also produced GM-CSF
during the EAE process (Mayo et al. 2014) that will polarize macrophage into M1 phenotype via
JAK2–STAT5 (Hamilton 2008). Upregulated expression of lactosylceramide (LacCer) has been
reported in the CNS of EAE mice, and it regulates the astrocytes transcriptional program (Mayo
et al. 2014). In the same study, LacCer in astrocytes regulates the chemokine CCL2 and GM-CSF
production in a non-cell autonomous manner, which modulates the recruitment of activated
microglia and CNS-infiltrating monocytes. JAK-STAT in astrocytes is undeniably the central
control system in switching between beneficial and detrimental functions during neuroinflamma­
tion. Thus, the JAK-STAT signaling pathway might be the candidate to manipulate astrocyte to
counteract the progression of EAE and MS.

6.1.6 JAK-STAT Signaling Pathway in Down Syndrome

Very little is known about the neuroinflammation in the Down syndrome (DS) brain. However,
inflammatory receptor genes are found overexpressed in both human and mouse models for DS
(Ferrando-Miguel et al. 2003; Amano et al. 2004; Wilcock and Griffin 2013; Ling et al. 2014).
Notably, pro-inflammatory receptor genes located on chromosome 21, including IFNAR1, IFNAR2,
and IFNGR2 are triplicated in DS samples (Wilcock and Griffin 2013), and they can activate JAK­
STAT signaling pathway when interferons bind to the receptor. Once activated, the JAK-STAT
signaling pathway promotes astrocytes differentiation (Bonni 1997). Besides, the analysis of protein
expression level demonstrated that there was an approximately 2-fold increment of IFNAR2 expres­
sion in human fetal DS brain at 19–21 weeks of gestational age (Ferrando-Miguel et al. 2003). The
increased expression levels of Ifnar1, Ifnar2, and Il10rb genes were also seen in a DS mouse model,
Ts1Cje. These trisomic genes were overexpressed about 1.5-fold in Ts1Cje mouse whole brain (Amano
et al. 2004). In addition to Ifn receptor, the downstream target, STAT1, was also found upregulated in
the P84 Ts1Cje cerebellum and cerebral cortex (Ling et al. 2014). Furthermore, western blot analysis
on fibroblast cell lines from individuals with trisomy 21 confirmed the upregulation of IFN receptor
proteins (Sullivan et al. 2016).
The number of astrocytes is increased in human fetuses with DS. Immunohistochemical staining
on the frontal lobe of DS human fetuses showed increased GFAP positive cells as compared to
the age-matched controls (Zdaniuk et al. 2011). The finding was also consistent with previous
studies on both human fetuses and Ts65Dn mice brain (another mouse model for DS) (Contest­
abile et al. 2007; Guidi et al. 2008). Astrocytes in DS are not only more proliferative and
abundant, but they also display altered processes (Dossi, Vasile, and Rouach 2018).
In addition, pro-inflammatory cytokines, such as IL-6, TNF-α, and TGF-β, are well-known
activators of the JAK-STAT signaling pathway. These cytokines were at least 2-fold higher in
autopsied human DS brain tissues (<40 years old) when compared to age-matched controls. In
contrast, it showed at least a 3-fold increase in Down syndrome with AD (>40 years old)
(Wilcock et al. 2015). Besides, the level of IFN-γ was significantly increased in the whole brain
of the trisomy 16 mouse (Hallam et al. 2000). The presence of the extra copy of IFN receptor and
the elevation of IFN level have been postulated to sensitize the cells to interferon interaction and
lead to activation of the JAK-STAT signaling pathway (Ling et al. 2014; Lee et al. 2016).
Furthermore, it has been indicated that TNF-α and IFN-γ bind to receptors on microglia and
astrocytes (Benveniste and Benos 1995), which can activate both cell type and potently induces
JAK-STAT signaling pathway in microglia and astrocytes. Activation of glial cells includes
triggering of the JAK-STAT signaling pathway that could lead to neuroinflammation via the
release of nitric oxide (NO) (Figure 6.5) (Kim et al. 2002; Lively and Schlichter 2018). A previous
study reported that the gene expression level of inducible nitric oxide synthase (iNOS) that
stimulates NO generation was higher in DS astroglia than in control astroglia (Chen et al. 2014).
94 JAK-STAT Signaling in Diseases

FIGURE 6.5 Neuroinflammation in Down Syndrome.

When TNF-α and IFN-γ bind to receptors on microglia and astrocytes, it is potently to activate JAK-STAT signaling
pathway. Besides, an extra copy of IFN receptors on glial cells sensitizes the cells to interferon interaction, which
further triggers the JAK-STAT signaling pathway. In consequence, the expression level of iNOS increased in and
released NO, which leads to neuroinflammation.

In addition, increased of reactive oxygen species (ROS) production was observed in primary
cultures of hippocampal neurons and astrocytes derived from Ts1Cje, a mouse model for DS
(Shukkur et al. 2006). The expression of STAT1 and STAT3 were then highly induced in response
to ROS (Simon et al. 1998). In consequence, chronic expression of STAT1 and STAT3 stimulate
an inflammatory environment in the brain and lead to neuroinflammation. This could be through
upregulation of downstream transcriptional targets of STATs, such as Jmjd3, Ccl5, Ezr, Ifih1, Irf7,
Uba7, and Pim1, which are the genes that code for inflammatory proteins (Przanowski et al.
2014). JAK inhibitors or JAKinibs are currently a type of pharmacological approach that aids in
inhibiting the activity of one or more enzymes of the JAK family, thereby interfering with the
JAK-STAT signaling pathway. The application of JAKinibs, targeting astroglial and microglial
activation states in Down syndrome, offers excellent therapeutic potential and warrant further
investigation.

6.2 Concluding Remarks

The JAK-STAT signaling pathway in response to a wide range of cytokines is now known as
one of the critical factors to promote neuroinflammation in neurodegenerative disease. However,
it is conditional by whether it is pro-inflammatory cytokines or anti-inflammatory cytokines
that trigger the JAK-STAT signaling pathway. In neurodegenerative disease, M1-reactive
JAK-STAT Pathway and Neuroinflammation 95

microglial or A1-reactive astrocyte secretes pro-inflammatory cytokines that create an environ­

ment conducive to inflammation. Cytokines act on the cells that secrete them, or nearby cells,
and stimulate JAK-STAT signaling pathways. Activated JAK-STAT signaling leads to more
microglia change into the reactive state as well as increase more pro-inflammatory cytokines and
upregulated inflammatory gene expression. JAKinib is the inhibitor to one or more of the JAK
family, thereby suppressing JAK-STAT signaling pathway that could reduce the releasing pro-
inflammatory cytokines. However, inhibition of JAKs may potentially reduce anti-inflammatory
response as the JAK-STAT signaling pathway also triggers by anti-inflammatory cytokines. The
cytokines released in the microenvironment conditions the detrimental or beneficial effect;
therefore, modulation of the balance between pro-inflammatory and anti-inflammatory cyto­
kines, which would affect the function of the JAK-STAT signaling pathway, could be another
way to reduce inflammation.

ACKNOWLEDGEMENTS
This work was supported by Malaysian Ministry of Science, Technology and Innovation Science-
fund (Project ID: 02-01-04-SF2336) awarded to K.-H.L. and National Natural Science Founda­
tion of China (Project ID: 81850410549) awarded to K.-L.T.

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