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Published in final edited form as:

Hepatology. 2021 November ; 74(5): 2684–2698. doi:10.1002/hep.32028.

Mast Cells Regulate Ductular Reaction and Intestinal

Inflammation in Cholestasis Through Farnesoid X Receptor
Signaling
Vik Meadows1,2, Lindsey Kennedy2, Burcin Ekser3, Konstantina Kyritsi2, Debjyoti Kundu2,
Tianhao Zhou2, Lixian Chen2, Linh Pham2,*, Nan Wu2, Jennifer Demieville4, Laura
Hargrove4, Shannon Glaser4, Gianfranco Alpini1,2, Heather Francis1,2
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1Richard L. Roudebush VA Medical Center, Indiana University School of Medicine, Indianapolis,

IN
2Divisionof Gastroenterology and Hepatology, Department of Medicine, Indiana University School
of Medicine, Indianapolis, IN
3Divisionof Transplant Surgery, Department of Surgery, Indiana University School of Medicine,
Indianapolis, IN
4Department of Medical Physiology, Texas A&M University College of Medicine, Bryan, TX.

Abstract
BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA)
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levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary
sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells
(MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In
bile-duct-ligated (BDL) MC-deficient mice (B6. Cg-KitW-sh/HNihrJaeBsmJ [KitW-sh]), ductular
reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection
exacerbates liver damage in normal mice.

APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary
FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased

ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Heather Francis, Ph.D., F.A.A.S.L.D., Indiana University,
Gastroenterology, Medicine, Richard L. Roudebush VA Medical Center, 702 Rotary Circle, Rm. 013C, Indianapolis, IN 46202-2859,
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heafranc@[Link], Tel.: +1-317-274-3514.

*Department of Science and Mathematics, Texas A&M University - Central Texas, Killeen, TX.
Author Contributions: V.M. was responsible for conceptualization, data curation, formal analysis, investigation, methodology,
validation, visualization, and writing (original draft, review, and editing). L.K. was responsible for data curation, formal analysis,
investigation, methodology, validation, and writing (review and editing). B.E. was responsible for resources and writing (editing).
K.K. was responsible for data curation and writing (editing). D.K. was responsible for data curation and writing (editing).
T.Z. was responsible for software and data curation. L.C. was responsible for data curation and writing (editing). L.P. was
responsible for data curation and writing (editing). N.W. was responsible for data curation and editing. J.D. was responsible for
data curation, formal analysis, investigation, methodology, and validation. L.H. was responsible for data curation, formal analysis,
investigation, methodology, and validation. S.G. was responsible for funding acquisition and resources. G.A. was responsible for
funding acquisition, resources, and writing (review and editing). H.F. was responsible for conceptualization, formal analysis, funding
acquisition, project administration, resources, supervision, visualization, and writing (original draft, review, and editing).
Potential conflict of interest: Nothing to report.
Supporting Information
Additional Supporting Information may be found at [Link]/doi/10.1002/hep.32028/suppinfo.
 Meadows et al. Page 2

MC number and biliary FXR expression in patients with liver injury compared with control.
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Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients
PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR,
inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and
histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters.
BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15,
and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-
FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following
FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4
knockout (Mdr2−/−) mice, models of PSC, displayed increased intestinal MC infiltration and
FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in
Mdr2−/− mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic
stellate cell activation.
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CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and
DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.

Cholangiocytes are the target cells of cholestatic liver diseases, such as primary sclerosing
cholangitis (PSC), which is characterized by increased hepatic fibrosis, ductular reaction
(DR)(1,2) and impaired bile acid (BA) secretion.(3,4) A significant subset of patients with
PSC present with inflammatory bowel disease (IBD), which increases their risk and
incidence of colorectal and hepatobiliary cancer.(5) During PSC, cholangiocytes exhibit
proliferative, inflammatory, fibrotic, and senescent phenotypes in response to liver injury,
and several studies have demonstrated the critical contribution of the gut-liver axis during
PSC.(6)

We have demonstrated that mast cells (MCs) infiltrate the liver during cholestatic liver injury
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and trigger biliary damage.(1) Following migration and activation, MCs induce DR(7) and
senescence(1) through paracrine interactions with cholangiocytes. The PSC mouse models,
multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2−/−) and
mice subjected to bile duct ligation (BDL), have elevated MC presence and serum histamine
(HA) levels, increasing biliary expression of H1 and H2 HA receptors (HR); however,
disruption of HA signaling ameliorates cholestatic liver injury.(8–10) Following BDL, MC-
deficient [Link]-KitW-sh/HNihrJaeBsmJ (KitW-sh) mice have reduced DR, inflammation, and
hepatic fibrosis compared with BDL wild type (WT), and introduction of MCs into KitW-sh
mice mimics cholestatic injury,(10) demonstrating the damaging role of MCs during liver
injury. Moreover, MCs induce DR through TGF-β1(11) and VEGF(8) signaling pathways.

BA synthesis and enterohepatic circulation is tightly regulated by farnesoid X receptor

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(FXR) and downstream targets including FGF15/FGF19 (FGF15, mouse; FGF19, human)
and cytochrome p450 (Cyp) family 7 and 27 subfamily a member 1 (Cyp7a1 and Cyp27a1).
(12) FXR signaling regulates the expression of many BA transporters such as Na+-dependent

taurocholate cotransport peptide and apical Na+ BA transporter (ASBT).(13) Furthermore,

FXR plays a protective role in intrahepatic cholestasis by antagonizing NF-κB–mediated
hepatic inflammation(12,14) while increasing liver injury in extrahepatic cholestasis through
dysregulation of BA transporter expression.(15) Fxr−/− mice have decreased intrahepatic bile

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duct mass (IBDM) and biliary proliferation following BDL, likely because of the reduced
bile flow and pressure, compared with WT mice.(16) Impairment of FGF15 signaling inhibits
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liver regeneration and exacerbates hepatosteatosis,(17) lending to its importance in hepatic

function.

Our studies found that stabilization of MCs in Mdr2−/− mice reduces total BA (TBA)
levels,(7) and we, among others, have shown that MCs express FXR, ASBT and FGF
receptors(18,19) and migrate into tissue in response to FGF stimulation.(20) In this study, we
evaluated the role of MC-FXR in regulation of BA circulation, intestinal inflammation and
liver damage in models of cholestasis.

Materials and Methods

MATERIALS
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All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated
otherwise. Z-guggulsterone (FXR antagonist)(21) was purchased from MilliporeSigma
(Burlington, MA). Mouse pan Fibroblast growth factor (FGF) enzyme immunoassay (EIA)
was purchased from R&D Systems (Minneapolis, MN). Mouse FGF15 EIA was purchased
from My BioSource, Inc. (San Diego, CA) and LifeSpan Biosciences, Inc. (Seattle, WA).
FGF19 EIA was purchased from Abcam (Cambrige, MA). HA EIA was purchased from
Cayman Chemicals (Ann Arbor, MI). Application, dilution, and vendor information for
all antibodies used in this study are detailed in Supporting Table S1. RNA was reverse
transcribed and amplified using Reaction Ready First Strand cDNA Synthesis kit and RT2
quantitative PCR (qPCR) Primer Assay obtained from Qiagen (Valencia, CA). All qPCR
primers are listed in Supporting Table S2. TBA colorimetric kits were purchased from Cell
BioLabs, Inc. (San Diego, CA). Positive immunoreactivity was quantified with Image-Pro
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from Media Cybernetics, Inc. (Rockville, MD).

For in vitro studies, we used immortalized murine intrahepatic cholangiocyte cell lines.
(22)Murine hepatic MCs (MC/9, American Type Culture Collection [ATCC] CRL-8306)
were purchased from ATCC (Manassas, VA). Human HSC (#5300) were obtained from
ScienCell (Carlsbad, CA). All cell lines were cultured and maintained by us following
vendor recommendations and as published.(8) Media and reagents were purchased from
Thermo Fisher (Waltham, MA).

IN VIVO MODELS
Commercially available homozygous KitW-sh (MC-deficient) 10 to 12-week-old male mice
were obtained from Jackson Laboratory along with sex and age-matched WT c57BL/6J
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mice. WT and KitW-sh mice both display healthy liver phenotype, and WT mice have
minimal MC presence.(10) To ascertain the role of MC-FXR in MC-induced damage,
KitW-sh received a single injection through tail vein of MCs (MC/9, ATCC CRL-8306,
5 × 106 cells/0.1–0.2 mL sterile 1× PBS) treated with vehicle 0.1% DMSO (MC) or
10 μM Z-guggulsterone (FXR inhibitor, MC-Gugg)(21,23) for 48 hours and tagged with
PKH26 Red Fluorescent Cell Linker before injection. Liver (costained with cytokeratin 19
[CK-19] to mark bile ducts), lung, and spleen were evaluated for MC presence by PKH26

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detection with confocal microscopy (LEICA TCS SP5 X system, Leica Microsystems,
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Inc., Buffalo Grove, IL). To confirm FXR inhibition, MC-FXR, FGF15, and small
heterodimer partner (SHP) expression and FGF secretion were measured by qPCR and
EIA, respectively. Serum, liver, isolated cholangiocytes, small intestine, and distal ileum
were collected from all animal groups (exact animal numbers are provided in the Supporting
Information). Isolated cholangiocytes were collected as described.(7) Immunohistochemistry
was performed on 4 to 6-μm formalin-fixed paraffin-embedded liver or small intestine
sections. Immunofluorescence was performed on 4 to 6-μm optical cutting media-embedded
liver, lung, or spleen.

To verify our findings in established models of cholestatic liver injury, male WT and KitW-sh
mice, 10 to 12-week-old, were subjected to BDL for 7 days before euthanasia along with
appropriate controls as described.(10) Additionally, Mdr2−/− mice, an established genetic
model of cholestatic liver damage, treated with saline or cromolyn sodium (MC stabilizer,
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24 mg/kg BW for 1 week) were used to confirm our findings.(7) Serum, liver, small intestine,
and isolated cholangiocytes and hepatocytes were collected from all mice (exact animal
numbers are provided in the Supporting Information).

All animal colonies were maintained following current Institutional Animal Care and Use
Committee protocols approved by the Indiana University School of Medicine Laboratory
Animal Resource Center, Indianapolis, IN, and Baylor Scott and White Health Animal
Facility, Temple, TX. All mice were given free access to drinking water and standard chow.
Animals were kept in a temperature-controlled environment with 12:12 hr light/dark cycles.

FXR/FGF19 SIGNALING AND MC ACTIVATION IN HUMAN PATIENTS

Human liver sections from healthy controls and patients with cholestatic liver diseases were
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used for immunohistochemistry for FXR costained with tryptase to determine the presence
of activated MCs. Healthy nondiseased liver tissues were purchased from Sekisui XenoTech,
LLC (Kansas City, KS). Healthy nondiseased, PSC and PSC with IBD comorbidity (PSC-
IBD) serum were deidentified and provided by Dr. Burcin Ekser following transplantation.
Liver sections (formalin-fixed, 4 to 5 μm thick) obtained by explant from deidentified
transplant patients from patients with PSC, PSC-IBD, primary biliary cholangitis (PBC),
biliary atresia, and NASH were provided by Dr. Burcin Ekser. Patient demographics are
detailed in Supporting Table S3. All samples were obtained under a protocol approved by
Indiana University Health; the protocol was approved by the Indiana University Institutional
Review Board. Serum FGF19 (Abcam, Cambrige, MA) and HA (Cayman Chemicals, Inc.,
Ann Arbor, MI) levels were measured by EIA following manufacturer protocols, and levels
of SHP were evaluated in total liver by qPCR from healthy nondiseased (control), PSC,
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and PSC-IBD samples. For all patient sample analysis, written informed consent was
obtained from each patient and the study protocol conformed to the ethical guidelines of
the 1975 Declaration of Helsinki as described and approved by the Institutional Review
Board, Indiana University. No donor organs were obtained from executed prisoners or other
institutionalized persons.

Further detailed information for experiments performed in vivo and in vitro are detailed in
the Supporting Methods.

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STATISTICAL ANALYSIS
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Data are presented as mean ± SEM. Groups were analyzed by the Student’s unpaired t test
when two groups were analyzed. Welch one-way ANOVA was used when more than two
groups are analyzed, followed by appropriate post hoc test with GraphPad Prism 9 (San
Diego, CA). P < 0.05 was considered significant.

Results
PATIENTS WITH CHOLESTATIC LIVER DISEASE HAVE INCREASED MC PRESENCE AND
FXR EXPRESSION
FXR agonists are currently being studied as therapeutic options for patients with cholestatic
liver disease(4,12); however, the effect of FXR on hepatic MC presence has not been defined.
In patients, FXR expression (brown; found in hepatocytes and cholangiocytes) increased in
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cholangiocytes from late-stage PSC, PSC-IBD, late-stage PBC, biliary atresia, and NASH
compared with control tissue, and the up-regulation of FXR is accompanied by elevated
expression of MC tryptase (red staining) found near bile ducts (Fig. 1A). Further, serum
levels of FGF19 (Fig. 1B) and mRNA expression of SHP (Fig. 1C) increased in patients
with PSC-IBD compared with controls. These results support the clinical implications
of MC infiltration during human cholestatic liver disease and the up-regulation of FXR
expression and activation of downstream signaling during the progression of chronic liver
injuries, including PSC-IBD.

TBA CONTENT AND HEPATIC FXR/FGF SIGNALING ARE DECREASED IN CHOLESTATIC

MODELS LACKING MCS OR HA SIGNALING
To establish a rationale for our study, we examined changes in TBA content and FXR/FGF
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signaling in established cholestatic models, including BDL and Mdr2−/− mice. With regard
to TBA content, we found that BDL WT mice had elevated serum TBA content compared
with WT mice; however, BDL KitW-sh mice had reduced serum TBA content compared
with BDL WT mice (Fig. 2A). BDL WT mice have significantly increased hepatic FXR
mRNA expression compared with WT mice (Fig. 2B), whereas BDL KitW-sh mice have
lower hepatic FXR expression compared with both WT and BDL WT mice, indicating
that MCs are involved in the regulation of the hepatic FXR expression. Furthermore, we
found that panFGF serum secretion was minimal in WT and KitW-sh mice (Fig. 2C);
however, panFGF serum levels increased in BDL WT mice compared with WT mice,
whereas BDL KitW-sh mice displayed decreased panFGF serum secretion compared with
BDL WT (Fig. 2C). Finally, we verified increased FGF15 expression and immunoreactivity
by immunofluorescence in bile ducts of BDL WT mice compared with WT mice, which
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was reduced in BDL KitW-sh mice (Fig. 2D). In Mdr2−/− mice, we found increased FXR
expression (Supporting Fig. S1A), biliary FGF15 immunoreactivity (Supporting Fig. S1B),
and panFGF serum content (Supporting Fig. S1C) that were reduced in Mdr2−/− mice treated
with cromolyn sodium.

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MC INFILTRATION ALTERS INTESTINAL FXR/FGF15 IN CHOLESTATIC MICE

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We found that BDL WT and Mdr2−/− mice have increased intestinal MC infiltration
compared with their WT controls shown by tryptase β−2 (Fig. 2E and Supporting Fig.
S1E). Along with increased MC presence, we found elevated expression of FXR and FGF15
in distal ileum from BDL WT mice compared with their WT controls (Fig. 2F,G). When
Mdr2−/− mice were treated with cromolyn sodium to reduce MC activation, intestinal MC
infiltration, FXR, and FGF15 expression were all reduced (Supporting Fig. S1D–F). In
distal ileum from BDL WT, we also found increased mRNA expression of FGF15 and
SHP, whereas ASBT decreased compared with WT mice (Supporting Fig. S2A), and, in
small intestine, stem cell factor (SCF), IL-1β, TGF-β1, and chemokine (C-C motif) ligand
(CCL) 3 mRNA expression are increased following BDL compared with WT mice. These
parameters were decreased in BDL KitW-sh mice lacking MCs (Supporting Fig. S2B)
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VALIDATION OF MC-FXR/FGF EXPRESSION AND MC INJECTION/MIGRATION AND

DAMAGE OF MODEL
Cultured murine MCs were treated with vehicle (MC) or Z-guggulsterone (MC-Gugg),
tagged with PKH26, and injected into KitW-sh mice through tail vein. MC-Gugg have
decreased FXR FGF15 and SHP (P < 0.07) mRNA expression (Fig. 3A–C) and panFGF
secretion (Fig. 3D) compared with vehicle-treated MCs. Similar to our previous findings,
(10) we found that MCs (marked with PKH26, red) migrate to the liver and reside in

close proximity to bile ducts (stained with CK-19, green) (Fig. 3E,F). Concurrent with our
previous work, we found minimal MC presence in lung and spleen from our MC-Gugg–
injected mice (data not shown).(10) By hematoxylin and eosin staining, we verified that MC
injection induces periportal inflammation and lobular damage that is ameliorated in KitW-sh
mice treated with MC-Gugg (Fig. 3G).
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MCS ALTER ENTEROHEPATIC FXR/FGF AXIS AND INTESTINAL INFLAMMATION

In KitW-sh mice injected with MCs, compared with WT, there was increased (1) liver and
serum TBA content (Fig. 4A,B) and (2) hepatic and biliary FXR expression (Fig. 4C,D)
that was reduced in KitW-sh mice injected with MC-Gugg. Downstream of FXR, hepatocyte
Cyp7a1 expression also increased in KitW-sh mice injected with MCs; however, hepatocyte
Cyp27a1 was reduced (Fig. 4E) compared with WT, and when KitW-sh mice were treated
with MC-FXR, Cyp7a1 was reduced, whereas Cyp27a1 remained unchanged (Fig. 4E).
Immunoreactivity of FGF15 (Fig. 4G) and liver and serum FGF15 secretion (Fig. 4H,I) all
increased in KitW-sh mice injected with MCs compared with WT and KitW-sh mice. All
of these parameters were decreased when KitW-sh mice were injected with MC-Gugg (Fig.
4G–I). There were minimal changes seen between WT and KitW-sh mice for TBA levels and
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FXR and FGF15 expression.

Interestingly, in accordance with our hepatic findings, distal ileum FXR and FGF15
expressions are minimal in WT and elevated in KitW-sh mice (Fig. 5A,B). MC injection
into KitW-sh mice increased intestinal FXR and FGF15 expression (Fig. 5A,B), which
was reduced when KitW-sh mice were injected with MCs lacking FXR (Fig. 5A,B),
demonstrating an impact of MC-FXR on hepatic and intestinal FXR/FGF15 signaling in
MC-deficient mice. We found that small intestine mRNA levels of SCF, IL-1β, TGF-β1, and

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CCL3 were elevated following MC injection in KitW-sh compared with WT and KitW-sh mice
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(Fig. 5C). These inflammatory markers decreased when FXR was inhibited in MCs before
injection (Fig. 5C). These findings indicate an important role for MCs in both FXR/FGF15
enterohepatic circulation and intestinal inflammation.

INHIBITION OF MC-FXR PREVENTS DR, INFLAMMATION, BILIARY SENESCENCE,

SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE, AND HEPATIC FIBROSIS
Introduction of MCs into KitW-sh mice increased IBDM (Fig. 6A) and F4/80-positive
Kupffer cells (Fig. 6B), which also confirmed by semiquantification (Fig. 6C,D), compared
with WT and KitW-sh mice. MC injection also enhanced hepatic inflammatory marker
expression of IL-1β, CCL5, chemokine (C-X-C motif) ligand (CXCL) 2, and CXCL5
(Fig. 6E). Inhibition of MC-FXR reduces IBDM and hepatic inflammation compared with
KitW-sh + MC mice (Fig. 6A–E). KitW-sh mice injected with MCs had increased biliary
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proliferation shown by Ki-67 (Supporting Fig. S3A) and biliary senescence demonstrated
by immunofluorescence for p16 in liver sections and mRNA expression of p18 and
p21 in isolated cholangiocytes (Supporting Fig. S3B,C) compared with WT and KitW-sh
mice. Inhibition of MC-FXR before injection into KitW-sh mice reduced these parameters
(Supporting Fig. S3A–C).

KitW-sh + MC mice display increased collagen deposition as shown by increased fast

green-sirius red stain and semiquantification compared with KitW-sh and WT mice 3 days
after injection; however, inhibition of MC-FXR resulted in reduced collagen deposition
and hepatic fibrosis compared with KitW-sh + MC mice (Supporting Fig. S4A). Further,
WT and KitW-sh mice have minimal HSC presence shown by desmin that is increased
in KitW-sh + MC and found in the surrounding bile ducts and portal area (Supporting
Fig. S4B). KitW-sh + MC mice display elevated expression of alpha smooth muscle actin
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(α-SMA) compared with WT and KitW-sh, whereas in KitW-sh + MC-Gugg, HSC presence
and α-SMA expression is reduced (Supporting Fig. S4B,C).

INHIBITION OF MC-FXR REDUCES BILIARY SENESCENCE-ASSOCIATED SECRETORY

PHENOTYPES AND HA/H1 SIGNALING
Ingenuity Pathway Analysis (IPA) analysis demonstrated FXR and HA signaling crosstalk
through inflammatory cytokines/senescence-associated secretory phenotypes (SASP), TGF-
β1 and IL-1β, and H1HR (Fig. 7A). Biliary mRNA TGF-β1 and IL-1β expression increased
in KitW-sh + MC compared with WT and KitW-sh mice, whereas inhibition of MC-FXR
reduced these parameters (Fig. 7B). In human patients with PSC and PSC-IBD, we found
increased HA secretion compared with control patients (Fig. 7C). Furthermore, WT and
KitW-sh mice have minimal serum HA levels that were elevated in KitW-sh + MC; however,
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when MC-FXR was inhibited, HA levels are reduced (Fig. 7D). We have shown that HA
activates cholangiocytes through paracrine interactions through H1HR signaling.(24) WT
and KitW-sh have minimal biliary mRNA expression of H1HR (Fig. 7E). Following MC
injection, KitW-sh have increased biliary H1HR mRNA expression compared with WT and
KitW-sh mice, indicating activation of biliary HA signaling; however, KitW-sh + MC-Gugg
mice have reduced biliary H1HR expression compared with KitW-sh + MC mice (Fig. 7E).
Combined, these results demonstrate that MC-FXR signaling has a synergistic relationship

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with systemic and autocrine HA signaling through regulation of biliary HA and SASP
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through H1HR.

INHIBITION OF MC-FXR BLOCKS BILIARY HA SIGNALING, IN VITRO

Similar to our in vivo findings, we found that in vitro murine cholangiocytes express
minimal mRNA expression of IL-1β, TGF-β1, and H1HR under basal conditions; however,
after stimulation with murine MC-vehicle supernatant, biliary IL-1β, TGF-β1 and H1HR
increased (Supporting Fig. S5A,B). Inhibition of MC-FXR reduced biliary expression
of IL-1β, TGF-β1, and H1HR (Supporting Fig. S5A,B). Correspondingly, HA secretion
increased in murine cholangiocytes treated with murine MC-vehicle supernatants, but
was reduced (not significant) when murine MCs were pretreated with the FXR inhibitor
(Supporting Fig. S5C). Next, we found that following murine MC-vehicle supernatant
stimulation, cholangiocytes have increased mRNA expression of FGF15 compared with
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basal cholangiocytes (Supporting Fig. S5D). Inhibition of MC-FXR reduced biliary

expression of FGF15 compared with murine cholangiocytes treated with MC-vehicle
supernatants (Supporting Fig. S5D). Taken together, these results confirm our IPA findings
and in vivo studies, wherein MC-FXR regulates systemic and hepatic MC activation
through activation of IL-1β, TGF-β1, and H1HR signaling. We conclude that MCs, through
activation of FXR, regulate biliary FXR/FGF15 signaling and IL-1β, TGF-β1, and H1HR
expression in a synergistic manner.

Cholangiocytes stimulated with MC-vehicle supernatant had increased proliferation

compared with basal cholangiocytes as measured by bromodeoxyuridine-positive
semiquantification and 3-(4,5-dimethylthiaz ol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophe nyl)-2H-tetrazolium assay (Supporting Fig. S6A,B). Treatment with MC-Gugg
supernatant resulted in decreased biliary proliferation (Supporting Fig. S6A,B). Taken
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together, we have found that MC-FXR is capable of activating an inflammatory and

proliferative cholangiocyte phenotype in vitro, mimicking hallmarks of in vivo DR seen
in cholestatic liver diseases.(25)

In the final sets of experiments, human HSCs stimulated with basal-treated MC

supernatants had increased α-SMA and fibronectin-1 mRNA expression compared with
basal treatment and inhibition of MC-FXR decreased these parameters (Supporting Fig.
S6C), demonstrating a direct interaction of MC-FXR and HSCs and further supporting our
in vivo studies.

Discussion
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We identified a signaling pathway wherein MC-specific FXR/FGF signaling regulates DR

and hepatic fibrosis, recapitulating cholestatic liver injury. We confirm previous work
establishing the role for MCs in the induction and development of cholestatic liver damage
characterized by DR, biliary senescence/SASP, and fibrosis in MC-deficient KitW-sh mice,
which are phenotypically normal. Our study reports the significant impact of MCs on TBA
signaling and biliary and intestinal FXR/FGF axis during liver injury.

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MCs are innate immune cells that, upon receptor activation, induce an inflammatory
response through various mediators, including HA and FGF.(26) Our group demonstrated
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that MCs infiltrate the liver following injury and perpetuate cholestatic damage(8,9,24) and
that stabilization of MCs in cholestatic models results in amelioration of DR, inflammation,
and hepatic fibrosis.(27) Aside from traditional receptors, MCs express a number of
other receptors and binding sites, including FXRβ and ASBT.(19) Our current work
demonstrates that MC-derived FXR/FGF signaling regulates biliary damage and hepatic
fibrosis. In support of this, during organ transplantation, MCs can produce a variety of both
proinflammatory and anti-inflammatory mediators as well as release factors like FGF-2,
which increases cell-to-cell interactions during regeneration.(28) In addition, FGF may
induce MC recruitment, as demonstrated in a study of prostate cancer in which the authors
found that stimulation of FGF-2 induced MC recruitment and promoted angiogenesis in
mice and inhibition of FGF-2 reduced tumor growth.(20)
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We found that inhibition of MC-FXR signaling ameliorates biliary senescence and SASP
through IL-1β/TGF-β1 and HA/H1HR signaling. Interestingly, most mediators secreted by
MCs are also SASP components, and several studies have implicated TGF-β1 in liver
disease progression.(9,11,29) We found that MCs induce IL-1β expression that was reduced
when FXR was inhibited; however, a study by Xiong et al. found that obeticholic acid
(OCA; FXR agonist), in combination with lipopolysaccharide, ameliorated liver damage and
inflammation by decreasing hepatic IL-1β.(30)

During canonical enterohepatic circulation, intestinal FXR/FGF15 signaling becomes

activated following an elevation of serum TBAs, which results in down-regulation of
ileal ASBT and inhibition of hepatic BA synthesis enzymes.(4) Fxr−/− mice have reduced
intrahepatic BA pressure following BDL surgery, resulting in lower IBDM compared with
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BDL WT.(31) We found that MC injection increased TBA content and hepatic FXR/FGF15
signaling in MC-deficient mice, and these features were blunted after inhibition of MC-
FXR, pinpointing MCs as critical regulators of TBA and hepatic FXR/FGF15 signaling.
Interestingly, we found that intestinal FXR/FGF15 expression was increased following
MC injection in MC-deficient mice and reduced in MC-FXR injected and untreated MC-
deficient mice. This could be due to KitW-sh phenotype, which lack melanocytes and
interstitial cells, but warrants further investigation. In contrast to our findings, Verbeke et al.
found that vehicle-treated BDL rats had low expression of SHP, a downstream target of FXR
activation, in ileum and jejunum that was rescued in ileum only following oral gavage of
OCA.(32) Nonetheless, in our current study, BDL WT and Mdr2−/− mice displayed elevated
intestinal FXR and FGF15 expression compared with WT controls, due to the complexity of
the intestinal FXR/FGF15 axis this also requires further investigation.
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Previous studies found that BAs regulate the release of specific mediators from MCs,
including the release of HA(33,34); however, our studies show that MCs influence hepatic
and intestinal BA signaling. Our findings are not surprising considering that MCs express
BA transporters and ligands,(19) which may interact with surrounding cells to induce
proliferation, senescence, or inflammation or alter BA synthesis. MC HA and intestinal
H1HR activation contribute to chenodeoxycholic acid (CDCA)–induced colonic chloride
secretion in rats, whereas MC-deficient rats had decreased CDCA-induced chloride

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 Meadows et al. Page 10

secretion,(33) supporting the role for MCs in intestinal BA signaling. Interestingly, BA

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activation of FXR in rat colon induced mucosal MC release of nerve growth factor,
which was reduced following FXR inhibition and silencing,(35) supporting our proposed
crosstalk between BA signaling, FXR, and MC mediators. Furthermore, human colonic
transcriptomic analysis showed up-regulation of BA signaling pathways in patients with
PSC-IBD compared with those with IBD and healthy controls(36); however, MC presence
and activation must be assessed in intestinal tissue from patients with PSC-IBD in order to
confirm MC and FXR intestinal crosstalk. It is unlikely that MCs synthesize BAs, and thus,
regulation of MC mediators like FGF15 or HA may act as a secondary mechanism to control
BA circulation and signaling.

We demonstrate that inhibition of MC-FXR reduces biliary FXR and FGF15 in vivo and in
vitro, which may result in reduced cholehepatic shunting of BAs; however, further analysis
is needed to confirm this. Our data show that inhibition of MC-FXR results in a reduction
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of TBA levels that are otherwise elevated following cholestatic liver injury.(37) Quist et
al. found that CDCA induces HA release from MCs in vitro, but cholic acid impeded
HA release, thus supporting the concept that BAs and MCs may interact to influence HA
signaling.(34)

It has been shown that there is dysregulation of FXR and FGF signaling in various
liver diseases including NAFLD, PBC and PSC.(16,38,39) OCA is currently being used
in clinical trials and shows potential for improved liver histology and lipid absorption
in patients with PSC and NASH, respectively, and provides clinical benefits to patients
with BA diarrhea(40–42); however, the overall benefits of FXR agonism are controversial
because of adverse events seen in patients with decompensated cirrhotic PSC and PBC.
(3) This may be due to the increased expression of biliary FXR in patients with liver
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injury, as shown in our study. If FXR is already enhanced in cholangiocytes, administering

OCA may be counterproductive by inducing biliary senescence or increasing damage
during cholestatic injury; however, further studies should be performed to investigate this
possibility. Furthermore, gut microbiota influences BA signaling and intestinal inflammation
and the subsequent positive or negative effects of FXR during diseases like NAFLD
or NASH.(43) In support of our study, in intestinal Fxr−/− knockout mice fed high-fat
diet (HFD) and treated with antibiotics, HFD-associated pathologies were reduced,(44)
further implicating the complex role of FXR signaling during liver disease. Interestingly,
OCA but not fexaramine (FXR agonist) ameliorated altered gut-barrier vascularization in
experimental cirrhotic mice (carbon tetrachloride or BDL), but both, OCA and fexaramine,
were able to reduce bacterial translocation from small intestine to the liver.(45) It has been
recently shown that MC presence and BA receptor increase in the duodenum of patients with
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irritable bowel syndrome, which, along with our study, indicates a role for MC regulation of
BA receptor expression throughout the intestinal tract.(46) The role of MCs in the regulation
of epithelial barrier, inflammation, and gut-liver axis during cholestatic liver injury is a
complicated future direction for this study.

In conclusion, we have generated a model of DR and hepatic fibrosis by injecting MCs into
phenotypically normal MC-deficient mice and confirmed our findings using the established
BDL and PSC mouse model. Our data demonstrate that MCs regulate biliary injury and

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 Meadows et al. Page 11

DR through alteration of TBA levels and FXR/FGF signaling through biliary SASP and
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HA/H1HR pathways (Fig. 8). In human samples, we found that both FXR in bile ducts
and MC presence are up-regulated in cholestatic liver damage, demonstrating the close
interaction between MCs and biliary FXR during liver disease. Although controversial,
our results demonstrate that specific inhibition of MC-FXR ameliorates cholestatic liver
injury by reducing TBA levels, biliary FXR/FGF15, and subsequent HA/H1HR signaling.
Our study also confirms a role for MC-FXR in the regulation of intestinal MC infiltration,
inflammation, and FXR/FGF15 in cholestatic mice (Fig. 8). Further investigation into the
contribution of MC mediators is needed to fully identify clinical therapies; however, our data
suggest that MCs may be a targetable link for patients with PSC alone or PSC coupled with
IBD.

Supplementary Material
Author Manuscript

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
Supported by the Hickam Endowed Chair, Gastroenterology, Medicine, Indiana University and the Indiana
University Health – Indiana University School of Medicine Strategic Research Initiative, the SRCS and RCS Award
and VA Merit awards to G.A. (5I01BX000574) and H.F. (1I01BX003031) from the United States Department
of Veteran’s Affairs, Biomedical Laboratory Research and Development Service, and NIH grants DK108959 and
DK119421 to H.F., DK11003 to S.G., and DK054811 to G.A. and S.G. This material is the result of work supported
by resources at the Central Texas Veterans Health Care System, Temple, TX, and Richard L. Roudebush VA
Medical Center, Indianapolis, IN. The content is the responsibility of the author(s) alone and does not necessarily
reflect the views or policies of the Department of Veterans Affairs or the United States Government.

Abbreviations:
Author Manuscript

α-SMA alpha smooth muscle actin

ASBT apical Na+bile acid transporter

ATCC American Type Culture Collection

BA bile acid

BDL bile duct ligation

CCL chemokine (C-C motif) ligand

CDCA chenodeoxycholic acid

CK-19 cytokeratin-19
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CXCL chemokine (C-X-C motif) ligand

Cyp cytochrome p450

DR ductular reaction

EIA enzyme immunoassay

FGF fibroblast growth factor

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 Meadows et al. Page 12

FXR farnesoid X receptor

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Gugg guggulsterone

HA histamine

HR histamine receptor

IBD inflammatory bowel disease

IBDM intrahepatic bile duct mass

IPA Ingenuity Pathway Analysis

KitW-sh [Link]-KitW-sh/HNihrJaeBsmJ

MC mast cell
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Mdr2−/− multidrug resistance 2/ATP-binding cassette family 2 member 4

knockout

OCA obeticholic acid

PBC primary biliary cholangitis

PSC primary sclerosing cholangitis

qPCR quantitative PCR

SASP senescence-associated secretory phenotype

SCF stem cell factor

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SHP small heterodimer partner

TBA total bile acid

WT wild type

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FIG. 1.
(A) Hepatic FXR expression and MC presence increase in human liver disease explant
patients. Livers from nondiseased control, PSC, PSC-IBD, PBC, biliary atresia, and NASH
were stained using immunohistochemistry for FXR (brown), costained with tryptase (red)
to detect activated MC presence. Enhanced biliary FXR immunoreactivity (marked by black
arrows) is present in all diseased tissue explants compared with control, with a subset
(PSC, PSC-IBD, and PBC) demonstrating increased hepatocyte FXR expression, indicating
activated hepatic FXR function. Tryptase expression also increased in all models of disease
compared with control. (B) Patients with PSC (n = 14) and PSC-IBD (n = 17) have
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increased serum FGF19 compared with healthy controls (n = 5). (C) Patients with PSC
(n = 3) and PSC-IBD (n = 4) also have increased hepatic SHP expression compared with
controls (n = 5). Data are mean ± SEM of n = 2 (serum) or n = 3 (qPCR) experiments per
patient sample. *P < 0.05 vs. control. Representative images are presented as ×20 and zoom
boxes are ×80.
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FIG. 2.
(A) MCs alter TBA and FXR/FGF signaling in an established model of cholestatic MC-
deficient mice. Serum TBA content is increased in BDL WT compared with WT mice, but
in BDL KitW-sh mice, TBA content is reduced. Serum TBA was unchanged between control
groups. Hepatic FXR is unchanged in WT and KitW-sh mice; however, expression is elevated
in BDL WT mice compared with WT and KitW-sh. (B) BDL KitW-sh mice exhibited reduced
hepatic FXR expression compared with BDL WT mice. (C) BDL WT mice have elevated
serum panFGF secretion compared with WT and KitW-sh, which is reduced in BDL KitW-sh
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mice; no significant changes were found between control groups. (D) Biliary FGF15 (green)
expression was elevated in BDL WT mice shown by immunofluorescence, costained with
CK-19 to mark bile ducts (red) but reduced in BDL KitW-sh mice with no changes between
control groups. (E) In BDL WT small intestine, there is increased MC presence detected by
tryptase β−2 (Tpsβ−2) immunoreactivity and marked by red arrows. In distal ileum from
BDL WT there was increased (F) FXR and (G) FGF15 immunoreactivity compared with
WT mice. Data are mean ± SEM of n = 4 experiments for qPCR, panFGF EIA, and TBA
from 4–6 mice per group. *P < 0.05 vs. WT; #P < 0.05 vs. BDL WT. Representative images
are presented as ×20 for FXR and ×40 for FGF15 and Tpsβ−2.
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FIG. 3.
Validation of model and hepatic damage in KitW-sh mice. Cultured MCs (MC/9, ATCC
CRL-8306) were stimulated with 0.1% DMSO (vehicle) or 10 μM Z-guggulsterone for 48
hours and (A) FXR, (B) FGF15, (C) SHP mRNA expression, and (D) panFGF secretion
were measured. Following treatment, MC-Gugg have reduced (A-C) FXR, FGF15, and
SHP mRNA levels, measured by qPCR, and (D) panFGF secretion, measured by EIA,
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compared with vehicle-treated MCs. (E) Cultured MCs were treated with 0.1% DMSO or
guggulsterone (Gugg) and tagged with PKH26 before injection into KitW-sh mice through
tail vein. MC migration to the liver was confirmed in KitW-sh mice injected with both (F)
vehicle-treated MCs and Z-guggulsterone–treated MCs (MC-Gugg) by immunofluorescence
imaging of PKH26 (red) and CK-19 to mark bile ducts (green). (G) Hematoxylin and
eosin (H&E) staining demonstrates increased hepatic damage and ductular inflammation in
KitW-sh + MC that is absent in WT and KitW-sh mice. Hepatic damage is reduced in KitW-sh
+ MC-Gugg mice. Data are mean ± SEM of n = 4 experiments from n = 2 biological
replicates for qPCR and panFGF EIA. *P < 0.05 vs. MC-vehicle. Representative images
are presented as ×20 for immunofluorescence, with ×40 zoom boxes, and ×10 for H&E.
Abbreviation: Inj, injury.
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FIG. 4.
MC-FXR regulates hepatic and biliary FXR/FGF signaling. Introduction of MCs in KitW-sh
mice increased (A) hepatic and (B) circulating TBA content; however, both TBA levels were
reduced with MC-FXR inhibition. (A,B) No significant changes were noted between WT
and KitW-sh groups. (C) Hepatic and (D) biliary FXR expression increased in KitW-sh mice
injected with MCs compared with WT and KitW-sh mice, and inhibition of MC-FXR reduces
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both hepatic and biliary FXR expression in KitW-sh + MC-Gugg mice. The expression of
(E) hepatocyte Cyp7a1 increased in both control KitW-sh mice and KitW-sh mice injected
with MCs compared with WT, whereas (F) hepatocyte Cyp27a1 decreased in all groups
compared with WT. (E,F) In KitW-sh mice injected with MC-Gugg, Cyp7a1 expression
decreased and Cyp27a1 levels remained unchanged compared with control. (G) Biliary
FGF15 (green) expression, costained with CK-19 to mark bile ducts (red), and (H) hepatic
and (I) serum FGF15 secretion increased in KitW-sh mice injected with MCs, that was
subsequently reduced in mice injected with MC-Gugg (G-I). Minimal changes were noted
between control groups (G-I). Data are mean ± SEM of n = 8 experiments for qPCR and n =
4 experiments for FGF15 and panFGF EIA from 6–8 mice and n = 4 experiments from 6–8
mice per group for serum and liver TBA. *P < 0.05 vs. WT, #P < 0.05 vs. KitW-sh + MC.
Representative images are presented as ×20 with ×40 zoom boxes. Abbreviation: Inj, injury.
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FIG. 5.
Intestinal FXR/FGF15 and inflammation are regulated by MC-FXR. Introduction of MCs
in KitW-sh mice increases ileal (A) FXR and (B) FGF15 expression compared with WT and
KitW-sh mice and inhibition of MC-FXR decreases (A) FXR and (B) FGF15 expression, as
shown in KitW-sh + MC-Gugg mice. (C) MC injection increased small intestine expression
of SCF, IL-1β, TGF-β1, and CCL3 in KitW-sh compared with WT and KitW-sh mice;
however, intestinal inflammation was reduced when MC-FXR was inhibited. Representative
images are presented as ×20. Data are mean ± SEM of n = 4 experiments for qPCR from 6–8
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mice (MC injection group). *P < 0.05 vs. WT and KitW-sh, #P < 0.05 vs. KitW-sh + MC, &P
< 0.05 vs. WT.
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FIG. 6.
Inhibition of MC-FXR reduces IBDM and inflammation. The effect of MC-FXR inhibition
on IBDM and liver inflammation was measured by immunohistochemistry for (A) CK-19
and (B) EGF-like module-containing mucin-like hormone receptor-like 1 mouse homolog
(F4/80), respectively, with (C,D) semiquantification. WT and KitW-sh mice display minimal
IBDM and F4/80 positive Kupffer cells that increased following MC injection in KitW-sh +
MC mice. Inhibition of MC-FXR reduced both IBDM and inflammation. (A) Red arrows
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indicate CK-19 positive bile ducts. (E) WT and KitW-sh mice have minimal expression
of hepatic inflammatory markers IL-1β, CCL5, CXCL2, and CXCL5, whereas KitW-sh
+ MC mice have increased hepatic inflammatory marker expression that is reduced in
KitW-sh + MC-FXR mice. Data are mean ± SEM of n = 10–15 representative images for
immunoreactivity semiquantification and of n = 4 experiments for qPCR from 6–8 mice
per group. *P < 0.05 vs. WT, #P < 0.05 vs. KitW-sh + MC. All representative images are
presented as ×10. Abbreviation: Inj, injury.
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FIG. 7.
FXR and HA signaling crosstalk through IL-1β, TGF-β1, and H1HR. (A) IPA demonstrated
a link between HA and FXR signaling crosstalk through IL-1β, TGF-β1, and H1HR. (B)
TGF-β1 and IL-1β expression increased in isolated cholangiocytes from KitW-sh + MC
compared with WT and KitW-sh mice by qPCR and inhibition of MC-FXR reduced biliary
TGF-β1 and IL-1β expression. (C) Serum HA increases in patients with PSC (n = 14) and
PSC-IBD (n = 17) compared with controls. (D) WT and KitW-sh have minimal HA serum
secretion, which is elevated in KitW-sh + MC mice. (D) Serum HA is reduced in KitW-sh
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+ MC-Gugg mice. (E) MC injection increases biliary H1HR expression in KitW-sh + MC

compared with WT and KitW-sh mice and inhibition of MC-FXR reduces biliary H1HR
expression. Data are mean ± SEM of n = 8 experiments for qPCR, n = 4 experiments for HA
EIA from 6–8 mice per group, and n = 2 experiments per patient sample for HA EIA. *P <
0.05 vs. control group, #P < 0.05 vs. KitW-sh + MC. Abbreviation: Inj, injury.
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Hepatology. Author manuscript; available in PMC 2022 July 29.

 Meadows et al. Page 23
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FIG. 8.
Graphical abstract demonstrating the role of MC-FXR during cholestatic liver damage.
Patients with cholestatic liver diseases, such as PSC, often present with comorbidities like
IBD. We found that MCs, which express FXR and secrete FGFs and HA, infiltrate the
liver and contribute to increased DR, biliary SASP, and FXR/FGF15 during cholestatic
liver injury. Furthermore, MCs can activate HSCs to increase α-SMA, fibronectin-1, and
collagen, exacerbating liver damage. In this study, we found that MCs infiltrate the intestine
of cholestatic liver injury mouse models, increasing intestinal inflammation and altering
the FXR/FGF15 axis, leading to dysregulated BA signaling. MCs may serve as an ideal
target for therapeutic interventions for patients presenting with cholestatic liver injury and
intestinal inflammation. Created with [Link].
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Hepatology. Author manuscript; available in PMC 2022 July 29.