The composition of the microbiome in health and disease has only recently become a major research... more The composition of the microbiome in health and disease has only recently become a major research focus. Although it is clear that an imbalance or dysbiosis in the microbiota is associated with disease, its interrelatedness to disease penetrance is largely unknown. Inflammatory bowel disease (IBD) is an excellent disease in which to explore these questions because of the extensive genetic studies identifying disease susceptibility loci and the ability to easily sample the intestinal microbiota in IBD patients due to the accessibility of stool samples. In addition, mouse models of IBD have contributed to our understanding of the interrelatedness of the gut microbiota and genes associated with IBD. The power of the mouse studies is that multiple colitis models exist that can be used in combination with genetically modified mice that harbour deficiencies in IBD susceptibility genes. Collectively, these studies revealed that bacterial dysbiosis does occur in human IBD and in mouse colitis models. In addition, with an emphasis on immune genes, the mouse studies provided evidence that specific immune regulatory proteins associated with IBD influence the gut microbiota in a manner consistent with disease penetrance. In this review, we will discuss studies in both humans and mice that demonstrate the impact of immunodeficiences in interleukin-10, interleukin-17, nucleotide-binding oligomerization domain (NOD) 2, NOD-like receptor proteins 3 and 6, Toll-like receptor or IgA have on the interrelatedness between the composition of the gut microbiota and disease penetrance of IBD and its mouse models.
The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immu... more The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG1 antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG3 were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell...
Journal of immunology (Baltimore, Md. : 1950), Jan 31, 2015
It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response ... more It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-) (/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbore...
Although classically B cells are known to play important roles in immune protection via humoral i... more Although classically B cells are known to play important roles in immune protection via humoral immunity, recently their regulatory mechanisms have been best appreciated in the context of autoimmunity. Several studies have identified different subsets of regulatory B cells that vary not only in their phenotype but also in their mechanism of action. Although the best-studied mechanism of B-cell immune regulation is IL-10 production, other IL-10-independent mechanisms have been proposed. These include maintenance of CD4(+)Foxp3(+) regulatory T cells; production of transforming growth factor-β, IL-35, IgM or adenosine or expression of PD-L1 (programmed death 1 ligand 1) or FasL (Fas ligand). Given that B-cell-targeted therapy is being increasingly used in the clinic, a complete understanding of the mechanisms whereby B cells regulate inflammation associated with specific diseases is required for designing safe and effective immunotherapies targeting B cells.
Regulatory T cells (Tregs) and B cells present in gut-associated lymphoid tissues (GALT) are both... more Regulatory T cells (Tregs) and B cells present in gut-associated lymphoid tissues (GALT) are both implicated in the resolution of colitis. However, how the functions of these cells are coordinated remains elusive. We used the dextran sulfate sodium (DSS)-induced colitis model combined with gene-modified mice to monitor the progression of colitis, and simultaneously examine the number of Tregs and B cells, and the production of IgA antibodies. We found that DSS-treated mice exhibited more severe colitis in the absence of B cells, and that the adoptive transfer of B cells attenuated the disease. Moreover, the transfer of IL-10(-/-) B cells also attenuated colitis, suggesting that B cells inhibited colitis through an interleukin-10 (IL-10)-independent pathway. Furthermore, antibody depletion of Tregs resulted in exacerbated colitis. Intriguingly, the number of GALT Tregs in B cell-deficient mice was significantly decreased during colitis and the adoptive transfer of B cells into these ...
B cells are thought to play a pathogenic role in multiple sclerosis (MS), an autoimmune disease a... more B cells are thought to play a pathogenic role in multiple sclerosis (MS), an autoimmune disease affecting the central nervous system (CNS). This idea is supported by the reduction of disease in MS patients undergoing antibody-mediated B cell depletion therapy. In contrast, in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, B cells have been shown to play a regulatory role. This is suggestive of a dual role for B cells in CNS autoimmunity. It is possible that a critical balance between the pathogenic and regulatory populations of B cells might be involved in the manifestation of the disease. Although in mice, different B cell subsets have been shown to exert immunoregulation through varied mechanisms, the phenotype of regulatory B cells in humans and factors affecting their function are not well known. Also, the origin and development of regulatory B cells is not known. It is important to thoroughly identify the different populations of B cells that might be involved in suppressing CNS autoimmunity, their mode of function and factors that regulate their immunosuppressive properties for using regulatory B cells as a therapy for MS. Here we present methods to study the phenotype and mechanisms of immune suppression by B cells in different mouse models of EAE.
Experimental and clinical studies often require highly purified cell populations. FACS is a techn... more Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.
MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous syst... more MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous system) historically characterized as an inflammatory and demyelinating disease. More recently, extensive neuronal pathology has lead to its classification as a neurodegenerative disease as well. While the immune system initiates the autoimmune response it remains unclear how it orchestrates neuronal damage. In our previous studies, using in vitro cultured embryonic neurons, we demonstrated that MBP (myelin basic protein)-specific encephalitogenic CD4 T-cells induce early neuronal damage. In an extension of those studies, here we show that polarized CD4 Th1 and Th17 cells as wells as CD8 T-cells and NK (natural killer) cells induce microtubule destabilization within neurites in a contact-independent manner. Owing to the cytotoxic potential of these immune cells, we isolated the luminal components of lytic granules and determined that they were sufficient to drive microtubule destabilization. Since lytic granules contain cytolytic proteins, we determined that the induction of microtubule destabilization occurred prior to signs of apoptosis. Furthermore, we determined that microtubule destabilization was largely restricted to axons, sparing dendrites. This study demonstrated that lymphocytes with cytolytic activity have the capacity to directly drive MAD (microtubule axonal destabilization) in a bystander manner that is independent of neuronal death. r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r
The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which con... more The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastrointestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature , but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5 + B1a cells possess some
The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migrati... more The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigenspecific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2 2/2 mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2 2/2 mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG 1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG 1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2 2/2 mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.
Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and... more Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in rob...
R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellul... more R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellular processes, including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro and has been associated with liver autoimmunity. We used Rras-deficient mice to study the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. Further investigation revealed that, during EAE, absence of R-Ras promoted the formation of MHC II(low) DC concomitant with a significant increase in proliferation of natural regulatory T cells, resulting in an increase in their cell numbers in the periphery. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of natural regulatory T cell numbers by inhibiting the development of MHCII(low) DC with tolerogenic potential.
The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells follo... more The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells following bone marrow transplantation (BMT), with CD45.1 and CD45.2 being efficiently distinguished by flow cytometry. During the analysis of control mixed BM chimera mice in which lethally irradiated recipients were transplanted with an equal number of BM cells from WT CD45.1 and WT CD45.2 mice, we observed an unequal reconstitution of specific B cell subsets in the bone marrow (BM), lymph node (LN) and spleen. Specifically, in the BM and LN, there was an increase in the percentage of CD45.2 mature B cells. In the spleen, an increase in the percentage of CD45.2 transitional (T) 1 and T2 cells was observed. In contrast, the percentage of splenic CD45.1 marginal zone (MZ) B cells was significantly increased. When we compared the percentage of B cell subsets in unmanipulated WT CD45.1 and WT CD45.2 mice, we found that WT CD45.2 mice had significantly more LN B cells while WT CD45.1 mice exhibited an increase in MZ B cells. These data indicate that the alteration in the ratio of CD45.1 and CD45.2 B cell subsets in mixed chimera mice is a cell-intrinsic effect. Thus whenever the CD45 congenic system is used to track two genetically distinct populations of immune cells WT chimeras must be generated to allow normalization of the experimental data to avoid the reporting of unintentionally skewed data.
The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin... more The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization. In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen. The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer. The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.
Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and T... more Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and TLR6, by 1Á8-fold and twofold, respectively, in peritoneal cavity B-2 cells from C57BL/6 mice, implicating that the co-expression of TLR2 and TLR6 occurs as a combinatorial repertoire in response to porin. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing the majority of bacterial products, TLR2 alone participates in porin recognition. TLR2 expression was increased on B-2 cells, whereas the expression of TLR4 remained unaffected. Besides TLRs, mRNA for myeloid differentiation factor 88 (MyD88), an effector molecule associated with the TLR-mediated response, was enhanced by twofold, suggesting its involvement in the activity of porin. The B-2 cells showed a 1Á8-fold increase in mRNA expression of the signalling molecule, nuclear factor-kappa B (NF-B), in the presence of porin. Porin treatment of B-2 cells selectively up-regulated the expression of the costimulatory molecule, CD86, by 44-fold. Porin induced the cell-surface expression of immunoglobulin (Ig)M, of IgG2a preferentially among the IgG subclasses, and of IgA, on B-2 cells. The porin-mediated inductions of IgG2a and IgA were augmented by interleukin-6 on B-2 cells, by 27-and 1Á6-fold, respectively.
Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38 000 ... more Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38 000 Da monomer of porin of Shigella dysenteriae type 1. Since human, the only natural host of S. dysenteriae type 1, recognized the protein through humoral immune response, it is of great significance to study the surface-exposed outer membrane antigen as an adjuvant. Porin treatment of CD11b þ peritoneal cavity (PerC) Mx of BALB/c mouse was found to up-regulate CD80 on cell surface and had no effect on CD86 expression. The surface expression of CD80 got increased by 1.6-fold in the presence of gamma interferon (IFN-Q) supporting selective regulation of the B7-1 (CD80) member of the B7 family. Mx released 7.25 pg of interleukin-12 (IL-12) in the presence of porin. The protein in combination with IFN-Q augmented profoundly the release of IL-12 by 2.6-fold. Porin-mediated induction of IL-12 release would therefore influence Th1type response, known to be preferentially triggered due to up-regulation of CD80 expression. Treatment of PerC Mx by the protein showed an increase of mRNA for both Toll-like receptor (TLR)2 and myeloid differentiation factor 88 (MyD88) by 2-and 2.3-fold respectively, emphasizing that TLR2 is essential for recognition of S. dysenteriae type 1 porin. Understanding the mechanism of adjuvanticity of porin of S. dysenteriae type 1 is a necessary step towards the development of a better adjuvant against shigellosis.
A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of th... more A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of the human autoimmune disease multiple sclerosis (MS), has been established. In the first role, B cells contribute to the pathogenesis of EAE through the production of anti-myelin antibodies that contribute to demyelination. On the contrary, B cells have also been shown to have protective functions in that they play an essential role in the spontaneous recovery from EAE. In this review, we summarize studies conducted in a number of species demonstrating the conditions under which B cells are pathogenic in EAE. We also discuss the phenotype and anti-inflammatory mechanisms of regulatory B cells.
Multiple sclerosis (MS) is considered to be a T cell-mediated autoimmune disease that results in ... more Multiple sclerosis (MS) is considered to be a T cell-mediated autoimmune disease that results in the presence of inflammatory lesions/plaques associated with mononuclear cell infiltrates, demyelination and axonal damage within the central nervous system (CNS). To date, FDA approved therapies in MS are thought to largely function by modulation of the immune response. Since autoimmune responses require many arms of the immune system, the direct cellular mechanisms of action of MS therapeutics are not definitively known. The mouse model of MS, experimental autoimmune encephalomyelitis (EAE), has been instrumental in deciphering the mechanism of action of MS drugs. In addition, EAE has been widely used to study the contribution of individual components of the immune system in CNS autoimmunity. In this regard, the role of B cells in EAE has been studied in mice deficient in B cells due to genetic ablation and following depletion with a B cell-targeted monoclonal antibody (mAb) (anti-CD20). Both strategies have indicated that B cells regulate the extent of EAE clinical disease and in their absence disease is exacerbated. Thus a new population of "regulatory B cells" has emerged. One reoccurring component of regulatory B cell function is the production of IL-10, a pleiotropic cytokine with potent anti-inflammatory properties. B cell depletion has also indicated that B cells, in particular antibody production, play a pathogenic role in EAE. B cell depletion in MS using a mAb to CD20 (rituximab) has shown promising results. In this review, we will discuss the current thinking on the role of B cells in MS drawing from knowledge gained in EAE studies and clinical trials using therapeutics that target B cells.
The composition of the microbiome in health and disease has only recently become a major research... more The composition of the microbiome in health and disease has only recently become a major research focus. Although it is clear that an imbalance or dysbiosis in the microbiota is associated with disease, its interrelatedness to disease penetrance is largely unknown. Inflammatory bowel disease (IBD) is an excellent disease in which to explore these questions because of the extensive genetic studies identifying disease susceptibility loci and the ability to easily sample the intestinal microbiota in IBD patients due to the accessibility of stool samples. In addition, mouse models of IBD have contributed to our understanding of the interrelatedness of the gut microbiota and genes associated with IBD. The power of the mouse studies is that multiple colitis models exist that can be used in combination with genetically modified mice that harbour deficiencies in IBD susceptibility genes. Collectively, these studies revealed that bacterial dysbiosis does occur in human IBD and in mouse colitis models. In addition, with an emphasis on immune genes, the mouse studies provided evidence that specific immune regulatory proteins associated with IBD influence the gut microbiota in a manner consistent with disease penetrance. In this review, we will discuss studies in both humans and mice that demonstrate the impact of immunodeficiences in interleukin-10, interleukin-17, nucleotide-binding oligomerization domain (NOD) 2, NOD-like receptor proteins 3 and 6, Toll-like receptor or IgA have on the interrelatedness between the composition of the gut microbiota and disease penetrance of IBD and its mouse models.
The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immu... more The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG1 antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG3 were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell...
Journal of immunology (Baltimore, Md. : 1950), Jan 31, 2015
It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response ... more It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-) (/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbore...
Although classically B cells are known to play important roles in immune protection via humoral i... more Although classically B cells are known to play important roles in immune protection via humoral immunity, recently their regulatory mechanisms have been best appreciated in the context of autoimmunity. Several studies have identified different subsets of regulatory B cells that vary not only in their phenotype but also in their mechanism of action. Although the best-studied mechanism of B-cell immune regulation is IL-10 production, other IL-10-independent mechanisms have been proposed. These include maintenance of CD4(+)Foxp3(+) regulatory T cells; production of transforming growth factor-β, IL-35, IgM or adenosine or expression of PD-L1 (programmed death 1 ligand 1) or FasL (Fas ligand). Given that B-cell-targeted therapy is being increasingly used in the clinic, a complete understanding of the mechanisms whereby B cells regulate inflammation associated with specific diseases is required for designing safe and effective immunotherapies targeting B cells.
Regulatory T cells (Tregs) and B cells present in gut-associated lymphoid tissues (GALT) are both... more Regulatory T cells (Tregs) and B cells present in gut-associated lymphoid tissues (GALT) are both implicated in the resolution of colitis. However, how the functions of these cells are coordinated remains elusive. We used the dextran sulfate sodium (DSS)-induced colitis model combined with gene-modified mice to monitor the progression of colitis, and simultaneously examine the number of Tregs and B cells, and the production of IgA antibodies. We found that DSS-treated mice exhibited more severe colitis in the absence of B cells, and that the adoptive transfer of B cells attenuated the disease. Moreover, the transfer of IL-10(-/-) B cells also attenuated colitis, suggesting that B cells inhibited colitis through an interleukin-10 (IL-10)-independent pathway. Furthermore, antibody depletion of Tregs resulted in exacerbated colitis. Intriguingly, the number of GALT Tregs in B cell-deficient mice was significantly decreased during colitis and the adoptive transfer of B cells into these ...
B cells are thought to play a pathogenic role in multiple sclerosis (MS), an autoimmune disease a... more B cells are thought to play a pathogenic role in multiple sclerosis (MS), an autoimmune disease affecting the central nervous system (CNS). This idea is supported by the reduction of disease in MS patients undergoing antibody-mediated B cell depletion therapy. In contrast, in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, B cells have been shown to play a regulatory role. This is suggestive of a dual role for B cells in CNS autoimmunity. It is possible that a critical balance between the pathogenic and regulatory populations of B cells might be involved in the manifestation of the disease. Although in mice, different B cell subsets have been shown to exert immunoregulation through varied mechanisms, the phenotype of regulatory B cells in humans and factors affecting their function are not well known. Also, the origin and development of regulatory B cells is not known. It is important to thoroughly identify the different populations of B cells that might be involved in suppressing CNS autoimmunity, their mode of function and factors that regulate their immunosuppressive properties for using regulatory B cells as a therapy for MS. Here we present methods to study the phenotype and mechanisms of immune suppression by B cells in different mouse models of EAE.
Experimental and clinical studies often require highly purified cell populations. FACS is a techn... more Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.
MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous syst... more MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous system) historically characterized as an inflammatory and demyelinating disease. More recently, extensive neuronal pathology has lead to its classification as a neurodegenerative disease as well. While the immune system initiates the autoimmune response it remains unclear how it orchestrates neuronal damage. In our previous studies, using in vitro cultured embryonic neurons, we demonstrated that MBP (myelin basic protein)-specific encephalitogenic CD4 T-cells induce early neuronal damage. In an extension of those studies, here we show that polarized CD4 Th1 and Th17 cells as wells as CD8 T-cells and NK (natural killer) cells induce microtubule destabilization within neurites in a contact-independent manner. Owing to the cytotoxic potential of these immune cells, we isolated the luminal components of lytic granules and determined that they were sufficient to drive microtubule destabilization. Since lytic granules contain cytolytic proteins, we determined that the induction of microtubule destabilization occurred prior to signs of apoptosis. Furthermore, we determined that microtubule destabilization was largely restricted to axons, sparing dendrites. This study demonstrated that lymphocytes with cytolytic activity have the capacity to directly drive MAD (microtubule axonal destabilization) in a bystander manner that is independent of neuronal death. r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r
The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which con... more The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastrointestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature , but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5 + B1a cells possess some
The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migrati... more The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigenspecific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2 2/2 mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2 2/2 mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG 1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG 1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2 2/2 mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.
Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and... more Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in rob...
R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellul... more R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellular processes, including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro and has been associated with liver autoimmunity. We used Rras-deficient mice to study the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. Further investigation revealed that, during EAE, absence of R-Ras promoted the formation of MHC II(low) DC concomitant with a significant increase in proliferation of natural regulatory T cells, resulting in an increase in their cell numbers in the periphery. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of natural regulatory T cell numbers by inhibiting the development of MHCII(low) DC with tolerogenic potential.
The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells follo... more The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells following bone marrow transplantation (BMT), with CD45.1 and CD45.2 being efficiently distinguished by flow cytometry. During the analysis of control mixed BM chimera mice in which lethally irradiated recipients were transplanted with an equal number of BM cells from WT CD45.1 and WT CD45.2 mice, we observed an unequal reconstitution of specific B cell subsets in the bone marrow (BM), lymph node (LN) and spleen. Specifically, in the BM and LN, there was an increase in the percentage of CD45.2 mature B cells. In the spleen, an increase in the percentage of CD45.2 transitional (T) 1 and T2 cells was observed. In contrast, the percentage of splenic CD45.1 marginal zone (MZ) B cells was significantly increased. When we compared the percentage of B cell subsets in unmanipulated WT CD45.1 and WT CD45.2 mice, we found that WT CD45.2 mice had significantly more LN B cells while WT CD45.1 mice exhibited an increase in MZ B cells. These data indicate that the alteration in the ratio of CD45.1 and CD45.2 B cell subsets in mixed chimera mice is a cell-intrinsic effect. Thus whenever the CD45 congenic system is used to track two genetically distinct populations of immune cells WT chimeras must be generated to allow normalization of the experimental data to avoid the reporting of unintentionally skewed data.
The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin... more The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization. In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen. The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer. The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.
Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and T... more Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and TLR6, by 1Á8-fold and twofold, respectively, in peritoneal cavity B-2 cells from C57BL/6 mice, implicating that the co-expression of TLR2 and TLR6 occurs as a combinatorial repertoire in response to porin. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing the majority of bacterial products, TLR2 alone participates in porin recognition. TLR2 expression was increased on B-2 cells, whereas the expression of TLR4 remained unaffected. Besides TLRs, mRNA for myeloid differentiation factor 88 (MyD88), an effector molecule associated with the TLR-mediated response, was enhanced by twofold, suggesting its involvement in the activity of porin. The B-2 cells showed a 1Á8-fold increase in mRNA expression of the signalling molecule, nuclear factor-kappa B (NF-B), in the presence of porin. Porin treatment of B-2 cells selectively up-regulated the expression of the costimulatory molecule, CD86, by 44-fold. Porin induced the cell-surface expression of immunoglobulin (Ig)M, of IgG2a preferentially among the IgG subclasses, and of IgA, on B-2 cells. The porin-mediated inductions of IgG2a and IgA were augmented by interleukin-6 on B-2 cells, by 27-and 1Á6-fold, respectively.
Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38 000 ... more Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38 000 Da monomer of porin of Shigella dysenteriae type 1. Since human, the only natural host of S. dysenteriae type 1, recognized the protein through humoral immune response, it is of great significance to study the surface-exposed outer membrane antigen as an adjuvant. Porin treatment of CD11b þ peritoneal cavity (PerC) Mx of BALB/c mouse was found to up-regulate CD80 on cell surface and had no effect on CD86 expression. The surface expression of CD80 got increased by 1.6-fold in the presence of gamma interferon (IFN-Q) supporting selective regulation of the B7-1 (CD80) member of the B7 family. Mx released 7.25 pg of interleukin-12 (IL-12) in the presence of porin. The protein in combination with IFN-Q augmented profoundly the release of IL-12 by 2.6-fold. Porin-mediated induction of IL-12 release would therefore influence Th1type response, known to be preferentially triggered due to up-regulation of CD80 expression. Treatment of PerC Mx by the protein showed an increase of mRNA for both Toll-like receptor (TLR)2 and myeloid differentiation factor 88 (MyD88) by 2-and 2.3-fold respectively, emphasizing that TLR2 is essential for recognition of S. dysenteriae type 1 porin. Understanding the mechanism of adjuvanticity of porin of S. dysenteriae type 1 is a necessary step towards the development of a better adjuvant against shigellosis.
A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of th... more A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of the human autoimmune disease multiple sclerosis (MS), has been established. In the first role, B cells contribute to the pathogenesis of EAE through the production of anti-myelin antibodies that contribute to demyelination. On the contrary, B cells have also been shown to have protective functions in that they play an essential role in the spontaneous recovery from EAE. In this review, we summarize studies conducted in a number of species demonstrating the conditions under which B cells are pathogenic in EAE. We also discuss the phenotype and anti-inflammatory mechanisms of regulatory B cells.
Multiple sclerosis (MS) is considered to be a T cell-mediated autoimmune disease that results in ... more Multiple sclerosis (MS) is considered to be a T cell-mediated autoimmune disease that results in the presence of inflammatory lesions/plaques associated with mononuclear cell infiltrates, demyelination and axonal damage within the central nervous system (CNS). To date, FDA approved therapies in MS are thought to largely function by modulation of the immune response. Since autoimmune responses require many arms of the immune system, the direct cellular mechanisms of action of MS therapeutics are not definitively known. The mouse model of MS, experimental autoimmune encephalomyelitis (EAE), has been instrumental in deciphering the mechanism of action of MS drugs. In addition, EAE has been widely used to study the contribution of individual components of the immune system in CNS autoimmunity. In this regard, the role of B cells in EAE has been studied in mice deficient in B cells due to genetic ablation and following depletion with a B cell-targeted monoclonal antibody (mAb) (anti-CD20). Both strategies have indicated that B cells regulate the extent of EAE clinical disease and in their absence disease is exacerbated. Thus a new population of "regulatory B cells" has emerged. One reoccurring component of regulatory B cell function is the production of IL-10, a pleiotropic cytokine with potent anti-inflammatory properties. B cell depletion has also indicated that B cells, in particular antibody production, play a pathogenic role in EAE. B cell depletion in MS using a mAb to CD20 (rituximab) has shown promising results. In this review, we will discuss the current thinking on the role of B cells in MS drawing from knowledge gained in EAE studies and clinical trials using therapeutics that target B cells.
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Papers by Avijit Ray