Cell Transfer Model Research Articles

Vagotomy affects the development of oral tolerance and increases susceptibility to develop colitis independently of the alpha-7 nicotinic receptor.

Vagotomy (VGX) increases the susceptibility to develop colitis suggesting a crucial role for the cholinergic anti-inflammatory pathway in the regulation of the immune responses. Since oral tolerance and the generation of regulatory T cells (Tregs) are crucial to preserve mucosal immune homeostasis, we studied the effect of vagotomy and the involvement of α7 nicotinic receptors (α7nAChR) at the steady state and during colitis. Therefore, the development of both oral tolerance and colitis (induced by dextran sulfate sodium (DSS) or via T cell transfer) was studied in vagotomized mice and in α7nAChR-/- mice. VGX, but not α7nAChR deficiency, prevented oral tolerance establishment. This effect was associated with reduced Treg conversion in the lamina propria and mesenteric lymphnodes. To the same extent, vagotomized mice, but not α7nAChR-/- mice, developed a more severe DSS colitis compared with control mice treated with DSS, associated with a decreased number of colonic Tregs. However, neither VGX nor absence of α7nAChR in recipient mice affected colitis development in the T cell transfer model. In line, deficiency of α7nAChR exclusively in T cells did not influence the development of colitis induced by T cell transfer. Our results indicate a key role for the vagal intestinal innervation in the development of oral tolerance and colitis, most likely by modulating induction of Tregs independently of α7nAChR.

T Cell Transfer Model of Colitis

T Cell Transfer Model of Colitis

Eomesodermin Expression in CD4+ T Cells Restricts Peripheral Foxp3 Induction

CD4(+) T cells polarize into effector Th subsets characterized by signature transcription factors and cytokines. Although T-bet drives Th1 responses and represses the alternative Th2, Th17, and Foxp3(+) regulatory T cell fates, the role of the T-bet-related transcription factor eomesodermin (Eomes) in CD4(+) T cells is less well understood. In this study, we analyze the expression and effects of Eomes in mouse CD4(+) T lymphocytes. We find that Eomes is readily expressed in activated CD4(+) Th1 T cells in vivo. Eomes(+) CD4(+) T cells accumulated in old mice, under lymphopenic conditions in a T cell transfer model of colitis, and upon oral Ag administration. However, despite its expression, genetic deletion of Eomes in CD4(+) T cells did not impact on IFN-γ production nor increase Th2 or Th17 responses. In contrast, Eomes deficiency favored the accumulation of Foxp3(+) cells in old mice, after in vivo differentiation of Eomes-deficient naive CD4(+) T cells, and in response to oral Ag in a cell-intrinsic way. Enforced Eomes expression during in vitro regulatory T cell induction also reduced Foxp3 transcription. Likewise, bystander Eomes-deficient CD4(+) T cells were more efficient at protecting from experimental autoimmune encephalitis compared with wild-type CD4(+) T cells. This enhanced capacity of Eomes-deficient CD4(+) T cells to inhibit EAE in trans was associated with an enhanced frequency of Foxp3(+) cells. Our data identify a novel role for Eomes in CD4(+) T cells and indicate that Eomes expression may act by limiting Foxp3 induction, which may contribute to the association of EOMES to susceptibility to multiple sclerosis.

Effects of Dietary Plant Sterols and Stanol Esters with Low- and High-Fat Diets in Chronic and Acute Models for Experimental Colitis.

In this study, we evaluated the effects of dietary plant sterols and stanols as their fatty acid esters on the development of experimental colitis. The effects were studied both in high- and low-fat diet conditions in two models, one acute and another chronic model of experimental colitis that resembles gene expression in human inflammatory bowel disease (IBD). In the first experiments in the high fat diet (HFD), we did not observe a beneficial effect of the addition of plant sterols and stanols on the development of acute dextran sulphate sodium (DSS) colitis. In the chronic CD4CD45RB T cell transfer colitis model, we mainly observed an effect of the presence of high fat on the development of colitis. In this HFD condition, the presence of plant sterol or stanol did not result in any additional effect. In the second experiments with low fat, we could clearly observe a beneficial effect of the addition of plant sterols on colitis parameters in the T cell transfer model, but not in the DSS model. This positive effect was related to the gender of the mice and on Treg presence in the colon. This suggests that especially dietary plant sterol esters may improve intestinal inflammation in a T cell dependent manner.

Fc Receptor-mediated Effector Function Contributes to the Therapeutic Response of Anti-TNF Monoclonal Antibodies in a Mouse Model of Inflammatory Bowel Disease.

Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease. We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model. The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis. These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies.

Ghrelin receptor modulates T helper cells during intestinal inflammation.

The orexigenic peptide ghrelin has anti-inflammatory properties in colitis, however, the mechanism of action and the immune cells targeted remain still to be elucidated. Here, we assessed the possible effect of ghrelin on T helper (Th) cells in a T cell transfer model of chronic colitis. Disease was induced in the recombination activating gene 1 knockout mice (Rag1(-/-) ) by adoptive transfer of naïve Th cells from ghrelin receptor knockout mice (GRLN-R(-/-) ) or littermate wild-type (WT) mice. The course and severity of colitis was assessed by monitoring body weight, diarrhea score, histological analysis, gene expression, and flow cytometry analysis. The possible effects of ghrelin on Th cell proliferation, polarization, and apoptosis was examined invitro. Our data showed that Rag1(-/-) mice injected with GRLN-R(-/-) Th cells displayed increased severity of colitis compared to mice injected with WT Th cells. In addition, Rag1(-/-) mice injected with GRLN-R(-/-) Th cells had significantly higher intestinal inflammation and increased accumulation of Th1 and Th17 cells in the colon. In vitro, ghrelin directly affected proliferation of Th cells and induced apoptosis whereas it did not influence Th cell polarization. Our observations suggest that ghrelin modulates Th effector cells in the gut controlling proliferation and inducing apoptosis. Our findings further support the use of ghrelin as a novel therapeutic option to treat intestinal inflammatory diseases.

Fructooligosaccharides exert intestinal anti-inflammatory activity in the CD4+ CD62L+ T cell transfer model of colitis in C57BL/6J mice.

Fructooligosaccharides (FOS) are used as functional foods due to their prebiotic effects. Intestinal anti-inflammatory activity has been established in most, but not all, studies in animal models of colitis, using mainly chemically induced inflammation. Our goal was to test the effect of FOS (degree of polymerization 2-8) in the chronic, lymphocyte-driven CD4+ CD62L+ T cell transfer model of colitis. Colitis was induced by transfer of CD4+ CD62L+ T cells to C57BL/6J Rag1(-/-) mice. FOS (75mgday(-1)) was administered by gavage as a post-treatment. Three groups were established: non-colitic (NC), colitic control (C, CD4+ CD62L+ transferred mice treated with vehicle) and colitic+FOS (C+FOS, similar but treated with FOS). Mice were killed after 13days. Treatment of mice with FOS ameliorated colitis, as evidenced by an increase in body weight, a lesser myeloperoxidase and alkaline phosphatase activities, a lower secretion of proinflammatory cytokines by mesenteric lymph node cells ex vivo (IFN-γ, IL-17, and TNF-α), and a higher colonic expression of occludin (C+FOS vs. C, p<0.05). Increased relative abundance of lactic acid bacteria was observed in FOS-treated mice (p<0.05). FOS exert intestinal anti-inflammatory activity in T lymphocyte-dependent colitis, suggesting it may be useful in the management of inflammatory bowel disease in appropriate conditions.

Foxp3+ regulatory T cells restrain pathogenic RORγt+ group 3 ILC-mediated colitis (MUC8P.728)

Abstract IL-10 receptor beta (IL-10Rb) is a common receptor chain for both IL-10 and IL-22. Since IL-10 is a protective cytokine that restrains inflammatory responses and IL-22 signaling is required for epithelial cell-dependent tissue repair, targeted deletion of Il10rb should greatly exacerbate inflammatory bowel disease (IBD). Unexpectedly, we found that Il10rb-/- mice are completely resistant to development of spontaneous colitis. In contrast, Il10-/- mice develop IBD by 12 weeks of age and harbor a large number of IL-22-producing RORgt+ innate lymphoid cells (ILCs). Using a CD45RBhi T cell transfer model, we found that induction of intestinal inflammation requires the presence of gut resident RORgt+ ILCs. The IL-22-producing ILCs are essential to perpetuate the disease process initiated by the T cells. Furthermore, Foxp3+ Tregs directly inhibit ILC expression of IL-23R and other tissue injury sensors, thereby suppressing innate cell-driven colitis. Our study has uncovered a novel immune network that maintains intestinal homeostasis.

The gut microbiota regulates K/BxN autoimmune arthritis independent of Th17 and IL-17 (BA6P.121)

Abstract The bacterial community that colonizes mucosal surfaces helps shape the development and function of the immune system. The effect of the microbiota on autoimmunity has important implications, although the mechanisms are not well understood. The K/BxN mouse model of rheumatoid arthritis is dependent on the microbiota, and particularly on segmented filamentous bacteria (SFB), for the autoimmune phenotype. Because SFB potently induce Th17 cell differentiation, previous studies have suggested Th17 cells as the link between microbiota and arthritis. In this study we sought to test directly the functional significance of Th17 and IL-17 in arthritis development by genetic approaches. We utilized mice deficient for either transcription factor RORγt (which controls the Th17 program), or the cytokine IL-17A (the major cytokine of the IL-17 family) in both cell transfer and spontaneous disease models. Unexpectedly, mice without Th17 or IL-17 were not protected from disease. Germinal center B and follicular helper T cell (Tfh) populations were normal, as were levels of serum IgG against self antigen. We found no evidence for compensation in production of the related cytokine IL-17F in IL-17A deficient mice. However, antibiotic treatment prevented disease in both IL-17 deficient and sufficient K/BxN mice, and resulted in defective Tfh and germinal center B cell differentiation. We conclude that the gut microbiota promotes autoimmune development by mechanisms independent of Th17 or IL-17.

Beyond innate immunity: the role of interferon regulatory factor 3 in CD4+ T cell function (IRM15P.462)

Abstract Interferon regulatory factor 3 (IRF3) is a transcription factor found in the cytoplasm of most nucleated cells. Upon pathogen recognition, IRF3 becomes activated and translocates to the nucleus where it mediates the production of type I interferon (IFN). Whereas IRF3 has been studied extensively in the context of type I IFN production and innate immunity, its role in adaptive immunity has remained largely unexplored. The objective of our research is to elucidate the role of IRF3 in CD4+ T cell function. Our findings demonstrate that IRF3-deficient CD4+ T cells show robust proliferation in response to TCR engagement, but are compromised in their ability to differentiate into specialized T helper cell subsets—most notably IL-17-producing Th17 cells. As such, CD4+ T cells lacking IRF3 expression fail to induce disease in the T cell transfer model of colitis. These studies reveal a novel, T cell-intrinsic function of IRF3 and extend its role beyond the innate immune response to pathogens.

Antigen specific tolerance and the prevention of autoimmune type 1 diabetes occurs through indirect antigen presentation and requires programmed death ligand 1 (THER5P.918)

Abstract Antigen-coupled ethylene carbodiimide-fixed cells is an attractive therapeutic platform for treating human patients with type 1 diabetes since these cells can prevent and reverse diabetes in the non-obese diabetic mouse model of diabetes. However, the mechanisms contributing to the induction of T cell tolerance in vivo following treatment are unclear. Therefore, we used the BDC2.5 T cell transfer model to study how p31-coupled cells inhibit T cell function in vivo. Our results demonstrate that indirect antigen presentation was critical for tolerance induction in vivo. We further demonstrate that PD-L1 on the host antigen presenting cells is required for tolerance induction. Importantly, using two-photon microscopy we show that BDC2.5 T cells arrest on CD11c+ dendritic cells during the induction of tolerance, suggesting that these cells are responsible for presenting coupled cell antigen and tolerance induction. To increase the feasibility of translating antigen coupled cells to the clinic, we demonstrate that whole blood preparations and frozen cells are sufficient for tolerance induction, a significant concern for donor coupled cell sources when considering translation to human patients. These data further our understanding of how antigen-coupled cells induce tolerance and how this tolerance protocol could be altered to increase clinical feasibility.

Selective deletion of estrogen receptor alpha in FoxP3+ CD4+ T cells influences peripheral T cell homeostasis (BA11P.139)

Abstract Being female confers a greater risk of developing an autoimmune disease than any other known genetic or environmental risk factor. Given that a loss of regulatory T cell (Treg) function contributes to autoimmunity and estrogen receptor alpha (ERα) promotes Tregs and suppresses experimental autoimmune encephalomyelitis (EAE), ERα-induced Tregs offers a new strategy to treat autoimmune disorders. The objective of these studies was to determine whether selective ablation of ERα in FoxP3+ Tregs dysregulates peripheral T cell homeostasis. To the end, mice harboring a floxed Esr1 gene (ERαf/f) were bred to mice expressing a YFP-Cre fusion protein driven by an IRES following foxp3 to obtain FoxP3+ Treg restricted deletion of Ers1. Thymic and peripheral CD4+ and CD8+ T cell frequency appeared unaffected with Esr1 ablation except for a sex-biased reduction in the total number of thymic and peripheral FoxP3+ Tregs and an increase in total number of splenocytes in ERαf/f FoxP3Cre mice compared to ERα+/+ FoxP3Cre mice. Spotaneous chronic inflammation and autoimmunity was undetected in ERαf/f FoxP3Cre mice. In a CD4+ T cell transfer model of experiment colitis, selective ablation of the ERα in FoxP3+ Tregs resulted in exacerbated inflammation compared to mice harboring ERα-sufficient Treg cells. Overall, these preliminary results suggest that FoxP3+ Treg-specific ERα is essential for the function of peripheral Tregs and immune homeostasis in a sex-biased manner.

Tregs expressing high Tl1a are proinflammatory and do not protect from T cell induced colitis. (CCR5P.204)

Abstract Patients with Crohn’s disease have increased mucosal expression of TL1A. TL1A co-stimulates T cells and can promote expansion of activated and regulatory T cells. Mice generated with sustained and elevated T cell expression of Tl1a (L-Tg) have increased Foxp3+ Treg cells yet develop worsened ileitis, proximal migration of colitis and intestinal fibrosis. The aim of this study was to investigate the role of Tl1a in the differentiation and function of Tregs.In the L-Tg mice, the CD4+ T cell population was comprised of GFPhigh and GFPlowcells that correlated with the level of Tl1a expression.Foxp3 expression and suppression function was significantly lower in the Tregs expressing high Tl1a. Indeed, addition of high levels of exogenous rTl1a under iTreg differentiation conditions advances the expansion of IL-13 producing effector T cells. The percentage of GFPhigh Foxp3+ Tregs co-expressing GATA3 were significantly higher explaining the possible Th2 polarization of Tregs expressing high Tl1a. Additionally, in the T cell transfer model of colitis, RAG mice cotransferred with GFPlowFoxp3+ Tregs were better protected from inflammation and weight loss compared to mice cotransferred with GFPhighFoxp3+ T cells.These data reveal a novel role for TL1A in regulating T cell mediated immune responses such that the level of Tl1a (high v. low) can differentially influence the expansion of effector and Treg cells leading to either a pro-inflammatory or protective mucosal environment.

Tumor progression locus 2 inhibits regulatory T cell development and immunosuppressive functions (LYM8P.640)

Abstract Regulatory T cells (Tregs) are a specialized subset of immunosuppressive T cells that function to maintain peripheral tolerance. We previously demonstrated that the serine-threonine kinase, tumor progression locus 2 (Tpl2, also designated Cot/Map3k8), regulates TCR signaling and inflammatory cytokine secretion in CD4+ T cells. Herein, we demonstrate that Tpl2 is preferentially expressed by Tregs and regulates their development and functions. Tpl2-/- mice exhibited increased proportions of thymic natural Tregs (nTregs) as well as peripheral splenic Tregs, in vivo. Enhanced Treg development was due to a T cell autonomous defect, since Treg development from tpl2-/- naïve T cells cultured in vitro was similarly enhanced, and peripheral Treg proportions were also increased within the tpl2-/- donor compartment of mixed bone marrow chimeras. This defect depended upon TCR signal strength and was enhanced at low antigen concentrations. Importantly, tpl2-/- Tregs had increased expression of the immunosupressive cytokines, IL-10 and IL-35, and provided better protection than wild-type Tregs in vivo in a T cell transfer model of colitis. These results demonstrate that Tpl2 has an important physiological role in limiting Foxp3 expression, Treg development and functions. Therefore, Tpl2 inhibition could potentially be used to deviate pathological immune responses in a variety of autoimmune diseases towards a protective, tolerogenic response through preferential Treg induction.

Tpl2 differentially regulates IFNγ and IL-17 production by CD4+ T cells in a T cell transfer model of colitis. (LYM8P.636)

Abstract Autoimmune diseases are approaching epidemic levels, estimated to affect 5-8% of the population. A number of autoimmune diseases are driven by autoreactive T cells, specifically T helper 1 (Th1) and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a T cell transfer model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2-/- naïve CD4+ T cells were impaired in IL-17A secretion under traditional Th17 inducing conditions of IL-6+TGF-β. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In vivo, transfer of Tpl2-/- T cells resulted in reduced proportions of CD4+ T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that Th17 cells alternatively induced by IL-6+IL-23 was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1-dependent responses, including Th1-mediated immunopathology during autoimmunity.

Tumor progression locus 2 differentially regulates IFNγ and IL-17 production by effector CD4+ T cells in a T cell transfer model of colitis.

Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2−/− naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2−/− T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.

First in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice

In this study three different dry powder vaccine formulations containing the model antigen ovalbumin were evaluated for their immune effects after nasal administration to C57Bl/6 mice in an adoptive cell transfer model. The formulations were chitosan nanoparticles in a mannitol matrix, chitosan microparticles and agarose nanoparticles in a mannitol matrix. Dry powder administration to mice was well tolerated and did not result in any adverse reactions. No translocation of the dry powder formulations to the lung could be detected. The local cellular immune response in the cervical lymph nodes was modest and only for the chitosan microparticles and the agarose nanoparticles was there a significant difference compared to s.c. injection of ovalbumin in alum. No humoral response could be measured after nasal administration. The results provide some evidence that nasal administration of dry powder formulations can stimulate an immune response, but the response was modest. This is probably due to a low antigen dose and low immunogenicity of the formulations. Further studies will aim at enhancing the antigen load and improving adjuvant activity.

Regulation of T cell lineage commitment by SMAR1 during inflammatory & autoimmune diseases.

Background & objectives:CD4+ T cells are involved in abnormal inflammatory responses causing adverse effects to the body. Th17 cells play a major role in immune disorders and the exact mechanism by which CD4+ T cells regulate its effector Th1 and Th17 phenotype at chromatin level is not clearly understood. This study was aimed to understand the role of matrix associated region (MAR) binding protein SMAR1 (scaffold/matrix attachment region binding protein 1) in T cell differentiation during inflammatory and autoimmune condition using SMAR1 transgenic mice as model.Methods:Wild type (C57BL/6J) and SMAR1 transgenic mice were used for isolation of T cells and further identification of different T cell lineages, along with histological analysis. Further, we studied autoimmune and inflammatory diseases using chemically induced and T cell transfer model of colitis and rheumatoid arthritis to better understand the role of SMAR1 in immune responses.Results:SMAR1 transgenic mice were resistant to dextran sodium sulphate (DSS) induced colitis with decreased expression of Th1 and Th17 specific cytokines. Overexpression of SMAR1 repressed Th17 response by negatively regulating RORγt and IL-17 expression. Downregulation of SMAR1 upregulated signal transducer and activator of transcription 3 (pSTAT3) and IL-17 expression that caused generation of more proinflammatory Th1 and Th17 cells leading to inflammation and disease.Interpretation & conclusions: Our results show an important role of SMAR1 in regulating CD4+ T cell differentiation during inflammatory disorders via regulation of both Th1 and Th17 signaling pathways. This study reveals a critical role of SMAR1 in maintaining the proinflammatory immune responses by repressing Th1 and Th17 cell function and it gives the novel insight into immune regulatory mechanisms.

P-165 Colonization of Lymphopenic Mice with Dysbiotic Microflora Markedly Increases the Severity and Incidence of Chronic Colitis Following T Cell Transfer

It is well-known that intestinal microbiota are required for the induction of chronic gut inflammation in the CD4+CD45RBhigh→ RAG-1-/- mouse model of chronic colitis. Following our relocation from LSU Health Sciences Center (LSUSHC) to Texas Tech University Health Sciences Center (TTUHSC), we observed a significant reduction in the incidence and severity of disease (30-40%) compared to our previous 15 year history of ∼85% at LSUHSC. The objectives of this study were to: a) ascertain whether differences in the gut microbiota may account for the differences in disease incidence and b) determine whether colonization of RAG-1-/- (RAG ko) mice with feces obtained from colitic mice increases the incidence and severity of colitis in the T cell transfer model. DNA from freshly frozen feces was isolated using standard protocols and 16S rRNA sequencing was performed using the Roche 454 platform. For some studies, mice were colonized (via gastric gavage) with 40 mg donor feces 1 week prior to adoptive transfer of naïve (CD4+CD45RBhigh) T-cells. Microbiome analysis revealed marked shifts in the relative abundance of several major bacterial communities present in feces obtained from LSUHSC versus TTUHSC mice. For example, the relative abundance of Firmicutes and Bacteroidetes was ∼70 and 20%, respectively in healthy TTUHSC-RAG ko mice whereas these same 2 phyla represented 48 and 50%, respectively in healthy LSUHSC-RAG ko animals. Interestingly, the fecal microbiota from TTUHSC-RAG ko mice revealed significantly higher abundances of Proteobacteria, Verrucomicrobia and Tenericutes when compared to the microbiota obtained from healthy LSUHSC-RAG ko mice. Not surprisingly, induction of chronic colitis in LSUHSC-RAG ko mice via adoptive transfer of naïve T cells, produced a marked dysbiosis most notably characterized by an increased abundance of Verrucomicrobia together with corresponding and marked decrease in Bacteroidetes. Adoptive transfer of naive T cells into healthy TTUHSC-RAG ko mice that were first colonized with healthy LSUHSC-RAG ko feces induced mild-to-moderate colitis in ∼80% of the mice at 8 weeks post T cell transfer. In addition, we found that T cell transfer into TTUHSC-RAG ko mice that were first colonized with feces from colitic LSUHSC-RAG ko mice induced robust colitis in >90% of these recipients (called LSUHSCc→TTUHSC mice). Furthermore, T cell transfer into TTUHSC-RAG ko mice that had been colonized with feces from colitic LSUHCc→TTUHSC mice induced moderate-to-severe colitis in virtually all (>98%) of these recipients (called TTUHSCc→TTUHSC mice). Importantly, colonization of healthy wild type mice or TTUHSC-RAG ko mice (in the absence of T cell transfer) with colitic feces from TTUHSCc→TTUHSC mice did not induce disease over the 8 week observation period. We conclude that colonization of healthy RAG ko mice with dysbiotic microbiota obtained from colitic mice markedly increases the severity and incidence of chronic colitis in the T cell transfer model. Our data also suggest that the increase in severity and incidence of disease is not due to the presence of intestinal pathogens and can only be produced in mice with a dysregulated immune system (supported by grants from the DOD-W81XWH-11-1-0666 and NIH- RO1 DK 091269).

P-158 Understanding how GATA3 Expression by Tregs Prevents Pathogenic Tregs

Although the etiology of inflammatory bowel disease is still unclear, current evidence suggests that dysregulated immune responses contribute to the disease state. One common approach to current and future therapies is to target mucosal inflammation by interfering with immune pathways. Regulatory T cells (Treg) are known to play a critical role in maintaining homeostasis of the gastrointestinal (GI) tract. While Tregs are critical for maintenance of the GI tract, their dysregulation, either through reduction in number and or function, has been associated with immunopathology. Furthermore, there is mounting evidence that human Treg can aberrantly secrete pro-inflammatory cytokines in settings of autoimmunity. Thus, understanding the factors that stabilize Treg function and survival in highly inflamed settings remains an outstanding question of high clinical relevance. We previously demonstrated that the canonical Th2 transcription factor GATA3 plays an integral role in promoting Treg physiology during inflammation within the gastrointestinal tract, and specifically in regulating inflammation during colitis. GATA3 does this through the dual role of promoting Treg accumulation at the site of inflammation and limiting excessive plasticity of Treg, namely the expression of Th1 (Tbet and IFNg) and Th17 (RORgt and IL-17A) factors. Thus GATA3 plays a multifaceted role in controlling the fate of Treg within inflamed tissues by enhancing both their stability and function. The overall aim of this proposal is to assess the contribution of Treg acquisition of effector function to immunopathology during gastrointestinal inflammation and the mechanism by which GATA3 limits dysregulation of Treg during colitis. Our studies include infection-induced inflammation of the gastrointestinal tract and the CD45RB T cell transfer model of colitis. We now show that Tregs can contribute to disease in infection-induced inflammation. Mice with Tregs that can maintain GATA3 expression have reduced immunopathology. Further, our work reveals a potential role for GATA3 in promoting IL-10 production by Treg during chronic GI infection. Taken together our studies show that Treg dysregulation can cause immunopathology directly. Future studies will examine mechanistically how GATA3 prevents pathogenic Tregs.