Transfer Colitis Model Research Articles

94 A Covalent Inhibitor of ITK and RLK Inhibits Th1 and Th17 Cell Differentiation and Prevents Disease Manifestation in an Adoptive Transfer Model of Colitis

94 A Covalent Inhibitor of ITK and RLK Inhibits Th1 and Th17 Cell Differentiation and Prevents Disease Manifestation in an Adoptive Transfer Model of Colitis

Mo1759 Reciprocal IL-22 Expression Masks IL-17 Activities in T-Cell Transfer Colitis Model in IL-17A-/- Mice

Mo1759 Reciprocal IL-22 Expression Masks IL-17 Activities in T-Cell Transfer Colitis Model in IL-17A-/- Mice

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.

Targeting human CD2 by the monoclonal antibody CB.219 reduces intestinal inflammation in a humanized transfer colitis model

The cell adhesion molecule CD2 facilitates antigen-independent T-cell activation and CD2 deficiency or blockade reduces intestinal inflammation in murine models. We here aimed to evaluate the therapeutic potential of monoclonal antibodies (mAb) specific for human CD2 in colitis treatment. Transfer colitis induced by naïve CD4+ T cells expressing human CD2 was treated with anti-human CD2 mAb. The mAb CB.219 protected from severe colitis in a preventive treatment regimen, while therapeutic treatment ameliorated intestinal inflammation. Diminished intestinal tissue damage was paralleled by a profound suppression of lamina propria lymphocytes to produce pro-inflammatory cytokines and tumor necrosis factor α as well as the neutrophil chemoattractant CXC motif ligand 1 and the CC chemokine ligand 3. Furthermore, infiltration with macrophages and T cells was low. Thus, reduced intestinal inflammation in our humanized colitis model by targeting CD2 on T cells with the mAb CB.219 suggests a novel approach for colitis treatment.

PTPN2 controls differentiation of CD4⁺ T cells and limits intestinal inflammation and intestinal dysbiosis.

Loss-of-function variants within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with increased risk for Crohn's disease (CD). A disturbed regulation of T helper (Th) cell responses causing loss of tolerance against self- or commensal-derived antigens and an altered intestinal microbiota plays a pivotal role in CD pathogenesis. Loss of PTPN2 in the T-cell compartment causes enhanced induction of Th1 and Th17 cells, but impaired induction of regulatory T cells (Tregs) in several mouse colitis models, namely acute and chronic dextran sodium sulfate colitis, and T-cell transfer colitis models. This results in increased susceptibility to intestinal inflammation and intestinal dysbiosis which is comparable with that observed in CD patients. We detected inflammatory infiltrates in liver, kidney, and skin and elevated autoantibody levels indicating systemic loss of tolerance in PTPN2-deficient animals. CD patients featuring a loss-of-function PTPN2 variant exhibit enhanced Th1 and Th17 cell, but reduced Treg markers when compared with PTPN2 wild-type patients in serum and intestinal tissue samples. Our data demonstrate that dysfunction of PTPN2 results in aberrant T-cell differentiation and intestinal dysbiosis similar to those observed in human CD. Our findings indicate a novel and crucial role for PTPN2 in chronic intestinal inflammation.

PGRN protects against colitis progression in mice in an IL-10 and TNFR2 dependent manner.

This study was aimed to determine the role and regulation of progranulin (PGRN) in the pathogenesis of inflammatory bowel diseases (IBD). Dextran sulfate sodium (DSS)−, picrylsulfonic acid (TNBS)-induced, bone marrow chimera and CD4+CD45Rbhi T cell transfer colitis model were established and analyzed in wild-type and several genetically-modified mice, including PGRN, IL-10 and TNFR2 deficient mice. Elevated levels of PGRN were found in colitis samples from human IBD patients and mouse colitis models in comparison to the corresponding controls. PGRN-deficient mice became highly susceptible to DSS- and TNBS-induced colitis, whereas recombinant PGRN ameliorated the pathology and reduced the histological score in both DSS and TNBS colitis models. In addition, hematopoietic-derived PGRN was critical for protection against DSS-induced colitis, and lack of PGRN signaling in CD4+ T cells also exacerbated experimental colitis. PGRN-mediated protective effect in colitis was compromised in the absence of IL-10 signaling. In addition, PGRN's effect was also largely lost in the TNFR2-deficient colitis model. Collectively, these findings not only provide the new insight into PGRN's anti-inflammatory action in vivo, but may also present PGRN and its derivatives as novel biological agent for treating IBD.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Active hexose correlated compound exerts therapeutic effects in lymphocyte driven colitis.

Active hexose correlated compound (AHCC) is a commercial extract of Basidiomycetes fungi enriched in oligosaccharides that is used as a human nutritional supplement for various purposes in humans. Our aim was to study the anti-inflammatory effect of AHCC in the CD4+ CD62L(+) T cell transfer model of colitis, considered one of the closest to the human disease. Colitis was induced by transfer of CD4(+) CD62L(+) T cells to recombination activating gene 1(-/-) mice. AHCC (75 mg/d) was administered by gavage as a post-treatment. Three groups were established: noncolitic, colitic (CD4(+) CD62L(+) transferred mice treated with vehicle), and AHCC (colitic treated with AHCC). AHCC improved colitis, as evidenced by a 24% lower colonic myeloperoxidase and a 21% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory genes assessed by RT-qPCR was observed, particularly TNF-α and IL-1β. Ex vivo mesenteric lymph node cells obtained from AHCC treated mice exhibited a fully normalized production of IL-6, IL-17, and IL-10 (p < 0.05). Also, AHCC treated mice exhibited decreased STAT4 and IκB-α phosphorylation in splenic CD4(+) cells. Our data provide validation of AHCC colonic anti-inflammatory activity in a chronic, T cell driven model of inflammatory bowel disease.

Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83+ T cells. In contrast, CD83− T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83− T cells were not able to upregulate CD83 despite activation. Furthermore, CD83+ T cells suppressed the proliferation and inflammatory cytokine release of CD83− T cells in vitro. Strikingly, stimulated CD83+ T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83+ T cells were able to suppress colitis symptoms while CD83− T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83+ T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations.

Mo1720 Relevance of Poly(ADP-ribose) Polymerase 1 (PARP1) in Experimental Colitis

Background: Poly(ADP-ribose) polymerases (PARPs) constitute a family of 18 cell signaling enzymes which are involved in the regulation of DNA-binding proteins and DNA repair. These enzymes catalyze the transfer of ADP-ribose units from NAD+ to a number of acceptor proteins and can also interact directly with their target proteins. Among the PARPs, PARP1 is the most abundant isoform and regulates the expression of diverse proinflammatory mediators, through its interaction with and modification of numerous transcription factors. Recent observations (including our group's) suggest that PARP1 is a cofactor for NF-κBdependent gene expression. Several studies in PARP1 KO mice have shown decreased expression of cytokines, chemokines and adhesion molecules, as well as reduced immune tissue infiltration in various models of inflammation, including streptomycin-induced diabetes and LPS-induced septic shock. Interestingly, in vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates the pathophysiological changes of experimental colitis. However, these studies failed to characterize the mechanism by which PARP1 inhibition is protective in colitis or the cell type in which Parp1 deletion has the most significant impact in IBD. Aim: Considering the essential role of PARP1 in the regulation of inflammation in IBD, we evaluated the effect of Parp1 deletion in T cellmediated colitis. Methods: The effect of Parp1 deficiency was evaluated in different models of colitis: (1) Adoptive T cell transfer: WT or Parp1-deficient CD4+CD45RBHigh T-cells were transferred into Rag2-/mice, or Rag2-/x Parp1-/double knockout (DKO) mice and (2) DSS-induced colitis in WT, Parp1-/-, Rag2-/-, and Rag2-/x Parp1-/DKO mice. In all models, we evaluated body weight loss, colonic morphology and expression of major pro-inflammatory cytokines. Results: In comparison to WT mice, Parp1-/were protected from the effects of DSS, with significantly lower weight loss, and greatly reduced proinflammatory cytokine expression. However, Parp1 deficiency in T cells was not protective in the CD4+CD45RBHigh T cell transfer model. Unexpectedly, Rag2-/x Parp1-/DKO showed only marginal improvement in both the WT CD4+CD45RBHigh T cell transfer and DSS models. Conclusions: Our preliminary data obtained with the adoptive T-cell transfer model of colitis indicate that Parp1 is dispensable in Naive and effector T-cells. While Parp1-/mice are protected from colitis resulting from DSS-induced epithelial damage, the lack of significant protection in the T cell transfer or DSS models of innate colitis in Rag2-/x Parp1-/DKO mice suggests that the pathogenic role of Parp1 in colitis may involve modulating interactions of more than one cell type, presumably innate immune cells (including colonic epithelial cells) and regulatory T cells.

Mo1721 CD39 Is a Surface Marker for Type 1 CD8+ T-Cells in Patients With Crohn's Disease

Background: Poly(ADP-ribose) polymerases (PARPs) constitute a family of 18 cell signaling enzymes which are involved in the regulation of DNA-binding proteins and DNA repair. These enzymes catalyze the transfer of ADP-ribose units from NAD+ to a number of acceptor proteins and can also interact directly with their target proteins. Among the PARPs, PARP1 is the most abundant isoform and regulates the expression of diverse proinflammatory mediators, through its interaction with and modification of numerous transcription factors. Recent observations (including our group's) suggest that PARP1 is a cofactor for NF-κBdependent gene expression. Several studies in PARP1 KO mice have shown decreased expression of cytokines, chemokines and adhesion molecules, as well as reduced immune tissue infiltration in various models of inflammation, including streptomycin-induced diabetes and LPS-induced septic shock. Interestingly, in vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates the pathophysiological changes of experimental colitis. However, these studies failed to characterize the mechanism by which PARP1 inhibition is protective in colitis or the cell type in which Parp1 deletion has the most significant impact in IBD. Aim: Considering the essential role of PARP1 in the regulation of inflammation in IBD, we evaluated the effect of Parp1 deletion in T cellmediated colitis. Methods: The effect of Parp1 deficiency was evaluated in different models of colitis: (1) Adoptive T cell transfer: WT or Parp1-deficient CD4+CD45RBHigh T-cells were transferred into Rag2-/mice, or Rag2-/x Parp1-/double knockout (DKO) mice and (2) DSS-induced colitis in WT, Parp1-/-, Rag2-/-, and Rag2-/x Parp1-/DKO mice. In all models, we evaluated body weight loss, colonic morphology and expression of major pro-inflammatory cytokines. Results: In comparison to WT mice, Parp1-/were protected from the effects of DSS, with significantly lower weight loss, and greatly reduced proinflammatory cytokine expression. However, Parp1 deficiency in T cells was not protective in the CD4+CD45RBHigh T cell transfer model. Unexpectedly, Rag2-/x Parp1-/DKO showed only marginal improvement in both the WT CD4+CD45RBHigh T cell transfer and DSS models. Conclusions: Our preliminary data obtained with the adoptive T-cell transfer model of colitis indicate that Parp1 is dispensable in Naive and effector T-cells. While Parp1-/mice are protected from colitis resulting from DSS-induced epithelial damage, the lack of significant protection in the T cell transfer or DSS models of innate colitis in Rag2-/x Parp1-/DKO mice suggests that the pathogenic role of Parp1 in colitis may involve modulating interactions of more than one cell type, presumably innate immune cells (including colonic epithelial cells) and regulatory T cells.

The role of adenosine in helper T cell differentiation and function (840.1)

Background and Aims: Extracellular signaling molecules, such as anti‐inflammatory adenosine, play a major role in restraining immune response and protecting tissue from inflammatory damage. Adenosine is generated from the ATP through the action of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) that sequentially dephosphorylates ATP to ADP to 5’‐AMP, which is then catabolized to adenosine by ecto‐5'‐nucleotidase (CD73, Nt5e). The aim of this study was to investigate the role of extracellular adenosine in control of helper T cell (Th) cell differentiation and cellular function. Results: Adenosine levels were controlled with a pharmacological approach using the non‐hydrolysable adenosine analog, NECA. Naïve CD4+ T cells were differentiated in vitro in the presence or absence of NECA in selective culture conditions. The presence of NECA suppressed the production of IL‐17a in cells isolated from Th17 conditions. We then explored a genetic approach to control adenosine levels in vivo, using an adoptive transfer colitis model. According to the model, the transfer of CD4+CD45RBhi cells, a subset that contains largely T effector (Teff) cells, will induce colitis when transferred to Rag1‐/‐ mice. Colitis can be ameliorated with the co‐transfer of CD4+CD45RBlo, a cell population with a primarily regulatory phenotype. T cells were isolated from Nt5e‐/‐ mice to investigate the function of Th cells compromised in the ability to produce adenosine. In our adoptive transfer studies, CD73 on both Treg and T effector proved to be necessary for Treg to prevent Teff‐induced colitis. Conclusions: Adenosine signaling has the ability to restrain Th17 response during differentiation through the direct reduction of the production of the pro‐inflammatory cytokine IL‐17a. The ability to produce adenosine is essential for effective Treg activity and compromising the adenosine pool changes the differentiation patterns of Th cells causing excessive inflammatory activation and tissue damage.Grant Funding Source: Supported By: T32 Gastrointestinal Training Grant

Role of Regulatory B Cells in Chronic Intestinal Inflammation

The role of regulatory B cells (Bregs) producing interleukin (IL)-10 in the pathogenesis of inflammatory bowel diseases remains unknown. We investigated IL-10 production in B cells from patients with inflammatory bowel diseases and immunoregulatory functions of Bregs in experimental colitis mouse models. CpG DNA-induced IL-10 production in peripheral blood B cells isolated from patients with inflammatory bowel diseases and control subjects was examined. CD19 and CD1d were used for evaluating possible cell surface markers of Bregs. Colitis models of severe combined immunodeficiency mice were established by adoptive transfer of whole CD4 T cells or regulatory T cell (Treg)-depleted T cells (CD4CD25) isolated from SAMP1/Yit mice and the function of Bregs in intestinal inflammation was elucidated by evaluating the effects of cotransfer of whole or Breg-depleted B cells. CpG DNA-induced IL-10 production was significantly decreased in B cells from patients with Crohn's disease (CD), as compared with those from healthy controls, whereas Bregs were found to be enriched in a population of CD19 and CD1d B cells isolated from both human and mouse samples. The severity of intestinal inflammation was significantly increased in the Breg-depleted mice, with similar results also found in adoptive transfer colitis model mice even after Treg depletion. Our findings show that Bregs, characterized by the cell surface markers CD19 and CD1d, significantly reduced experimental colitis regardless of the presence or absence of Tregs. These results suggest that a deficiency or decrease of Bregs function exacerbates intestinal inflammation, which may be associated with the pathogenesis of CD.

Enteroantigen (eAg)-binding B lymphocytes in the mouse - phenotype, distribution, function and eAg-specific antibody secretion

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co-transplanted with Treg-depleted (CD4+CD25-) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg-presenting cells than unfractionated splenocytes or eAg- B cells in causing proliferation of eAg-specific T cells. In comparison with eAg- B cells, eAg+ B cells secrete a significant amount of IL-10 in vitro, suggesting an anti-inflammatory potential. Compared with wild-type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme-specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19(+) B cells by eAg and hybridization with hypoxanthine-aminopterin-thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg-specific antibodies.

P-204 Anti-inflammatory Effects of the Chinese Herbal Formula FAHF-2 in Experimental and Human IBD

Crohn’s disease (CD) is a chronic inflammatory disease with increasing incidence in children. Current medications have potentially serious side effects, hence increasing interest in alternative therapies. TNF-α and other pro-inflammatory cytokines play important roles in the pathogenesis of CD. FAHF-2 is derived from Wu-Mei-Wan used in China to treat colitis. We investigated its potential anti-inflammatory effects. FAHF-2 efficacy was tested in vivo in the CD45Rbhi RAG1-/- transfer colitis model. Weight loss, colonic histology and cytokine production were assessed. Peripheral blood mononuclear cells (PBMCs) and colonic biopsies were obtained from children newly diagnosed with CD and controls and cultured with or without FAHF-2. Cytokine levels were measured by ELISA and immunoassay. The effect of FAHF-2 on TNF-α producing cells was determined by flow cytometry. NFκB signaling was investigated in human lamina propria mononuclear cells upon FAHF-2 treatment by In-Cell Western. FAHF-2 treated mice had decreased weight loss (P < 0.05), improved histology (P < 0.05) and reduced TNF-α (P < 0.01), IL-17 (P < 0.05), IL-6 (P < 0.01), and IFN-γ (P < 0.05) production from MLNs. Treated human PBMCs produced less TNF-α (P < 0.001), IFN-γ (P < 0.05) and IL-12 (P < 0.01). FAHF-2 reduced the TNF-α producing monocytes (P < 0.05) and T-cells (P < 0.05) in PBMCs. Inflamed CD biopsies produced less TNF-α (P < 0.01), IL-1β (P < 0.05), IL-6 (P < 0.001), and IL-17 (P < 0.001). These effects are due to decreased NFκB activation. FAHF-2 was effective in halting progression of colitis in a murine model. In human samples, FAHF-2 inhibited both adaptive and innate immune pro-inflammatory cytokine responses in PBMCs and inflamed CD mucosa due in part to blockage of NFκB activation. This study shows that FAHF-2 has potential as a novel treatment for CD.

P-207 The Role of IL-17C and IL-17RE in the Development of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a complex polygenic disorder causing a chronic relapsing inflammation and thought to result from a dysregulated and aberrant immune response to intestinal flora in a context of genetic predisposition. Recently, human IL17REL, a member of interleukin-17 receptor family, was identified through a genome-wide association study for ulcerative colitis. However, it is unknown how IL-17REL is involved in the development of ulcerative colitis. On the basis of the substantial homology between human IL-17REL and the extracellular domains of IL-17RE, it is likely that IL-17REL binds to the ligand of IL-17RE. We recently demonstrated IL-17RE is induced in a subset of CD4 helper T cells, Th17 cells, and is a receptor for a cytokine IL-17C. IL-17C-IL-17RE signaling plays a critical role to promote inflammation in chronic autoimmune disease. In addition, we reported IL-17C is crucial for the regulation of an acute experimental colitis by controlling the expression of the tight junction molecule occludin by colonic epithelial cells. Therefore, we hypothesize during chronic intestinal inflammation, induction of IL-17C in the inflamed intestine recruits Th17 cells via IL-17RE and IL-17C-IL-17RE signaling modulates Th17 cell function and play a pathogenic role in mediating inflammation. First, we found out that the expression of IL-17C and IL-17RE is increased in colon and small intestine of mice during chronic colitis. IL-17C expression was found in colonic epithelial cells and it was reduced upon antibiotic treatment, suggesting IL-17C expression is maintained by microbiota in the colon. IL-17RE expression, on the other hand, was not only found in Th17 cells but also in regulatory T cells isolated from colon and small intestine. Therefore, we adopted a CD4 transfer colitis model, where Il17re deficient CD45RBhi CD4+ cells or Il17re deficient CD25+CD4+ cells were transferred to Rag deficient mice to address the biological role of IL-17RE in Th17 cells and regulatory T cells. This study is directed to understand the contribution of novel cytokine-receptor pair, IL-17C-IL-17RE, in the pathogenesis of chronic inflammation in colon. Furthermore, this study will help us to understand complex nature of IBD development and provide a new platform to design therapy to block proinflammatory cytokine.

Induced and thymus‐derived Foxp3+ regulatory T cells share a common niche

Foxp3⁺ regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymus-derived, tTreg cells) and in the periphery, where they are converted from conventional CD4⁺ T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4⁺ T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3⁺ Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1⁺ tTreg cells outcompeted Nrp1⁻ iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.

Miltefosine Suppresses Inflammation in a Mouse Model of Inflammatory Bowel Disease

The repertoire of immunomodulators that can be used for the treatment of inflammatory bowel disease is limited. The use of these drugs is further restricted by the occurrence of side effects in a proportion of patients. Miltefosine (hexadecylphosphocholine) is a lipid drug developed in the 1980s for the treatment of cancer but is nowadays best known for its application in the oral treatment of leishmaniasis. Although the exact mechanism of action of miltefosine has yet to be elucidated, the drug has previously been shown to inhibit phospholipases and protein kinase C, both key components of proproliferative signal transduction in T cells. Stimulated peripheral blood lymphocyte were treated with miltefosine, and proliferation was measured. We use the CD45RB T-cell transfer colitis model to investigate the effect of miltefosine treatment on intestinal inflammation. Effects on the severity of colitis were studied by histochemical and immunohistochemical staining, and cytokine levels were determined using a cytokine bead array. Miltefosine inhibited T-cell proliferation in vitro. In the transfer model, miltefosine significantly ameliorated the severity of colitis as measured by clinical, (immuno)histochemical, and biochemical parameters. Miltefosine inhibits T-cell proliferation and effectively reduces inflammation in the T-cell transfer model. The drug may therefore be a candidate immunomodulator for inflammatory bowel disease.

IL-23R+ innate lymphoid cells induce colitis via interleukin-22-dependent mechanism.

Polymorphisms of interleukin (IL)-23R and signaling components are associated with several autoimmune diseases, including inflammatory bowel diseases (IBD). Similar to T helper type 17 (Th17) lineage, type 3 innate lymphoid cells (ILCs) express retinoic acid-related orphan receptor γt (Rorγt) and IL-23R and hence, produce Th17-type cytokines. Recent reports implicated type 3 ILCs in IBD; however, how IL-23R signaling in these cells contributes to pathogenesis is unknown. IL-22, produced in copious amounts by type 3 ILCs, was reported to have both beneficial and pathogenic effects in adaptive, yet only a protective role in innate colitis models. Herein, by employing chronic CD45RB(high) CD4(+) T-cell transfer and anti-CD40 antibody-induced acute innate colitis models in Rag1(-/-) mice, we demonstrated opposite roles for IL-23R in colitogenesis: in the former a protective, and in the latter a pathogenic role. Furthermore, we show that IL-23R signaling promotes innate colitis via IL-22 as neutralization of IL-22 protected mice from colitis and adding back of IL-22 to IL-23R-deficient animals restored the disease. Collectively, our results reveal that similar to its controversial role during chronic or adaptive colitis, IL-22 may also have opposite roles in innate colitis pathogenesis in a context and insult-dependent manner.

Epigenetic licensing of Th17 and Treg cell differentiation (P1197)

Abstract Naïve CD4+ T helper (Th) cells acquire a range of fates depending on the cytokine milieu and anatomical location. For example, IL-17 producing Th17 and Foxp3+ regulatory (Treg) cells are dependent upon TGFβ1 and are critical for intestinal homeostasis. G9a is a histone lysine methyltransferase with di-methyl activity towards histone H3 lysine 9. H3K9me2 is a highly conserved histone modification and is commonly linked to transcriptional repression. In this study we examine the role of G9a in the control of Th17 and Treg cell differentiation. Using chromatin immunoprecipitation (ChIP) we found that there is a concomitant loss H3K9me2 and increase in H3K9Ac (an activating histone modification) at lineage specific genes of WT Th17 and Treg cells compared to naive. Meanwhile, naïve G9a-/- T cells have low levels H3K9me2 that results in enhanced sensitivity to TGFβ1 and an increase in the differentiation of both Th17 and Treg cells in vitro. Using the T cell transfer colitis model, we found that transfer of G9a-deficient T cells fails to cause the weight loss and colonic inflammation typical of the model. Furthermore, T effector-cell production of IFNγ was significantly reduced and IL-17 was enhanced when G9a-/- T cells were transferred. In addition, compared to WT controls, a higher proportion of G9a-deficient T cells express Foxp3. We conclude that G9a-dependent H3K9me2 is a homeostatic epigenetic checkpoint that controls the magnitude of Th17 and Treg cell responses.