T-cell Transfer Model Of Colitis Research Articles

Protective Role of β-arrestin2 in Colitis Through Modulation of T-cell Activation.

β-arrestin2 (β-arr2), identified as a scaffolding protein in G-protein-coupled receptor desensitization, is a negative regulator of inflammation in polymicrobial sepsis. In this study, we wanted to investigate the role of β-arr2 in intestinal inflammation, a site of persistent microbial stimulation. In the absence of β-arr2, mice exhibited greater extent of mucosal inflammation determined by cellular infiltration and expression of inflammatory mediators even under homeostatic conditions. Furthermore, β-arr2-deficient mice were more susceptible to dextran sulfate sodium-induced colitis as demonstrated by greater body weight loss, higher disease activity index, and shortened colon as compared with wild-type mice. We also show that T cells from β-arr2 knockout mice exhibit altered activation status under both basal and colitic conditions, implicating their involvement in disease induction. Further assessment of the role of β-arr2 in intrinsic T-cell differentiation confirmed its importance in T-cell polarization. Using the T-cell transfer model of colitis, we demonstrate that T-cell-specific β-arr2 is important in limiting colitic inflammation; however, it plays a paradoxical role in concurrent systemic wasting disease. Together, our study highlights a critical negative regulatory role of β-arr2 in intestinal inflammation and demonstrates a distinct role of T-cell-specific β-arr2 in systemic wasting disease.

Heightened Expression of CD39 by Regulatory T Lymphocytes Is Associated with Therapeutic Remission in Inflammatory Bowel Disease

To evaluate whether changes in expression of CD39 by regulatory T lymphocytes (Treg) impact treatment response in inflammatory bowel disease. To then define the biological role of expression of CD39 on Treg in an animal model of colitis. A prospective study of consecutive patients commencing anti-tumor necrosis factor therapy with infliximab (IFX) or adalimumab (ADA), who were then followed for 12 months. Treatment responses were defined both symptomatically and by endoscopy showing mucosal healing. Peripheral blood Tregs were quantified by flow cytometry. Functional importance of CD39 expression by Treg was determined in an adoptive T-cell transfer model of colitis. Forty-seven patients (ulcerative colitis, n = 22; Crohn's disease, n = 25) were recruited; 16 patients were complete responders and 13 nonresponders to anti-tumor necrosis factor. CD39 expression by Treg was lower in active inflammatory bowel disease and increased significantly after treatment in responders (CD39Treg/total Treg; 8% at baseline to 22.5% at late time point, P < 0.001). Responders were more likely to have therapeutic drug levels and in multivariate analysis therapeutic drug levels were associated with higher expression of CD39 by FoxP3 Treg and lower frequencies of interleukin 17A expressing cells. Tregs with genetic deletion of CD39 exhibit decrements in potential to suppress intestinal inflammation in a murine (CD45RB) T-cell transfer model of colitis in vivo, when compared with wild-type Treg. Increased expression of CD39 by peripheral blood Treg is observed in the setting of clinical and endoscopic remission in inflammatory bowel disease. Deficiency of CD39 expression by Treg can be linked to inability to suppress experimental colitis.

Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.

Effector CD4+CD25- T cells from Stearoyl CoA Desaturase deficient mice drive chronic colitis in RAG-1KO mice (MUC5P.761)

Abstract Fatty acids in lipid bilayer substantially influence membrane fluidity and thus dictate cellular functions. Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme that assists in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayer. Accordingly SCD-1KO mice display substantially reduced oleate in cell membranes. We investigated the colitogenic capacity of effector T-cells from SCD-1KO mice and their WT littermates in adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age and gender matched WT and SCD-1KO mice were isolated by cell sorting using magnetic beads and flow cytometry. RAG-1KO (n=5) were administered 5.0x105 CD4+CD25- cells/mouse intraperitoneally and monitored for body weights and colitis development. RAG-1KO mice which received CD4+CD25- cells from SCD-1KO mice displayed colomegaly, splenomegaly, elevated colonic, systemic lipocalin-2, a general inflammatory marker and hallmarks of colitis at histologic level. Interestingly, colonic TNFα and IL-10 gene transcripts were significantly reduced in SCD-1KO CD4+CD25- recipients whereas the opposite is true for iNOS and lipocalin-2. Further, systemic sIL-1Ra was significantly reduced in SCD-1KO CD4+CD25- recipients when compared to WT T cell recipients. Our results demonstrate that effector T-cells from oleate poor environment attain colitogenic properties and may be associated with increased immunoreactivity to gut microbiotal antigens.

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

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.

TH17 Cell Induction and Effects of IL-17A and IL-17F Blockade in Experimental Colitis

T helper (TH) 17 cells are believed to play a pivotal role in development of inflammatory bowel disease, and their contribution to intestinal inflammation has been studied in various models of colitis. TH17 cells produce a range of cytokines, some of which are potential targets for immunotherapy. However, blockade of IL-17A alone with secukinumab was not effective in Crohn's disease. In this regard, the pathogenic impact of IL-17A versus IL-17F during intestinal inflammation is still unresolved. Development of IFN-γ-producing, IL-17A-producing, and IL-17F-producing CD4 T cells was analyzed in the CD4CD25 T-cell transfer model of colitis at varying degrees of colitis. The pathogenic roles of IL-17A and IL-17F were investigated by treating colitic mice with neutralizing antibodies against these 2 cytokines. We found that colitis development was associated with an increase in IL-17A-producing TH17 cells in spleen, mesenteric lymph nodes, and lamina propria. In contrast, the relative abundance of IFN-γ-producing TH1 cell was stable in all 3 organs during progression of colitis, and the frequency of IFN-γIL-17A double-positive cells declined in spleen and mesenteric lymph node but not in lamina propria. IL-17F was coexpressed in TH17 cells and IFN-γIL-17A double positive but not in TH1 cells and its expression inversely correlated with colitis development. In vivo neutralization of both IL-17A and IL-17F ameliorated colitis in particular at early administration, whereas neutralization of IL-17A or IL-17F alone was inefficient. TH17 cell development correlates with colitis progression, and concurrent neutralization of their cytokine products IL-17A and IL-17F ameliorates intestinal inflammation. These findings suggest combined IL-17A and IL-17F blockade as a potential strategy in inflammatory bowel disease therapy.

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.

Leukocyte Recruitment Alters Pathological Angiogenesis Gene Expression During DSS Colitis

Experimental models of colitis in mice have been extensively used to study molecular events that occur during inflammatory bowel disease (IBD) development. However, direct and comprehensive comparison of mouse and human inflamed colon tissue at the genome level remains largely unknown and to what extent such differences reflect changes associated with human IBD. Here we report comparative functional genomic analysis between experimental colitis models and pediatric IBD. Two sets of pediatric IBD microarray data (GEO accession number GSE9686 and GSE10616) and experimental colitis microarray data (DSS colitis model GEO number GSE22307 and T-cell transfer colitis model GEO number 27302) were used for comparative functional genome expression pattern analysis. All microarray data were obtained using Affymetrix GeneChip arrays providing the most comprehensive coverage of transcribed genomes of human or mouse. Microarray data were input into Genesifter software and normalized for comparison using the Robust Multichip Averaging (RMA) method. Threshold value for significant differences in gene expression was set at 2 fold and the Benjamini-Hochberg test used to diminish false discovery rates. Identified genes were uploaded into Ingenuity software for network analysis, and cREMaG (cis-Regulatory Elements in the Mammalian Genome) software for transcription factor/promoter analysis. Microarray analysis revealed that chitinase 3-like 1(cartilage glycoprotein-39, CHI3L1), Cysteine-rich, angiogenic inducer 61 (CYR61, or CCN1), and Chemokine (C-C motif) ligand 2 (CCL2) were up regulated in both pediatric CD and UC. Ingenuity network analysis of differentially expressed genes from pediatric CD was clustered into 8 networks and pediatric UC into 25 networks. By comparison of pediatric IBD and experimental colitis microarray data, we found the following common features: 1) CXCL9 and S100A8 were abundantly up regulated; 2) cytokine-cytokine receptor networks were dysregulated; and 3) IRF-1 transcription binding sites were over represented in the promoter region of up regulated genes, while HNF1A and Lhx3 binding sites were over represented in the promoter region of the down regulated genes. Together, our study provides a comprehensive comparative view of genome expression and transcription factor profile changes between pediatric IBD and experimental colitis models revealing common pathways that may be useful for future therapeutics.

Deletion of Foxp3+ regulatory T cells in genetically targeted mice supports development of intestinal inflammation.

BackgroundMice lacking Foxp3+ regulatory T (Treg) cells develop severe tissue inflammation in lung, skin, and liver with premature death, whereas the intestine remains uninflamed. This study aims to demonstrate the importance of Foxp3+ Treg for the activation of T cells and the development of intestinal inflammation.MethodsFoxp3-GFP-DTR (human diphtheria toxin receptor) C57BL/6 mice allow elimination of Foxp3+ Treg by treatment with Dx (diphtheria toxin). The influence of Foxp3+ Treg on intestinal inflammation was tested using the CD4+ T-cell transfer colitis model in Rag−/− C57BL/6 mice and the acute DSS-colitis model.ResultsContinuous depletion of Foxp3+ Treg in Foxp3-GFP-DTR mice led to dramatic weight loss and death of mice by day 28. After 10 days of depletion of Foxp3+ Treg, isolated CD4+ T-cells were activated and produced extensive amounts of IFN-γ, IL-13, and IL-17A. Transfer of total CD4+ T-cells isolated from Foxp3-GFP-DTR mice did not result in any changes of intestinal homeostasis in Rag−/− C57BL/6 mice. However, administration of DTx between days 14 and 18 after T-cell reconstitution, lead to elimination of Foxp3+ Treg and to immediate weight loss due to intestinal inflammation. This pro-inflammatory effect of Foxp3+ Treg depletion consecutively increased inflammatory cytokine production. Further, the depletion of Foxp3+ Treg from Foxp3-GFP-DTR mice increased the severity of acute dSS-colitis accompanied by 80% lethality of Treg-depleted mice. CD4+ effector T-cells from Foxp3+ Treg-depleted mice produced significantly more pro-inflammatory cytokines.ConclusionIntermittent depletion of Foxp3+ Treg aggravates intestinal inflammatory responses demonstrating the importance of Foxp3+ Treg for the balance at the mucosal surface of the intestine.

A2BAR expression in non-immune cells plays an important role in the development of murine colitis

BackgroundAdenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A2BAR plays a pro-inflammatory role during colitis. AimsOur goals were to determine if A2BAR expression was necessary on immune cells/non-immune cells during colitis and if A2BAR was a suitable target for treating intestinal inflammation. MethodsWild-type and A2BAR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A2BAR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A2BAR/RAG1 double knockout recipients. Finally, A2BAR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. ResultsWild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A2BAR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. ConclusionsOur results suggest that A2BAR on non-immune cells plays an important role for the induction of colitis and targeting A2BAR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.

Tu1860 Inverse Expression of IL-33 in TH1 Versus TH2 Mediated Experimental Colitis

Background: Interleukin (IL)-33 is a pro-inflammatory cytokine that has been associated with induction of human T helper 2 (TH2)-type inflammatory responses through activation of the ST2 receptor. Recent research has shown that IL-33 is upregulated in active ulcerative colitis (UC), but the role of IL-33 in regulation of intestinal mucosal immunologic reactions is uncertain. Aim: The aim of the present study was to describe the regulation of IL-33 in a TH1 and a TH2 model of experimental colitis, to determine if IL-33 is also regulated according to the T helper cell profile in the inflamed intestinal mucosa. Methods: Expression of IL-33 was investigated in dextran sodium sulphate (DSS)-induced colitis (a TH2-type of inflammation) and in the T-cell transfer model of colitis (SCID colitis) (a TH1-type of inflammation). The colonic IL-33 mRNA expression was analyzed by a quantitative realtime polymerase chain reaction (qRT-PCR). IL-33 protein expression and its localization were investigated by immunoblotting and by standard immunohistochemical procedures. The association between IL-33 expression and clinical outcome measures including the disease activity index (DAI), colon length, and histological inflammation scores was determined. Results: The colonic IL-33 mRNA expression was significantly up-regulated in the experimental DSS colitis model (p<0.05), and significantly down-regulated in the SCID colitis (p<0.01). Our results showed an increased number of IL-33 positive cells in DSS colitis localized to lamina propria inflammatory cells, whereas IL-33 protein expression in SCID colitis was abolished. IL-33 expression correlated positively with the DAI-score (p<0.001), colon length (p<0.001), and the histological inflammation score (p<0.001) of the DSS model. Conclusions: The present study demonstrates that colonic IL-33 expression in mice is highly dependent on the type of mucosal inflammatory response induced, being high in TH2-type inflammatory conditions, and low in TH1-type inflammatory responses. IL-33 might be a key determinant of the nature of the colonic mucosal inflammation and thus be of importance for the development of inflammatory bowel disease. Inhibition of IL33 signalling could in this way be a potential therapeutic target in UC which is driven by a TH2-type inflammatory reaction.

Tu1861 The IBD Associated Gene, Protein Tyrosine Phosphate Non Receptor Type 22, Modulates Interferon-γ Induced Cytokine Responses in Human Monocytes

Background: Interleukin (IL)-33 is a pro-inflammatory cytokine that has been associated with induction of human T helper 2 (TH2)-type inflammatory responses through activation of the ST2 receptor. Recent research has shown that IL-33 is upregulated in active ulcerative colitis (UC), but the role of IL-33 in regulation of intestinal mucosal immunologic reactions is uncertain. Aim: The aim of the present study was to describe the regulation of IL-33 in a TH1 and a TH2 model of experimental colitis, to determine if IL-33 is also regulated according to the T helper cell profile in the inflamed intestinal mucosa. Methods: Expression of IL-33 was investigated in dextran sodium sulphate (DSS)-induced colitis (a TH2-type of inflammation) and in the T-cell transfer model of colitis (SCID colitis) (a TH1-type of inflammation). The colonic IL-33 mRNA expression was analyzed by a quantitative realtime polymerase chain reaction (qRT-PCR). IL-33 protein expression and its localization were investigated by immunoblotting and by standard immunohistochemical procedures. The association between IL-33 expression and clinical outcome measures including the disease activity index (DAI), colon length, and histological inflammation scores was determined. Results: The colonic IL-33 mRNA expression was significantly up-regulated in the experimental DSS colitis model (p<0.05), and significantly down-regulated in the SCID colitis (p<0.01). Our results showed an increased number of IL-33 positive cells in DSS colitis localized to lamina propria inflammatory cells, whereas IL-33 protein expression in SCID colitis was abolished. IL-33 expression correlated positively with the DAI-score (p<0.001), colon length (p<0.001), and the histological inflammation score (p<0.001) of the DSS model. Conclusions: The present study demonstrates that colonic IL-33 expression in mice is highly dependent on the type of mucosal inflammatory response induced, being high in TH2-type inflammatory conditions, and low in TH1-type inflammatory responses. IL-33 might be a key determinant of the nature of the colonic mucosal inflammation and thus be of importance for the development of inflammatory bowel disease. Inhibition of IL33 signalling could in this way be a potential therapeutic target in UC which is driven by a TH2-type inflammatory reaction.

Temporal Genome Expression Profile Analysis During T-cell-Mediated Colitis: Identification of Novel Targets and Pathways

T cells critically regulate inflammatory bowel disease (IBD), with T-cell-dependent experimental colitis models gaining favor in identifying potential pathogenic mechanisms; yet limited understanding of specific pathogenic molecules or pathways still exists. In this study we sought to identify changes in whole genome expression profiles using the CD4CD45Rbhi T-cell transfer colitis model compared to genome expression differences from Crohn's disease (CD) tissue specimens. Colon tissue was used for histopathological and genome expression profiling analysis at 0, 2, 4, or 6 weeks after adoptive T-cell transfer. We identified 1775 genes that were significantly altered during disease progression, with 361 being progressively downregulated and 341 progressively upregulated. Gene expression changes were validated by quantitative real-time polymerase chain reaction (qRT-PCR), confirming genome expression analysis data. Differentially expressed genes were clearly related to inflammation/immune responses but also strongly associated with metabolic, chemokine signaling, Jak-STAT signaling, and angiogenesis pathways. Ingenuity network analysis revealed 25 unique network associations that were associated with functions such as antigen presentation, cell morphology, cell-to-cell signaling and interaction, as well as nervous system development and function. Moreover, many of these genes and pathways were similarly identified in CD specimens. These findings reveal novel, complex, and dynamic changes in gene expression that may provide useful targets for future therapeutic approaches.

Lack of TNFR2 expression by CD4+ T cells exacerbates experimental colitis

TNF plays fundamental roles in the induction and perpetuation of inflammation. The effects of TNF are mediated through TNF receptor (TNFR) 1 or 2. As these two receptors mediate different functions, selective targeting of one receptor may represent a more specific treatment for inflammatory disorders than the complete blocking of TNF. TNFR2 expression is up-regulated in inflammatory bowel disease. Hence, we directly assessed the role of TNFR2 signaling in the CD4(+) T-cell transfer model of colitis using TNFR2(-/-) or WT mice as donors of colitogenic CD4(+)CD45RB(hi) T cells for transfer into syngeneic RAG2(-/-) or RAG2(-/-)TNFR2(-/-) recipient mice. Although the absence of TNFR2 expression by non-lymphoid cells of the recipient mice does not influence the course of colitis, transfer of TNFR2(-/-) CD4(+) T cells leads to an accelerated onset of disease and to more severe signs of inflammation. The enhanced colitogenic potential of TNFR2(-/-) CD4(+) T cells is associated with reduced activation-induced cell death, resulting in an increased accumulation of TNFR2(-/-) CD4(+) T cells. Hence, TNFR2 signaling is crucial for the TNF-dependent contraction of the disease-inducing T cells. Therefore, a selective blocking of TNFR2 may lead to exacerbation rather than attenuation of T-cell-mediated inflammatory disorders.

Gpr83 expression is not required for the maintenance of intestinal immune homeostasis and regulation of T‐cell‐dependent colitis

Regulatory T (TR) cells are integral to the maintenance of intestinal homeostasis, where an intricate balance between tolerance and immunity must be maintained. Recently, studies have focused on the identification of molecules involved in the function and/or development of TR cells. One such molecule, the G-protein coupled receptor Gpr83, has been identified through gene expression analysis as being overexpressed within thymic and peripheral naturally arising regulatory T (nTR) cell populations. The aim of this study was to further define the characteristics of Gpr83 expression and to investigate the role of Gpr83 in TR-cell development and function through the generation and analysis of Gpr83-deficient mice. Following activation, naïve CD4+ T cells induce Gpr83 expression in a transforming growth factor (TGF)-β dependent manner. Rather than being a general marker of activation, Gpr83 expression could only be detected in cells also expressing forkhead/winged helix transcription factor (Foxp3), further supporting the association of Gpr83 with the regulatory cell phenotype. Mice deficient in Gpr83 expression developed normally and did not display signs of inflammatory disease. Thymic nTR-cell development was unaffected by a lack of Gpr83 expression and peripheral nTR-cell homeostasis was normal when compared with that of wild-type mice. Gpr83 expression was dispensable for the regulatory activity of nTR cells as Gpr83-deficient nTR cells could suppress the development of disease in a T-cell transfer model of colitis. These results suggest a redundant role for Gpr83 in the function of TR cells in this model of disease. Further studies are required to determine the role of Gpr83 in TR-cell biology.

T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance

Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins. It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-beta1 (refs 2-4), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-beta1. Furin-deficient T regulatory (Treg) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type Treg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-beta1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance.

Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice

Galectins have recently emerged as central regulators of the immune system. We have previously demonstrated that carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Here, we investigate the potential therapeutic effect of galectin-2 in experimental colitis. Galectin-2 expression and its binding profile were determined by immunohistochemistry. Acute and chronic colitis was induced by dextran sodium sulfate administration and in a T-cell transfer colitis model. Apoptosis was assessed by TdT-mediated dUTP-biotin nick-end labeling, and cytokine secretion was determined by cytometric bead array. We show that galectin-2 was constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated. Our study provides evidence that galectin-2 induces apoptosis in vivo and ameliorates acute and chronic murine colitis. Furthermore, galectin-2 has no significant toxicity over a broad dose range, suggesting that it may serve as a new therapeutic agent in the treatment of inflammatory bowel disease.