T-cell Transfer Model Research Articles

Hcp and VgrG1 are secreted components of theHelicobacter hepaticustype VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T-cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS-dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T-cell-transferred C57BL/6 Rag2(-/-) mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild-typeH. hepaticus as compared with mice infected with VgrG1-deficient isogenic bacteria. In addition, competitive assays between both wild-type and T6SS-deficient H. hepaticus, and between wild-type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro-inflammatory potential mediated by the T6SS. Cellular innate pro-inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro-inflammatory effect of the chronic H. hepaticus infection.

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.

Bone loss and renal Ca2+ wasting in experimental colitis is accompanied by downregulation of TRPV5 in renal distal convoluted tubules

Dysregulated Ca2+homeostasis likely contributes to the etiology of IBD‐associated bone loss. Experimental colitis leads to decreased expression of Klotho, a protein which supports renal Ca2+ reabsorption by stabilizing TRPV5 channel on the apical membrane of distal tubule epithelial cells. We investigated whether colitis leads to changes in renal Ca2+ handling and expression of TRPV5 utilizing TNBS, and two adoptive T‐cell transfer models (CD4+CD45RBHi or CD4+IL10−/−). Colitic mice had normal serum levels of Ca2+ and PTH. μCT analysis demonstrated changes in bone structure along with changes in serum sRANKL, IL‐6, osteocalcin, TNF, and total serum tDPD crosslinks. Increased fractional Ca2+excretion was accompanied by reduction of TRPV5 protein expression, with concomitant increase in its sialylation status. In mIMCD3 cells transduced with Ad‐TRPV5, proinflammatory cytokines reduced TRPV5 surface expression followed by its degradation. TNF induced interaction of TRPV5 with UBR4 ubiquitin ligase and siRNA‐mediated UBR4 knockdown prevented TNF induced TRPV5 degradation. The cytokine effect on TRPV5 was not observed in cells stably transfected with membrane‐bound Klotho. We propose that TNF and IFNγ act to decrease Klotho expression and activity, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin‐1, and ultimately to UBR4‐dependent endocytosis and degradation of TRPV5.

Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease

OBJECTIVES:Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD).METHODS:Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2−/−) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients.RESULTS:We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3β (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3β/γ 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls.CONCLUSIONS:These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.

Regulatory T cells expanded by rapamycin in vitro suppress colitis in an experimental mouse model

To provide rapid immunosuppression without side effects, we analyzed whether rapamycin alone, and regulatory T cells (Tregs) expanded ex vivo by rapamycin, suppressed colitis in a mouse model. Severe combined immunodeficiency (SCID) mice reconstituted with naive CD4(+) T cells were treated with or without intraperitoneal rapamycin. Body weight was evaluated. CD4(+) T cells were cultured in the presence of rapamycin for three 7-day rounds of stimulation. The ratio of Tregs to CD4(+) T cells was analyzed by flow cytometry. Naive CD4(+) T cells were transferred into SCID mice with CD4(+) T cells expanded in the presence or absence of rapamycin. Clinical symptoms of colitis, histological changes, and cytokine expression were investigated. Systemic rapamycin partially prevented the development of colonic inflammation in a transfer model of colitis, but decreased body weight in control mice. With rapamycin, stimulated CD4(+) T cells expanded eightfold in 3 weeks in vitro, and the proportion of Tregs increased to about 40%. Without rapamycin, CD4(+) T cells expanded 20-fold in 3 weeks, but the proportion of Tregs remained at about 15%. CD4(+) T cells expanded with rapamycin prevented the development of colitis in a naïve CD4(+) T-cell transfer model, in association with the downregulation of Th1 and Th17 responses. We demonstrated, for the first time, that CD4(+) T cells expanded with rapamycin in vitro suppressed colitis. Therefore, rapamycin-expanded Treg transfer therapy is expected to be efficacious for inflammatory bowel disease.

Constitutive TL1A Expression under Colitogenic Conditions Modulates the Severity and Location of Gut Mucosal Inflammation and Induces Fibrostenosis

Intestinal fibrostenosis is a hallmark of severe Crohn's disease and can lead to multiple surgeries. Patients with certain TNFSF15 variants overexpress TL1A. The aim of this study was to determine the effect of TL1A overexpression on intestinal inflammation and the development of fibrostenosis. We assessed the in vivo consequences of constitutive TL1A expression on gut mucosal inflammation and fibrostenosis using two murine models of chronic colitis. In the dextran sodium sulfate (DSS) and adoptive T-cell transfer models, there was proximal migration of colonic inflammation, worsened patchy intestinal inflammation, and long gross intestinal strictures in Tl1a transgenic compared to wild-type littermates. In the DSS model, myeloid- and T-cell-expressing Tl1a transgenic mice had increased T-cell activation markers and interleukin-17 expression compared to wild-type mice. In the T-cell transfer model, Rag1(-/-) mice receiving Tl1a transgenic T cells had increased interferon-γ expression but reduced T-helper 17 cells and IL-17 production. Narrowed ureters with hydronephrosis were found only in the Tl1a transgenic mice in all chronic colitis models. In human translational studies, Crohn's disease patients with higher peripheral TL1A expression also exhibited intestinal fibrostenosis and worsened ileocecal inflammation with relative sparing of rectosigmoid inflammation. These data show that TL1A is an important cytokine that not only modulates the location and severity of mucosal inflammation, but also induces fibrostenosis.

Theiler's Murine Encephalomyelitis Virus as a Vaccine Candidate for Immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.

A humanized monoclonal antibody targeting the β7 integrin selectively blocks intestinal homing of T lymphocytes

rhuMAb Beta7 is a humanized anti-human β7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the β7 subunit of the integrins α4β7 and αEβ7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7. We assessed the effect of murine anti-Beta7 on lymphocyte homing in mouse models of autoimmune disease. We also compared the effect of rhuMAb Beta7 on circulating mucosal-homing versus peripheral-homing T cells in naïve non-human primates. In cynomolgus monkeys, occupancy of β7 integrin receptors by rhuMAb Beta7 correlated with an increase in circulating β7(+) mucosal-homing lymphocytes, with no apparent effect on levels of circulating β7(-) peripheral-homing lymphocytes. rhuMAb Beta7 also inhibited lymphocyte homing to the inflamed colons of severe combined immunodeficient mice in CD45RB(high) CD4(+) T-cell transfer models. Consistent with a lack of effect on peripheral homing, in a mouse model of experimental autoimmune encephalomyelitis, anti-β7 treatment resulted in no amelioration of CNS inflammation. The results presented here suggest that rhuMAb Beta7 selectively blocks lymphocyte homing to the gastrointestinal tract without affecting lymphocyte trafficking to non-mucosal tissues. rhuMAb Beta7 provides a targeted therapeutic approach with the potential for a more attractive benefit:risk ratio than currently available inflammatory bowel disease therapies.

VEGF164 isoform specific regulation of T-cell-dependent experimental colitis in mice

Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC), two widespread diseases of unknown, multifactorial etiology. Colitis pathology involves a pathological angiogenic response where increases in vascular density participate in colitis histopathology. Vascular endothelial growth factor-A (VEGF-A) is a potent angiogenesis stimulator known to be involved in pathological angiogenesis in several diseases including colitis. However, the pathogenic importance of specific VEGF-A isoforms during T-cell-mediated experimental colitis remains largely unknown. The CD4⁺ CD45RB(high) T-cell transfer model of experimental colitis was used for these studies. The VEGF lac-Z transgenic reporter mouse was used to examine specific cellular sources of VEGF-A production. The VEGF₁₆₄ aptamer (Macugen), adenoviral VEGF₁₆₄, and the VEGF Trap were used to evaluate pathological importance. VEGF lac-Z reporter mice experiments showed that both infiltrating T cells and local tissue cells produce VEGF-A in the colon during disease. Inhibition of VEGF₁₆₄ using a highly selective RNA aptamer significantly attenuated CD4⁺ CD45RB(high) T-cell-dependent experimental colitis by reducing pathological angiogenesis and inflammatory pathology. Conversely, broad-spectrum VEGF inhibition with VEGF Trap did not attenuate disease, nor did adenoviral VEGF₁₆₄ overexpression significantly alter colitis pathology. VEGF₁₆₄ is actively produced by multiple cell types during T-cell-mediated colitis. Importantly, specific inhibition of the VEGF₁₆₄ isoform during T-cell-mediated colitis dose-dependently attenuated disease progression, while broad-scale inhibition of all VEGF-A isoforms was not therapeutically beneficial.

S1776 Chronic Lung and Liver Inflammation in a Mouse Model of Crohn's Colitis

Recent population-based studies demonstrate that in addition to the more common joint and hepatobiliary extraintestinal manifestations observed in patients with one of the inflammatory bowel diseases (IBD), pulmonary inflammation is a very common comorbidity increased in Crohn's disease. Because few studies have reported the presence of extraintestinal inflammation in mouse models of IBD, we undertook a systematic histopathological study to document the presence (or absence) of lung and liver inflammation in a mouse model of CD. Chronic colitis was induced in RAG-1-deficient (RAG-/-) mice by the adoptive transfer of naive (CD4+CD45RBhigh) T-cells (5x105 cells per mouse) obtained from the spleens of healthy donor mice. PBS-injected RAG-/mice were used as controls. Colons, lungs and liver as well as heart, kidneys, and brain were excised, fixed in formalin, embedded, sectioned and stained with H&E. Blinded histopathological analysis was performed on each organ by a pathologist not familiar with the experimental groups. We found that at 6-8 weeks post Tcell transfer, the majority of T-cell injected mice (80%) became moribund and were euthanized whereas PBS-injected animals appeared healthy throughout the entire 8week observation period. Blinded histopathological examination of the colons obtained from RAG-/mice injected with naive T-cells revealed moderate-to-severe colitis characterized by dramatic transmural T-cell and PMN infiltration, bowel wall thickening, Goblet cell dropout and epithelial cell erosions. Intracellular cytokine analysis of T-cells obtained from the colon and MLNs of colitic mice showed enhanced production of both IFNγ and IL-17. Surprisingly, we found that virtually all mice injected with naive T-cells presented with chronic bronchitis/ pulmonary inflammation. In many cases (>60%) confluent inflammation merged together between small and medium sized bronchi. Varying degrees of perivascular lymphocytic cuffing were also observed in virtually all of the mice however few PMNs were observed and vasculitis was absent. Lung parenchyma involvement was mild and patchy. In addition to gut and lung inflammation, we also observed significant hepatocellular necrosis with periportal and lobular lymphocytic inflammation. There was no evidence of bile ductal damage. No significant inflammation was observed in the heart, kidneys or brain of the colitic animals and no inflammation was observed in any organ system of the PBS controls. Taken together, these data demonstrate, for the first time, that pulmonary and liver inflammation is a occurrence in the T-cell transfer model of chronic colitis (Supported by NIH PO1 43785).

Blood flow rate in a T-cell transfer model of Inflammatory Bowel Disease in mice

Blood flow rate in a T-cell transfer model of Inflammatory Bowel Disease in mice

Association between blood flow and inflammatory state in a T-cell transfer model of inflammatory bowel disease in mice

Adoptive transfer of naive T-lymphocyte subsets into lymphopenic mice initiates chronic gut inflammation that mimics several aspects of inflammatory bowel disease (IBD). Patients with IBD can have profound alterations in intestinal blood flow, but whether the same is true in the T-cell transfer model has yet to be determined. In the current study, chronic intestinal inflammation was induced in recombinase-activating gene-1-deficient (RAG(-/-)) mice by adoptive transfer of CD4(+) T-lymphocytes obtained from interleukin-10 deficient (IL-10(-/-)) mice. Four weeks later, widespread colonic inflammation was observed in the reconstituted recipients, in contrast to 2 control sets of mice injected with a different subset of lymphocytes or with vehicle alone. We observed that the resulting pathology induced in the reconstituted RAG(-/-) mice was divided distinctly into 2 subsets: 1 with blood flow near normal with very high inflammation scores, and the other with severely attenuated blood flow but with much lower signs of inflammation. Colonic and ileal blood flow rates in the latter subset of CD4(+) mice averaged only approximately 30% compared to the mice with higher inflammation scores. The lower blood flow rates were associated with greatly reduced red blood cell concentrations in the tissue, suggesting a possible loss of vascular density. In this model of chronic intestinal inflammation, mild inflammation was associated with significant decreases in blood flow.

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.

Dexamethasone/1α‐25‐dihydroxyvitamin D3‐treated dendritic cells suppress colitis in the SCID T‐cell transfer model

Autoantigen-presenting immunomodulatory dendritic cells (DCs) that are used for adoptive transfer have been shown to be a promising therapy for a number of autoimmune diseases. We have previously demonstrated that enteroantigen-pulsed DCs treated with interleukin-10 (IL-10) can partly protect severe combined immunodeficient (SCID) mice adoptively transferred with CD4+ CD25(-) T cells from the development of wasting disease and colitis. We therefore established an in vitro test that could predict the in vivo function of DCs and improve strategies for the preparation of immunomodulatory DCs in this model. Based on these in vitro findings, we here evaluate three methods for DC generation including short-term and long-term IL-10 exposure or DC exposure to dexamethasone in combination with vitamin D3 (Dex/D3). All DCs resulted in lower CD4+ CD25(-) T-cell enteroantigen-specific responses in vitro, but Dex/D3 DCs had the most prominent effect on T-cell cytokine secretion. In vivo, Dex/D3 DCs most efficiently prevented weight loss and gut pathology upon CD4+ CD25(-) T-cell transfer in SCID mice, although the effect on gut pathology was antigen independent. Our data in the SCID T-cell transfer model illustrate some correlation between in vitro and in vivo DC function and document that prevention of experimental inflammatory bowel disease by transfer of immunosuppressive DCs is possible.

Genome‐wide Gene Expression Profiling of SCID Mice with T‐cell‐mediated Colitis

Inflammatory bowel disease (IBD) is a multifactorial disorder with an unknown aetiology. The aim of this study is to employ a murine model of IBD to identify pathways and genes, which may play a key role in the pathogenesis of IBD and could be important for discovery of new disease markers in human disease. Here, we have investigated severe combined immunodeficient (SCID) mice, which upon adoptive transfer with concanavalin A-activated CD4(+) T cells develop inflammation of the colon with predominance in rectum. Mice with increasing level of inflammation was studied. RNA from rectum of transplanted and non-transplanted SCID mice was investigated by a genome-wide gene expression analysis using the Affymetrix mouse expression array 430A (MOE430A) including 22,626 probe sets. A significant change in gene expression (P = 0.00001) is observed in 152 of the genes between the non-transplanted control mice and colitis mice, and among these genes there is an overrepresentation of genes involved in inflammatory processes. Some of the most significant genes showing higher expression encode S100A proteins and chemokines involved in trafficking of leucocytes in inflammatory areas. Classification by gene clustering based on the genes with the significantly altered gene expression corresponds to two different levels of inflammation as established by the histological scoring of the inflamed rectum. These data demonstrate that this SCID T-cell transfer model is a useful animal model for human IBD and can be used for suggesting candidate genes involved in the pathogenesis and for identifying new molecular markers of chronic inflammation in human IBD.

Sequential Role of Plasmacytoid Dendritic Cells and Regulatory T Cells in Oral Tolerance

Orally induced tolerance to environmental allergens prevents deleterious, systemic, delayed-type hypersensitivity responses via immune suppression mechanisms believed to include either anergy/deletion of specific effector T cells or active suppression by CD4(+)CD25(+) regulatory T cells (Tregs). The aim of this study was to investigate whether and how antigen (Ag) penetration through the gut orchestrates these 2 distinct mechanisms. Using a model of allergic contact dermatitis (ACD) mediated by hapten-specific cytolytic CD8(+) T cells and a T-cell transfer model of contact hypersensitivity in CD3epsilon-deficient mice, we studied the outcome of Ag gavage on CD8(+) effectors and Tregs. Full protection from ACD by gavage with the relevant allergen required 2 coordinated events taking place first in gut-associated lymphoid tissues and then systemically. Allergen gavage induced deletion by plasmacytoid dendritic cells of a large fraction of Ag-specific CD8(+) T cells in liver and mesenteric lymph nodes and also triggered the suppressive function of Treg of secondary lymphoid organs. Residual Ag-specific CD8(+) T cells conditioned during this mucosal step are fully susceptible to suppression by activated Treg, which completely prevented their differentiation into ACD effectors, upon re-exposure to the allergen via the skin. Thus, oral tolerance initiated in gut-associated lymphoid tissues by the plasmacytoid dendritic cells-mediated deletion of Ag-specific T cells is completed systemically by CD4(+)CD25(+) T cells. Biotherapies able to increase the susceptibility of effector T cells to the suppressive function of Treg may be valuable for the treatment of autoimmune and inflammatory diseases.

Blockage of the neurokinin 1 receptor and capsaicin-induced ablation of the enteric afferent nerves protect SCID mice against T-cell-induced chronic colitis

The neurotransmitter substance P (SP) released by, and the transient receptor potential vanilloid (TRPV1), expressed by afferent nerves, have been implicated in mucosal neuro-immune-regulation. To test if enteric afferent nerves are of importance for the development of chronic colitis, we examined antagonists for the high-affinity neurokinin 1 (NK-1) SP receptor and the TRPV1 receptor agonist capsaicin in a T-cell transfer model for chronic colitis. Chronic colitis was induced in SCID mice by injection of CD4(+)CD25(-) T cells. The importance of NK-1 signaling and TRPV1 expressing afferent nerves for disease development was studied in recipient SCID mice systemically treated with either high-affinity NK-1 receptor antagonists or neurotoxic doses of capsaicin. In addition, we studied the colitis-inducing effect of NK-1 receptor deleted CD4(+)CD25(-) T cells. Treatment with the NK-1 receptor antagonist CAM 4092 reduced the severity of colitis, but colitis was induced by NK-1 receptor-deleted T cells, suggesting that SP in colitis targets the recipient mouse cells and not the colitogenic donor T cells. Capsaicin-induced depletion of nociceptive afferent nerves prior to CD4(+)CD25(-) T-cell transfer completely inhibited the development of colitis. Our data demonstrate the importance of an intact enteric afferent nerve system and NK-1 signaling in mucosal inflammation and may suggest new treatment modalities for patients suffering from inflammatory bowel disease.

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.