TL1A (TNFSF15) Regulates the Development of Chronic Colitis by Modulating Both T-Helper 1 and T-Helper 17 Activation

Background & Aims: TL1A is a tumor necrosis factor–like molecule that mediates a strong costimulation of T-helper (TH) 1 cells. Expression of TL1A is increased in the mucosa of Crohn's disease patients and murine models of ileitis. The aim of this study was to determine the possible role of TL1A in chronic intestinal inflammation. Methods:We used dextran sodium sulfate (DSS)-induced chronic colitis to investigate the effects of TL1A on the development of colitis. The cytokine profile in the gut-associated lymphoid tissue (GALT) was measured. Neutralizing anti-TL1A antibodies were injected intraperitoneally into DSS-induced chronic colitis and G protein αi2−/− T-cell transfer colitis models. Severity of colitis was evaluated by body weight, colon length, histology, and cytokine production. Results:DSS-induced chronic colitis was characterized by the infiltration of CD4+ T cells. TL1A, death receptor 3, interferon (IFN)-γ, and interleukin (IL)-17 were increased significantly in GALT of DSS-treated mice. TL1A up-regulated both IFN-γ production from TH1 cells and IL-17 production from TH17 cells in GALT CD4+ T cells. Furthermore, IFN-γ and IL-17 production from CD4+ T cells, induced by IL-12 and IL-23 respectively, was enhanced synergistically by combination with TL1A. Anti-TL1A antibody prevented chronic colitis and attenuated established colitis by down-regulation of both TH1 and TH17 activation. Conclusions:Our results reveal that TL1A is an important modulator in the development of chronic mucosal inflammation by enhancing TH1 and TH17 effector functions. The central role of TL1A represents an attractive, novel therapeutic target for the treatment of Crohn's disease patients. Background & Aims: TL1A is a tumor necrosis factor–like molecule that mediates a strong costimulation of T-helper (TH) 1 cells. Expression of TL1A is increased in the mucosa of Crohn's disease patients and murine models of ileitis. The aim of this study was to determine the possible role of TL1A in chronic intestinal inflammation. Methods:We used dextran sodium sulfate (DSS)-induced chronic colitis to investigate the effects of TL1A on the development of colitis. The cytokine profile in the gut-associated lymphoid tissue (GALT) was measured. Neutralizing anti-TL1A antibodies were injected intraperitoneally into DSS-induced chronic colitis and G protein αi2−/− T-cell transfer colitis models. Severity of colitis was evaluated by body weight, colon length, histology, and cytokine production. Results:DSS-induced chronic colitis was characterized by the infiltration of CD4+ T cells. TL1A, death receptor 3, interferon (IFN)-γ, and interleukin (IL)-17 were increased significantly in GALT of DSS-treated mice. TL1A up-regulated both IFN-γ production from TH1 cells and IL-17 production from TH17 cells in GALT CD4+ T cells. Furthermore, IFN-γ and IL-17 production from CD4+ T cells, induced by IL-12 and IL-23 respectively, was enhanced synergistically by combination with TL1A. Anti-TL1A antibody prevented chronic colitis and attenuated established colitis by down-regulation of both TH1 and TH17 activation. Conclusions:Our results reveal that TL1A is an important modulator in the development of chronic mucosal inflammation by enhancing TH1 and TH17 effector functions. The central role of TL1A represents an attractive, novel therapeutic target for the treatment of Crohn's disease patients. The pathogenesis of Crohn's disease and ulcerative colitis relates to an inappropriate and exaggerated mucosal immune response to constituents of the intestinal flora.1Xavier R.J. Podolsky D.K. Unravelling the pathogenesis of inflammatory bowel disease.Nature. 2007; 448: 427-434Crossref PubMed Scopus (3107) Google Scholar, 2Sartor R.B. Microbial influences in inflammatory bowel diseases.Gastroenterology. 2008; 134: 577-594Abstract Full Text Full Text PDF PubMed Scopus (1395) Google Scholar Antigen-presenting cells (APCs) such as dendritic cells (DCs) are likely to play a central role in the host response to intestinal flora, both in innate responses to bacteria and by shaping the character of the host's adaptive immune response.3Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu Rev Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1130) Google Scholar, 4Elson C.O. Cong Y. McCracken V.J. et al.Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota.Immunol Rev. 2005; 206: 260-276Crossref PubMed Scopus (402) Google Scholar Furthermore, CD4+ T cells activated by APCs also have been shown to be involved in the pathogenesis of inflammatory bowel disease.3Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu Rev Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1130) Google Scholar A dysregulated T-cell response leads to alterations in mucosal cytokine expression. Crohn's disease (CD) has been characterized as having a T-cell helper (TH) 1 and TH17 cytokine pattern, and antibodies to both interferon (IFN)-γ and p40 (interleukin [IL]-12/23) can treat subsets of CD patients.5Hommes D.W. Mikhajlova T.L. Stoinov S. et al.Fontolizumab, a humanised anti-interferon gamma antibody, demonstrates safety and clinical activity in patients with moderate to severe Crohn's disease.Gut. 2006; 55: 1131-1137Crossref PubMed Scopus (191) Google Scholar, 6Mannon P.J. Fuss I.J. Mayer L. et al.Anti-interleukin-12 antibody for active Crohn's disease.N Engl J Med. 2004; 351: 2069-2079Crossref PubMed Scopus (740) Google Scholar TL1A, a recently identified tumor necrosis factor–like factor, is the only known ligand for death receptor (DR) 3 that is expressed primarily on activated lymphocytes.7Migone T.S. Zhang J. Luo X. et al.TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.Immunity. 2002; 16: 479-492Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar TL1A is expressed on endothelial cells, lymphocytes, plasma cells, monocytes, and DCs.8Prehn J.L. Thomas L.S. Landers C.J. et al.The T cell costimulator TL1A is induced by Fc{gamma}R signaling in human monocytes and dendritic cells.J Immunol. 2007; 178: 4033-4038PubMed Google Scholar, 9Bamias G. Mishina M. Nyce M. et al.Role of TL1A and its receptor DR3 in two models of chronic murine ileitis.Proc Natl Acad Sci U S A. 2006; 103: 8441-8446Crossref PubMed Scopus (135) Google Scholar TL1A can induce IFN-γ production of IL-12 and IL-18 primed gut-homing receptor CCR9+ T cells, but not CCR9− T cells, by the interaction of TL1A/DR3.10Papadakis K.A. Zhu D. Prehn J.L. et al.Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes.J Immunol. 2005; 174: 4985-4990PubMed Google Scholar In contrast, TL1A does not enhance IL-4 production from TH2 cells.11Prehn J.L. Mehdizadeh S. Landers C.J. et al.Potential role for TL1A, the new TNF-family member and potent costimulator of IFN-gamma, in mucosal inflammation.Clin Immunol. 2004; 112: 66-77Crossref PubMed Scopus (129) Google Scholar Therefore, the interaction of TL1A and DR3 may be important in TH1-mediated responses of the intestine. In fact, we and others have shown up-regulation of both TL1A and DR3 in rheumatoid arthritis12Cassatella M.A. da Silva G.P. Tinazzi I. et al.Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis.J Immunol. 2007; 178: 7325-7333PubMed Google Scholar and inflammatory bowel disease (IBD), particularly CD.11Prehn J.L. Mehdizadeh S. Landers C.J. et al.Potential role for TL1A, the new TNF-family member and potent costimulator of IFN-gamma, in mucosal inflammation.Clin Immunol. 2004; 112: 66-77Crossref PubMed Scopus (129) Google Scholar, 13Bamias G. Martin 3rd, C. Marini M. et al.Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease.J Immunol. 2003; 171: 4868-4874PubMed Google Scholar, 14Papadakis K.A. Prehn J.L. Landers C. et al.TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells.J Immunol. 2004; 172: 7002-7007PubMed Google Scholar Furthermore, recent genome-wide association studies revealed a highly significant association of single nucleotide polymorphism haplotypes of the TL1A gene with CD in Japanese, European, and US cohorts.15Picornell Y. Mei L. Taylor K. et al.TNFSF15 is an ethnic-specific IBD gene.Inflamm Bowel Dis. 2007; 13: 1333-1338Crossref PubMed Scopus (74) Google Scholar, 16Yamazaki K. McGovern D. Ragoussis J. et al.Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease.Hum Mol Genet. 2005; 14: 3499-3506Crossref PubMed Scopus (371) Google Scholar Bamias et al9Bamias G. Mishina M. Nyce M. et al.Role of TL1A and its receptor DR3 in two models of chronic murine ileitis.Proc Natl Acad Sci U S A. 2006; 103: 8441-8446Crossref PubMed Scopus (135) Google Scholar showed an association of increased TL1A and DR3 in expression in ileitis models, but no direct proof of the role of TL1A in mucosal inflammation. Taken together, these results suggest that the interaction of increased APC-derived TL1A and lymphocytic DR3 is involved in TH1-mediated intestinal inflammation as is seen in human CD. The precise role of TL1A in IBD has not been elucidated. In this study, we have investigated the function of TL1A in 2 different mouse models of chronic colitis resembling human CD. We show that TL1A enhances cytokine production from both TH1 and TH17 CD4+ T cells in intestinal mucosa and that neutralization of TL1A attenuates chronic colitis. These results suggest that TL1A is a central immune modulator in activation of mucosal TH1/TH17 CD4+ T cells and that it is critical for the development of chronic colitis. Our findings suggest that neutralization of TL1A could be a novel, highly specific approach for therapeutic intervention in CD. C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions in the Animal Care Facility at Cedars-Sinai Medical Center. Gαi2−/− (129/Sv background) mice were housed at the University of California, Los Angeles Animal Care Facility. RAG2−/− mice (129/Sv background) were obtained from the University of California, Los Angeles Department of Radiation Oncology or purchased from Taconic Farms (Germantown, NY). Mice used in all experiments were handled according to the guidelines and approved protocols of the Cedars-Sinai Medical Center and the University of California, Los Angeles Animal Care and Use Committees. Chronic colitis was induced by multiple-cycle administration of dextran sodium sulfate (DSS) drinking water.17Yoshihara K. Yajima T. Kubo C. et al.Role of interleukin 15 in colitis induced by dextran sulphate sodium in mice.Gut. 2006; 55: 334-341Crossref PubMed Scopus (80) Google Scholar Female mice of 8 weeks of age received either regular drinking water (control) or 3% (wt/vol) DSS drinking water (molecular weight, 40,000–50,000) (MP Biomedicals, Irvine, CA) on days 1–5, 8–12, 15–19, and 22–26. Histology was used to evaluate inflammation, extent, regeneration, crypt damage, and percentage involvement. In the Gαi2−/− T-cell transfer model, colitis was induced by colitigenic Gαi2−/− T cells.18Wei B. Velazquez P. Turovskaya O. et al.Mesenteric B cells centrally inhibit CD4+ T cell colitis through interaction with regulatory T cell subsets.Proc Natl Acad Sci U S A. 2005; 102: 2010-2015Crossref PubMed Scopus (157) Google Scholar The histologic scoring for colitis was based on an established scale using inflammatory and epithelial parameters, as previously reported.19Aranda R. Sydora B.C. McAllister P.L. et al.Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients.J Immunol. 1997; 158: 3464-3473PubMed Google Scholar Disease severity was determined by a combination of macroscopic (grade 0, normal; grade 1, mild; grade 2, moderate; and grade 3, severe) and histologic scores (grade 0, histologic score 0–1; grade 1, 2–4, grade 2, 5–7; grade 3, 8–10; and grade 4, 11–14). See the supplementary materials (see supplementary materials online at www.gastrojournal.org). Mononuclear cells from mesenteric lymph node (MLN) and lamina propria (LP) were isolated. CD4+ T cells were isolated from MLN and LP by negative selection (StemCell Technologies Inc, Vancouver, BC, Canada). Cells were cultured under the conditions indicated in the figure legends. In experiments in Figure 1, for IL-12 and IL-23 detection, cells were cultured with or without 1 μg/mL lipopolysaccharide.Figure 1Development of DSS-induced chronic colitis. Chronic colitis induced by 4 cycles of DSS was evaluated and compared with untreated mice (n = 7 per group). All data represent the mean ± SD. (A) Body weights as a percentage of the initial weight at day 1 are shown. ○, Control; ■, DSS. (B) Colon length from cecum to rectum. (C) Total numbers of mononuclear cells in GALT. *P < .01. (D) Cellular composition of MLN and LP cells was analyzed by flow cytometry and expressed as a percentage of the whole cell population. (E) Cytokine mRNA expression was determined in MLN and colon by real-time polymerase chain reaction. Data were normalized to the expression of β-actin mRNA. P < .01. (F) Mononuclear cells from GALT were cultured with or without anti-CD3ε and anti-CD28 Abs for IFN-γ and IL-17 production and with or without lipopolysaccharide for IL-12 and IL-23 production. *P < .01. Cytokine productions were measured by enzyme-linked immunosorbent assay. (B–F) □, Control; ■, DSS.View Large Image Figure ViewerDownload Hi-res image Download (PPT) See the Supplementary Materials (see supplementary materials online at www.gastrojournal.org). Cells were stained with antibodies against murine CD4 (RM4-5), B220 (RA3-6B2), CD45RB (16A), CD11c (HL3), major histocompatibility complex class II (IA/IE, clone 114.15.2), CD16/CD32 (clone 2.4G2, to block nonspecific FcR binding) (BD Biosciences, San Jose, CA), and TL1A (CoGenesys Inc, Rockville, MD). Intracellular staining was performed with antibodies against IFN-γ and IL-17 (BD Biosciences). See the supplementary materials (see supplementary materials online at www.gastrojournal.org). Total RNA was isolated from MLN cells and whole colons using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into complementary DNA (cDNA) with the Omniscript RT kit (Qiagen, Valencia, CA). cDNA was quantified by real-time polymerase chain reaction using the iCycler Thermal cycler (Bio-Rad, Hercules, CA). TL1A, DR3, IFN-γ, IL-17, tumor necrosis factor-α, and β-actin TaqMan probes and primers were designed using Beacon Design 4.0 (Premier Biosoft International, Palo Alto, CA). See the supplementary materials (see supplementary materials online at www.gastrojournal.org). Neutralizing hamster anti-mouse TL1A monoclonal antibody (mAb) (19E06A hybridoma) was obtained from CoGenesys Inc. Neutralizing anti-mouse IL-23 receptor (IL-23R) mAb was a gift from Dr Charles Elson (University of Alabama, Birmingham, AL). For DSS-induced chronic colitis, 500 μg of anti-TL1A mAb or control immunoglobulin (Ig)G (Leico Technologies, Inc, St Louis, MO) was administered intraperitoneally into mice once per week (days 1, 8, 15, and 22) or twice per week (days 15, 18, 22, and 25). A total of 100 μg of anti–IL-23R mAb or control IgG was administered intraperitoneally once per week (days 1, 8, 15, and 22) or twice per week (days 15, 18, 22, and 25). Mice were killed at day 29 and colitis was evaluated by a trained pathologist blinded to the treatment of the mice. For Gαi2−/− T-cell transferred colitis, 500 μg anti-TL1A mAb or control IgG was injected intraperitoneally twice per week after transfer of colitigenic Gαi2−/− T cells. The Student t test was used for statistical analysis. Differences were considered significant at a P value of less than .05. DSS-induced acute colitis has been known as a T-cell–independent model.20Dieleman L.A. Ridwan B.U. Tennyson G.S. et al.Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice.Gastroenterology. 1994; 107: 1643-1652Abstract PubMed Google Scholar However, in chronic colitis induced by multiple cycles or in the recovery phase of DSS, adaptive immunity plays an important role in the disease process.21Dieleman L.A. Palmen M.J. Akol H. et al.Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.Clin Exp Immunol. 1998; 114: 385-391Crossref PubMed Scopus (825) Google Scholar, 22Kabashima K. Saji T. Murata T. et al.The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut.J Clin Invest. 2002; 109: 883-893Crossref PubMed Scopus (402) Google Scholar Chronic colitis was induced by administration of 4 cycles of 3% DSS drinking water to C57BL/6 mice. Loss of body weight was first observed at day 5. Maximum weight loss was seen at day 12 and mice regained body weight after 2 cycles of DSS (Figure 1A). Mice were killed at day 29. Cecum and colon of DSS-treated mice showed signs of severe colitis including significantly shortened colon length (Figure 1B). Mononuclear cell numbers from MLN and LP were increased owing to infiltration of inflammatory cells in the DSS-treated mice (Figure 1C). CD4+CD45RBlow memory T cells and B cells were increased significantly in lamina propia mononuclear cells of DSS-treated mice (Figure 1D). In MLN, CD4+ T cells, especially CD4+CD45RBlow T cells, also were increased; however, the percentage of CD4+CD45RBlow cells in total CD4+ cells in MLN was less than in LP. IFN-γ from TH1 cells and IL-17 from TH17 cells are both key mediators in several autoimmune diseases and IBD.5Hommes D.W. Mikhajlova T.L. Stoinov S. et al.Fontolizumab, a humanised anti-interferon gamma antibody, demonstrates safety and clinical activity in patients with moderate to severe Crohn's disease.Gut. 2006; 55: 1131-1137Crossref PubMed Scopus (191) Google Scholar, 6Mannon P.J. Fuss I.J. Mayer L. et al.Anti-interleukin-12 antibody for active Crohn's disease.N Engl J Med. 2004; 351: 2069-2079Crossref PubMed Scopus (740) Google Scholar, 23Qian Y. Liu C. Hartupee J. et al.The adaptor Act1 is required for interleukin 17-dependent signaling associated with autoimmune and inflammatory disease.Nat Immunol. 2007; 8: 247-256Crossref PubMed Scopus (426) Google Scholar, 24Ito R. Shin-Ya M. Kishida T. et al.Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice.Clin Exp Immunol. 2006; 146: 330-338Crossref PubMed Scopus (242) Google Scholar In DSS-treated mice, IFN-γ, IL-17, and tumor necrosis factor-α messenger RNA (mRNA) (Figure 1E), and protein (Figure 1F) expressions were significantly higher than in untreated mice. However, IL-4 produced by TH2 cells rarely was detectable in this model (data not shown). In addition to production of IFN-γ and IL-17, IL-12 and IL-23 production also were increased in DSS-induced chronic colitis (Figure 1F). To evaluate the expression of TL1A and its receptor DR3 in DSS-induced chronic colitis, we performed real-time polymerase chain reaction in MLN and colon. TL1A mRNA and DR3 mRNA were increased significantly in DSS-treated mice (Figure 2A). FACS analysis revealed that MLN CD11chigh major histocompatibility complex class II+ DCs derived from DSS-treated mice expressed TL1A (Figure 2B), as Bamias et al9Bamias G. Mishina M. Nyce M. et al.Role of TL1A and its receptor DR3 in two models of chronic murine ileitis.Proc Natl Acad Sci U S A. 2006; 103: 8441-8446Crossref PubMed Scopus (135) Google Scholar showed previously. Because the interaction of APC-derived TL1A and DR3, mainly expressed on CD4+CD45RBlow memory T cells,9Bamias G. Mishina M. Nyce M. et al.Role of TL1A and its receptor DR3 in two models of chronic murine ileitis.Proc Natl Acad Sci U S A. 2006; 103: 8441-8446Crossref PubMed Scopus (135) Google Scholar was shown to induce T-cell activation in the mucosa, we investigated the effect of TL1A on gut-associated lymphoid tissue (GALT) CD4+ cells. IFN-γ and IL-17 produced in DSS-treated mice was enhanced greatly by the stimulation with TL1A (Figure 2C). These results reveal that the interaction of TL1A with DR3 leads to the activation of both TH1 and TH17 cells on CD4+ memory T cells in the GALT of chronic colitis. To determine the role that TL1A might play in both IFN-γ and IL-17 production in the mucosa of DSS-treated mice, we examined the synergistic effect of TL1A with IL-12 or IL-23 in GALT on the production of IFN-γ and IL-17. CD4+ T cells were stimulated with TL1A, IL-12, IL-23, IL-12 plus TL1A, or IL-23 plus TL1A. Stimulation with IL-12 plus TL1A enhanced IFN-γ production (Figure 3A). Interestingly, IL-23 plus TL1A could significantly up-regulate IL-17 production compared with IL-23 alone. The enhancing effects of TL1A on the production of IFN-γ or IL-17 were much more pronounced in the lamina propia mononuclear cells (Figure 3A). To confirm the effect of TL1A on CD4+ T cells, we performed intracellular staining of lamina propia mononuclear cells under conditions activating either TH1 or TH17 cells. TL1A increased the numbers of both IFN-γ–producing cells and IL-17–producing cells (Figure 3B). This result reveals that TL1A itself could up-regulate IFN-γ and IL-17 production in both TH1 and TH17 cells even in the absence of cytokines known to activate these T-cell populations. Moreover, IL-12 plus TL1A, and IL-23 plus TL1A, further increased numbers of IFN-γ– and IL-17–producing cells, respectively (Figure 3B). To further investigate this finding, we examined the effect of IL-23 and IL-23 plus TL1A on IFN-γ production, and the effect of IL-12 and IL-12 plus TL1A on IL-17 production. Our data showed that IL-23 and IL-23 plus TL1A could up-regulate IFN-γ production; however, IL-12 slightly decreased IL-17 production compared with control and IL-12 plus TL1A significantly inhibited IL-17 production compared with TL1A (Figure 3C). Consistent with these findings, IL-17 production was reduced when stimulated with IL-23 plus IL-12, and TL1A could not neutralize the inhibitory effect IL-12 has on IL-17 production (supplementary Figure 1; see supplementary materials online at www.gastrojournal.org). These findings suggest that IL-23 and IL-23 plus TL1A can work synergistically to activate both TH1 and TH17 cells, and to enhance both IFN-γ and IL-17 production from TH17 cells, as shown in Figures 3B and C. TL1A alone is sufficient to induce IL-17 production from lamina propia mononuclear cells but exerts its maximal synergistic effect in the presence of IL-23 (Supplementary Figure 1, Supplementary Figure 2; see supplementary materials online at www.gastrojournal.org).Figure 3TL1A synergizes with IL-12 or IL-23 in the development of a TH1 or TH17 response during chronic colitis. CD4+ T cells from GALT of untreated or DSS-treated mice were cultured with IL-12 or IL-23 with or without the addition of TL1A (10 ng/mL) for 72 hours. (A) IFN-γ and IL-17 production were measured by enzyme-linked immunosorbent assay (n = 5 per group). Data presented are means ± SD. ☐, Control; ▩, DSS. *P < .01. (B) Fluorescence-activated cell sorter analysis of intracellular IFN-γ and IL-17 production by CD4+ T cell isolated from the LP of DSS-treated mice. One representative experiment out of 3 independent experiments is shown. (C) IFN-γ and IL-17 production in LP of DSS-treated mice was measured by enzyme-linked immunosorbent assay (n = 5 per group). *P < .01. (D) IL-6 production from GALT stimulated with or without anti-CD3ε and anti-CD28 was measured by enzyme-linked immunosorbent assay. ☐, Control; ▩, DSS. *P < .01. (E) IL-6 production from GALT CD4+ T cells stimulated with or without TL1A. ☐, No stimulation; ▩, TL1A 10 ng/mL. *P < .01. (F) IL-6 production from GALT CD4+ T cells of DSS-treated mice stimulated with IL-12 or IL-23 with or without the addition of TL1A. *P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IL-6 has been shown to play an important role in the pathogenesis of chronic IBD.25Yamamoto M. Yoshizaki K. Kishimoto T. et al.IL-6 is required for the development of Th1 cell-mediated murine colitis.J Immunol. 2000; 164: 4878-4882Crossref PubMed Scopus (348) Google Scholar, 26Atreya R. Mudter J. Finotto S. et al.Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo.Nat Med. 2000; 6: 583-588Crossref PubMed Scopus (1045) Google Scholar Recent studies have shown that IL-23–dependent TH17 CD4+ T cells produced both IL-17 and IL-6.27Langrish C.L. Chen Y. Blumenschein W.M. et al.IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3173) Google Scholar, 28Yen D. Cheung J. Scheerens H. et al.IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6.J Clin Invest. 2006; 116: 1310-1316Crossref PubMed Scopus (1238) Google Scholar IL-6 production from T cells was increased significantly in DSS-treated mice (Figure 3D). TL1A itself up-regulated IL-6 production in the GALT (Figure 3E). Furthermore, IL-23, but not IL-12, induced IL-6 production and TL1A significantly enhanced IL-23–induced IL-6 production in DSS-treated mice (Figure 3F). In addition, IL-6 production was increased in IL-12 plus TL1A conditions, yet IL-12 itself did not enhance IL-6 production, confirming that TL1A could enhance IL-6 production. These findings indicated that TH17 cells produced both IL-17 and IL-6 in response to IL-23 and that TL1A enhanced IL-6 production from IL-23–primed TH17 cells. It has been reported that IL-6 plays a crucial role in the differentiation of TH17 cells from naive T cells.29Veldhoen M. Hocking R.J. Atkins C.J. et al.TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (2954) Google Scholar, 30Bettelli E. Carrier Y. Gao W. et al.Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature. 2006; 441: 235-238Crossref PubMed Scopus (5433) Google Scholar To exclude the possibility that the increase of IL-17 was induced indirectly via the up-regulation of IL-6 secretion by TL1A, we neutralized endogenous IL-6 by anti–IL-6Rα chain (CD126) antibodies. Endogenous IL-6 did not directly affect IL-17 production by TH17 cells in our experiments (data not shown). To investigate the role of TL1A in this TH1/TH17-mediated chronic colitis, we used anti-mouse TL1A mAb (provided by CoGenesys Inc) in our mouse model. The neutralizing effect of anti-TL1A mAb was assessed in vitro using MLN and co-culturing cells with IL-12 or IL-23 plus TL1A with increasing concentrations of neutralizing anti-TL1A mAb. A total of 10 μg/mL of anti-TL1A mAb completely neutralized the enhancing effect of TL1A on IFN-γ and IL-17 production (supplementary Figure 3; see supplementary material online www.gastrojournal.org). Because our data showed that TL1A enhanced mucosal T-cell activation and IFN-γ, IL-17, and IL-6 production in DSS-induced chronic colitis, neutralizing anti-TL1A mAb were administered once per week in DSS-treated mice starting at day 1 (Figure 4A). Monoclonal Ab were administered by repeated intraperitoneal injections of 500 μg on days 1, 8, 15, and 22. Disease severity and cytokine production were evaluated at day 29. Administration of anti-TL1A mAb led to significant protection, as indicated by significant attenuation of weight loss and colon shortening (Figure 4A and B). Histologic sections of the cecum and colon revealed reduced mucosal inflammation and injury in anti-TL1A mAb-treated mice (Figure 4C). The histologic scores and cell numbers in GALT as well as memory CD4+ T cells (data not shown) were reduced significantly in the anti-TL1A mAb treatment group (Figure 4D and E). IFN-γ, IL-17, and IL-6 produced from GALT, were decreased significantly by anti-TL1A mAb treatment (Figure 4F). Thus, in addition to a significant modulation of chronic colitis, anti-TL1A mAb suppressed the production of IFN-γ (TH1) and IL-17/IL-6 (TH17) cytokines from mucosal T cells.Figure 4Attenuation of TH1 and TH17 responses during DSS-induced chronic colitis by the administration of neutralizing TL1A mAb. Once per week, 500 μg anti-TL1A mAb or control IgG as control were injected intraperitoneally into mice receiving DSS (n = 10 per group). (A) Body weights as a percentage of the initial weight at day 1 are shown. ●, Anti-TL1A; ■, control IgG; ↓, intraperitoneal injection. (B) Left panel: representative colons from DSS-treated mice receiving anti-TL1A mAb or control. Right panel: colon length from cecum to rectum. ☐, Control IgG; ■, anti-TL1A. (C) H&E staining of cecum and colon (original magnification, 100×). (D) Evaluation of histologic score. (E) Mononuclear cell numbers of GALT. (F) IFN-γ, IL-17, and IL-6 productions from GALT. (D–F) ☐, Control IgG; ■, anti-TL1A. *P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we evaluated whether anti-TL1A treatment prevents the deve