Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages.

Listeria monocytogenes induces apoptosis in vitro and in vivo in a variety of cell types. However, the mechanism of cell death in L. monocytogenes-infected macrophages was initially reported to be distinct from apoptosis. Here, we studied the mechanism of L. monocytogenes-induced cell death using sensitive fluorescent techniques. We found that caspase-1 activation preceded cell death of macrophages infected with L. monocytogenes, using fluorogenic substrates. Caspase-1 activation was diminished after infection with wild-type L. monocytogenes when cells were treated with NH(4)Cl, or if they were infected with a listeriolysin mutant that cannot escape from the phagolysosome. Mitochondrial membrane integrity was preserved during the infection. A particular mechanism of cell death, recently termed 'pyroptosis', is associated with infection by intracellular microorganisms, and has an inherent pro-inflammatory character, due to involvement of caspase-1 activation with consequent IL-1 beta and IL-18 production. Cell death through caspase-1 activation would constitute a defence mechanism of macrophages which induces cell death to eliminate the bacteria's intracytosolic niche and recruits early host's defences through the secretion of inflammatory cytokines.

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening.

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.

Immunization with dendritic cells loaded with alpha-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria.

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.

Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

Butyrate suppresses hypoxia-inducible factor-1 activity in intestinal epithelial cells under hypoxic conditions.

Interaction between the products of intestinal bacteria and the intestinal epithelial cells is a key event in understanding the biological, physiological, and pathological functions of the intestinal epithelium. Here, we examined the effect of butyrate, one of the major intestinal bacterial products, on hypoxia-inducible factor-1 (HIF-1) activity under hypoxic conditions in intestinal epithelial cells. HIF-1 activity was assessed by luciferase assay using cytoplasmic extracts of intestinal epithelial cells, Caco-2, and IEC-6 cells. These cells were transiently transfected with hypoxia response element (HRE)-luciferase reporter plasmids and cultured under hypoxic conditions in the presence or absence of sodium butyrate (NaB). The effect of NaB on HRE DNA binding activity in Caco-2 cells under hypoxic conditions was assessed by electrophoretic mobility shift assay. Expression of a hypoxia-responsive gene encoding intestinal trefoil factor (ITF) in Caco-2 cells after NaB treatment was assessed using reverse-transcription PCR. The barrier function of Caco-2 cells under hypoxic conditions was also evaluated by transepithelial electrical resistance measurement. NaB suppressed up-regulation of HIF-1 transcriptional activity under hypoxic conditions in Caco-2 and IEC-6 cells. In parallel, NaB reduced HRE DNA binding activity under the same conditions. Furthermore, NaB down-regulated enhanced transcription of ITF gene. Addition of NaB under hypoxic conditions delayed recovery of transepithelial electrical resistance of the monolayers after hypoxia-reoxygenation treatment. These findings indicate that NaB suppresses HIF-1 transcriptional activity on hypoxia-responsive genes by reducing the HRE DNA binding activity under hypoxic conditions in intestinal epithelial cells.

Cytotoxic T-lymphocyte-, and helper T-lymphocyte-oriented DNA vaccination.

DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th). DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases. So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology. DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies. Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses. In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection. From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered.

Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo.

We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation. Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge. These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo.

Enhancement of protective immunity against intracellular bacteria using type-1 polarized dendritic cell (DC) vaccine.

The development of effective vaccine strategies for intracellular bacteria, including tuberculosis, is one of the major frontiers of medical research. Our previous studies showed that dendritic cell (DC) vaccine is a promising approach for eliciting protective immunity against intracellular bacteria. However, it has been reported that standard fully mature DCs show reduced ability to produce IL-12p70 upon subsequent interaction with antigen (Ag)-specific T cells, limiting their in vivo performance for vaccines. Recently, we found that such "DC exhaustion" could be prevented by the presence of IL-4 and IFN-γ during the maturation of mouse DCs (type-1 polarization), resulting in improved induction of anti-tumor immunity in cancer. Here we show that such type-1 polarized DCs promote dramatic enhancement of protective immunity against an intracellular bacterium, Listeria monocytogenes. Murine bone marrow-derived DCs were cultured and matured with LPS, IL-4 and IFN-γ (type-1 polarized DCs), and with LPS alone (non-polarized DCs). DCs were loaded with listeriolysin O (LLO) 91-99, H2-K(d)-restricted epitope of L. monocytogenes, and were injected into naïve BALB/c mice intravenously. Type-1 polarized DCs produced significantly higher levels of IL-12p70 than non-polarized DCs in vitro, and this vaccine strongly enhanced LLO 91-99-specific CD8(+) T cells exhibiting epitope-specific cytotoxic activity and IFN-γ production, leading to significant induction of protective immunity against L. monocytogenes. Type-1 polarized DCs are potential candidates for enhancing protective immunity in the design of effective vaccination strategies against intracellular bacteria.

A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70.

Heat shock protein 70 (HSP70) is a member of a highly conserved superfamily of intracellular chaperones called stress proteins that can activate innate and adaptive immune responses. We evaluated the effect of a fusion DNA vaccine that encoded mycobacterial HSP70 and MPT51, a major secreted protein of Mycobacterium tuberculosis. Spleen cells from mice immunized with fusion DNA of full-length HSP70 and MPT51 produced a higher amount of interferon-γ (IFN-γ) in response to the CD4+, but not the CD8+ T-cell epitope peptide on MPT51 than those from mice immunized with MPT51 DNA. Furthermore, because HSP70 comprises the N-terminal ATPase domain and the C-terminal peptide-binding domain, we attempted to identify the domain responsible for its enhancing effect. The fusion DNA vaccine that encoded the C-terminal domain of HSP70 and MPT51 induced a higher MPT51-specific IFN-γ production by CD4+ T cells than the vaccine that encoded MPT51 alone, whereas that with the N-terminal domain did not. Similar results were obtained by immunization with the fusion proteins. These results suggest that the DNA vaccine that encodes a chimeric antigen molecule fused with mycobacterial HSP70, especially with its C-terminal domain, can induce a stronger antigen-specific T-helper cell type 1 response than antigen DNA alone.

Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis using MPT51 overlapping peptides screening and DNA vaccination.

We identified a novel HLA-DR4-restricted CD4+ T-cell epitope on a secreted antigen of Mycobacterium tuberculosis, MPT51, in 004149-MM HLA-DR4-transgenic mice which express HLA-DRB1*0401, but not murine MHC class II molecules. The mice were immunized with plasmid DNA encoding MPT51 using gene gun and interferon (IFN)-gamma production from the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, only one peptide, p191-210, stimulated the splenocytes to produce IFN-gamma. Further analysis using flow cytometry and computer-assisted algorithm, ProPred, narrowed down the region of CD4+ T-cell epitope to p191-202. The CD4+ T-cell epitope would be feasible for vaccine design against tuberculosis as well as for analysis of MPT51-specific T-cells in M. tuberculosis infection.

Chemokine receptor-mediated delivery of mycobacterial MPT51 protein efficiently induces antigen-specific T-cell responses.

Here we evaluated the effects of immunization with a DNA vaccine encoding a fusion protein consisting of macrophage inflammatory protein-1alpha (MIP-1alpha) and MPT51 (a major secreted protein from Mycobacterium tuberculosis) on induction of specific CD8+ T cells. The DNA vaccine encoding the fusion protein could induce significantly higher number of the antigen-specific CD8+ T cells in mice than DNA vaccine encoding MPT51 alone. Also, splenocytes from mice immunized with the fusion DNA vaccine expressed higher level of IFN-gamma mRNA and protein upon stimulation with an epitope peptide derived from MPT51 than those from mice immunized with a mixture of two DNA vaccines encoding either MPT51 or MIP-1alpha. These results suggest that DNA vaccine encoding MIP-1alpha-antigen fusion protein is able to be efficiently internalized into antigen-presenting cells via the chemokine receptor and induce higher level of antigen-specific CD8+ T-cell responses.

In vivo hierarchy of individual T-cell epitope-specific helper T-cell subset against an intracellular bacterium.

Cellular immunity is indispensable for efficient protection against intracellular bacterial infection. CD4+ and CD8+ T cells specific for a variety of antigenic peptides derived from particular bacteria are induced after the infection. T cells recognizing different antigenic peptides have been speculated to have different functions in terms of the protective immunity. We here induced individual CD4+ T cells specific for each antigenic peptide derived from Listeria monocytogenes independently with DNA vaccines using gene gun bombardment and compared the CD4+ T-cell populations for their ability on the specific protective immunity against lethal listerial challenge and analyzed their characteristics.

Intratracheal administration of third-generation lentivirus vector encoding MPT51 from Mycobacterium tuberculosis induces specific CD8+ T-cell responses in the lung.

The present study evaluates the potential of improved third-generation lentivirus vector with respect to their use as an in vivo-administered T-cell vaccine against tuberculosis. Intratracheal administration of the lentivirus vector encoding MPT51 of Mycobacterium tuberculosis could induce MPT51-specific CD8+ T cells in the mediastinal lymph nodes 2 weeks after the administration. The vaccination could generate MPT51-specific memory CD8+ T cells in the lung, but not in the lymph nodes. Further, a single intratracheal immunization of MPT51 lentiviral vaccine decreased significantly the number of virulent M. tuberculosis in the lung after intratracheal challenge of the bacillus. These findings suggest that intratracheal immunization of the third-generation lentiviral vaccines is a promising vaccination strategy against pulmonary tuberculosis.

Immunization with a gene encoding granulocyte-macrophage colony-stimulating factor inserted with a single helper T-cell epitope of an intracellular bacterium induces a specific T-cell subset and protective immunity.

We evaluated here the effect of immunization with a gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted with a helper T cell (Th) epitope, listeriolysin O (LLO) 215-226 derived from Listeria monocytogenes on induction of a specific Th by gene gun bombardment. Immunization of C3H/He mice with pGM215m plasmid encoding murine GM-CSF inserted with LLO 215-226 Th epitope gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pGM215m-immunized mice showed significant protective immunity against lethal listerial challenge. These results suggest that immunization of a gene for GM-CSF inserted with a Th epitope is useful for eliciting a specific Th subset in vivo.

Immunization with dendritic cells retrovirally transduced with mycobacterial antigen 85A gene elicits the specific cellular immunity including cytotoxic T-lymphocyte activity specific to an epitope on antigen 85A.

In the present study, we evaluated antigen 85A (Ag85A) gene-transduced dendritic cells (DCs) vaccine against Mycobacterium tuberculosis. Murine bone marrow-derived DCs were retrovirally transduced with mycobacterial Ag85A gene and injected to BALB/c mice intravenously. The DC vaccine was capable of inducing purified protein derivative (PPD)- and the antigen-specific spleen cell proliferation and IFN-gamma production from both CD4+ and CD8+ T cells in spleens of the immune mice. In addition, the DC vaccination induced cytotoxic T-lymphocytes (CTL) and IFN-gamma-producing cells specific for a 9-mer CTL epitope on Ag85A molecule. This eliciting cellular immunity led to protection against wasting disease due to M. tuberculosis infection and induction of moderate bacterial clearance.

Curcumin maintenance therapy for ulcerative colitis: randomized, multicenter, double-blind, placebo-controlled trial.

BACKGROUND & AIMS: Curcumin is a biologically active phytochemical substance present in turmeric and has pharmacologic actions that might benefit patients with ulcerative colitis (UC). The aim in this trial was to assess the efficacy of curcumin as maintenance therapy in patients with quiescent ulcerative colitis (UC). METHODS: Eighty-nine patients with quiescent UC were recruited for this randomized, double-blind, multicenter trial of curcumin in the prevention of relapse. Forty-five patients received curcumin, 1g after breakfast and 1g after the evening meal, plus sulfasalazine (SZ) or mesalamine, and 44 patients received placebo plus SZ or mesalamine for 6 months. Clinical activity index (CAI) and endoscopic index (EI) were determined at entry, every 2 months (CAI), at the conclusion of 6-month trial, and at the end of 6-month follow-up. RESULTS: Seven patients were protocol violators. Of 43 patients who received curcumin, 2 relapsed during 6 months of therapy (4.65%), whereas 8 of 39 patients (20.51%) in the placebo group relapsed (P=.040). Recurrence rates evaluated on the basis of intention to treat showed significant difference between curcumin and placebo (P=.049). Furthermore, curcumin improved both CAI (P=.038) and EI (P=.0001), thus suppressing the morbidity associated with UC. A 6-month follow-up was done during which patients in both groups were on SZ or mesalamine. Eight additional patients in the curcumin group and 6 patients in the placebo group relapsed. CONCLUSIONS: Curcumin seems to be a promising and safe medication for maintaining remission in patients with quiescent UC. Further studies on curcumin should strengthen our findings.

IFN-gamma overcomes low responsiveness of myeloid dendritic cells to CpG DNA.

Dendritic cells (DC) are professional APC that have an extraordinary capacity to prime naive T cells. It has been reported that human DC subsets express distinct toll-like receptor (TLR), which influences their function. In mice, we observed that plasmocytoid DC (pDC) express a higher level of TLR9 compared with myeloid DC (mDC) cultured with GM-CSF. However, we demonstrated that stimulation with IFN-gamma is capable of upregulating TLR9 expression in mDC to a level comparable with expression in pDC. Consistent with this observation, IL-12 p40 and IL-6 mRNA expression and IL-12 p70 secretion in response to CpG-oligodeoxynucleotides are enhanced in mDC pretreated with IFN-gamma compared with untreated cells. Therefore, TLR-mediated responses of DC subsets may be influenced not only by signals delivered by pathogens but also by regulatory signals from cytokines such as IFN-gamma.

Curcumin prevents and ameliorates trinitrobenzene sulfonic acid-induced colitis in mice.

BACKGROUND & AIMS: Curcumin is known to have a variety of pharmacologic effects, including antitumor, anti-inflammatory, and anti-infectious activities. The pleiotropic effects of curcumin are attributable at least in part to inhibition of transcriptional factor nuclear factor kappaB (NF-kappaB). However, the effect of curcumin on intestinal inflammation has hitherto not been evaluated. The aim of this study was to determine whether treatment with curcumin prevents and ameliorates colonic inflammation in a mouse model of inflammatory bowel disease. METHODS: Mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with 0.5%, 2.0%, or 5.0% curcumin in the diet, and changes in body weight together with histologic scores were evaluated. Colonic T-cell subsets were characterized, and NF-kappaB in colonic mucosa was detected by immunohistochemistry. NF-kappaB activity in the colonic mucosa was evaluated using electrophoretic mobility shift assay. Cytokine messenger RNA expression in colonic tissue was assessed by semiquantitative reverse-transcription polymerase chain reaction. RESULTS: Treatment of mice with curcumin prevented and improved both wasting and histopathologic signs of TNBS-induced colonic inflammation. Consistent with these findings, CD4(+) T-cell infiltration and NF-kappaB activation in colonic mucosa were suppressed in the curcumin-treated group. Suppression of proinflammatory cytokine messenger RNA expression in colonic mucosa was also observed. CONCLUSIONS: This study has shown for the first time that treatment with curcumin can prevent and improve murine experimental colitis. This finding suggests that curcumin could be a potential therapeutic agent for the treatment of patients with inflammatory bowel disease.

Induction of protective immunity to Listeria monocytogenes with dendritic cells retrovirally transduced with a cytotoxic T lymphocyte epitope minigene.

In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8+ T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens.

Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice.

BACKGROUND AND AIMS: The imbalance between helper T (Th)1/Th2 cytokines has been observed in human inflammatory bowel disease and various animal models. Because interleukin (IL)-12 and interferon-gamma (IFN-gamma) productions are known to be a hallmark of Th1-dominant intestinal inflammation such as 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we strictly addressed the roles of IFN-gamma and IL-12 in the development of colitis, employing knockout mice with IFN-gamma receptor (IFN-gammaR) or IL-12 p40 gene disruptions and mice administered with neutralizing monoclonal antibodies (mAbs) against IFN-gamma or IL-12. METHODS: To induce colitis, 2.5 mg of the hapten reagent TNBS in 50% ethanol was administered into the colon. Two milligrams of rat anti-mouse IFN-gamma mAb, rat anti-mouse IL-12 mAb, or control rat IgG were administered intraperitoneally into mice before TNBS administration. Change in the body weight of mice was evaluated and the degree of inflammation of the colon of these mice was investigated histologically. Immunohistochemical and immunofluorescence analyses were performed to detect CD4+ T cells, macrophages and IL-12 in TNBS-induced colitis lesions. The profile of Th1 and Th2 cytokine expressions in colonic tissues was examined by cytokine-specific semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Single rectal administration of TNBS developed significant colitis in IFN-R-/- mice and anti-IFN-gamma mAb-pretreated mice, as well as control wild-type mice. Conversely, administration of TNBS produced no signs of colitis in IL-12 p40-/- and anti-IL-12 mAb-pretreated mice. CONCLUSIONS: IL-12, but not IFN-gamma, plays a pivotal role in the pathogenesis of TNBS-induced colitis.