NOD2 downregulates colonic inflammation by IRF4-mediated inhibition of K63-linked polyubiquitination of RICK and TRAF6.

It is well established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15) gene, a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses. Here we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine-threonine kinase). We then show that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and thus to downregulation of nuclear factor (NF)-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD.

Transcription of RORγt in developing Th17 cells is regulated by E-proteins.

In the present study we investigated the molecular mechanisms regulating the expression of RAR-related orphan receptor gamma t (RORγt), the central factor controlling interleukin (IL)-17 transcription and Th17 differentiation. In key studies, we found that cells from mice with major deletions of E-protein transcription factors, E2A and HEB, display greatly reduced RORγt/IL-17 expression and that E-protein-deficient mice exhibit greatly diminished IL-17-dependent inflammation in experimental allergic encephalitis models. In additional studies, we unexpectedly found that cells from mice with deletion of Id3, a protein that inhibits E-protein binding to DNA, display diminished RORγt/IL-17 expression and mice deficient in this protein exhibit decreased Th17-mediated inflammation in a cell-transfer colitis model. The explanation of these initially paradoxical findings came from studies showing that Id3 deficiency leads to increased IL-4-induced GATA-3 expression, the latter a negative regulator of RORγt transcription; thus, increased Id3 expression likely has a net positive effect on RORγt expression via its inhibition of IL-4 production. Finally, we found that both E-proteins and Id3 are upregulated in tandem by the cytokines that induce Th17 differentiation, transforming growth factor-ß, and IL-6, implying that these transcription factors are critical regulators of Th17 induction.

Successful granulocyte-colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin-10-producing T cells and increased lamina propria plasmacytoid dendritic cells.

Granulocyte-colony stimulating factor (G-CSF) has proved to be a successful therapy for some patients with Crohn's disease. Given the known ability of G-CSF to exert anti-T helper 1 effects and to induce interleukin (IL)-10-secreting regulatory T cells, we studied whether clinical benefit from G-CSF therapy in active Crohn's disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohn's patients were treated with G-CSF (5 microg/kg/day subcutaneously) for 4 weeks and changes in cell phenotype, cytokine production and dendritic cell subsets were measured in the peripheral blood and colonic mucosal biopsies using flow cytometry, enzyme-linked immunosorbent assay and immunocytochemistry. Crohn's patients who achieved a clinical response or remission based on the decrease in the Crohn's disease activity index differed from non-responding patients in several important ways: at the end of treatment, responding patients had significantly more CD4(+) memory T cells producing IL-10 in the peripheral blood; they also had a greatly enhanced CD123(+) plasmacytoid dendritic cell infiltration of the lamina propria. Interferon-gamma production capacity was not changed significantly except in non-responders, where it increased. These data show that clinical benefit from G-CSF treatment in Crohn's disease is accompanied by significant induction of IL-10 secreting T cells as well as increases in plasmacytoid dendritic cells in the lamina propria of the inflamed gut mucosa.

The role of IL-13 and NK T cells in experimental and human ulcerative colitis.

Recent studies that have evaluated the immunologic factors that mediate the development of the two forms of inflammatory bowel disease, namely Crohn's disease and ulcerative colitis (UC), have suggested that these diseases are because of disparate immune responses. Although Crohn's disease has been characterized as a dysregulation of the T helper (Th)1/Th17 pathways more recent evidence has emerged that UC pathogenesis is associated with a nonclassical NK (natural killer) T cell producing an atypical Th2 (interleukin (IL)-13) response. In the following review the insights gained from both animal models and human studies as to the function that IL-13 and NK T cells have in the pathogenesis of UC will be discussed.

Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis.

Trinitrobenzene sulfonic acid (TNBS)-induced colitis is an IL-12-driven, Th1 T cell-mediated colitis that resembles human Crohn's disease. In the present study, we showed initially that the oral administration of recombinant subunit B of cholera toxin (rCT-B) at the time of TNBS-induced colitis by intrarectal TNBS instillation inhibits the development of colitis or, at later time when TNBS-induced colitis is well established, brings about resolution of the colitis. Dose-response studies showed that a majority of mice (68%) treated with rCT-B at a dose of 100 microg (times four daily doses) exhibited complete inhibition of the development of colitis, whereas a minority (30%) treated with rCT-B at a dose of 10 microg (times four daily doses) exhibited complete inhibition; in both cases, however, the remaining mice exhibited some reduction in the severity of inflammation. In further studies, we showed that rCT-B administration is accompanied by prevention/reversal of increased IFN-gamma secretion (the hallmark of a Th1 response) without at the same time causing an increase in IL-4 secretion. This decreased IFN-gamma secretion was not associated with the up-regulation of the secretion of counterregulatory cytokines (IL-10 or TGF-beta), but was associated with a marked inhibition of IL-12 secretion, i.e., the secretion of the cytokine driving the Th1 response. Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation. From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can conceivably be applied to the treatment of Crohn's disease.

Treatment of experimental (Trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-beta1 plasmid: TGF-beta1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor beta2 chain downregulation.

In this study, we show that a single intranasal dose of a plasmid encoding active transforming growth factor beta1 (pCMV-TGF-beta1) prevents the development of T helper cell type 1 (Th1)-mediated experimental colitis induced by the haptenating reagent, 2,4, 6-trinitrobenzene sulfonic acid (TNBS). In addition, such plasmid administration abrogates TNBS colitis after it has been established, whereas, in contrast, intraperitoneal administration of rTGF-beta1 protein does not have this effect. Intranasal pCMV-TGF-beta1 administration leads to the expression of TGF-beta1 mRNA in the intestinal lamina propria and spleen for 2 wk, as well as the appearance of TGF-beta1-producing T cells and macrophages in these tissues, and is not associated with the appearances of fibrosis. These cells cause marked suppression of interleukin (IL)-12 and interferon (IFN)-gamma production and enhancement of IL-10 production; in addition, they inhibit IL-12 receptor beta2 (IL-12Rbeta2) chain expression. Coadministration of anti-IL-10 at the time of pCMV-TGF-beta1 administration prevents the enhancement of IL-10 production and reverses the suppression of IL-12 but not IFN-gamma secretion. However, anti-IL-10 leads to increased tumor necrosis factor alpha production, especially in established colitis. Taken together, these studies show that TGF-beta1 inhibition of a Th1-mediated colitis is due to: (a) suppression of IL-12 secretion by IL-10 induction and (b) inhibition of IL-12 signaling via downregulation of IL-12Rbeta2 chain expression. In addition, TGF-beta1 may also have an inhibitory effect on IFN-gamma transcription.

Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice.

BACKGROUND & AIMS: Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a T-helper 1 (Th1) T cell-mediated inflammation that is rapidly reversed by administration of anti-interleukin (IL) 12. This study sought to define the mechanism of this curative effect. METHODS: Cells and tissue from mice with TNBS colitis receiving treatment with anticytokines were analyzed for phenotype, cytokine production, and apoptosis. RESULTS: In initial studies, we found that treatment of mice with TNBS-induced colitis with anti-IL-12 was more effective than with anti-interferon (IFN)-gamma, and that anti-IL-12 led to complete normalization of IFN-gamma production by lamina propria T cells ex vivo, whereas anti-IFN-gamma did not. These data suggesting that anti-IL-12 leads to reversal of colitis by elimination of the Th1 T cells were substantiated by studies showing that anti-IL-12 treatment led to increased numbers of apoptotic cells in the lamina propria and spleen by both TUNEL staining of tissues and dispersed spleen cell populations. Finally, we found that the observed apoptosis was mediated by the Fas pathway because (1) MRL/MpJ-lpr(fas) mice lacking Fas function develop colitis that responds poorly to treatment with anti-IL-12; and (2) SJL/J mice with TNBS colitis that received Fas-Fc to block the Fas pathway were resistant to anti-IL-12 treatment. CONCLUSIONS: These studies show that a main mechanism of action of anti-IL-12 in TNBS-induced colitis is the induction of Fas-mediated apoptosis of the Th1 T cells, causing inflammation.

Intestinal lymphangiectasia, a disease characterized by selective loss of naive CD45RA+ lymphocytes into the gastrointestinal tract.

Intestinal lymphangiectasia (InL) is a disease characterized by hypoproteinemia and lymphocytopenia resulting from blocked intestinal lymphatics and loss of lymph fluid into the gastrointestinal tract. This leads to immunologic abnormalities including hypogammaglobulinemia, skin anergy and impaired allograft rejection. In the present study, we evaluated whether the above immunologic abnormalities are secondary to a quantitative or qualitative disorder of T cells. In initial studies we demonstrated that adult InL patients' peripheral blood contain strikingly (and significantly) reduced numbers of CD4+/CD45RA+ T cells, whereas the numbers of CD4+/CD45RO+ T cells were only moderately (and not significantly) reduced. In addition, the CD4+/CD45RO+ T cell population contained an increased percentage of highly differentiated and previously sensitized cells, as demonstrated by decreased CD27 and CD31 expression and increased HLA-DR and CD69 expression. In subsequent functional studies, we showed that the InL CD4+/CD45RO+ T cells, when stimulated in vitro, proliferate fivefold less than control CD4+/CD45RO+ T cells and produce fourfold more IL-4 and threefold less IFN-gamma and IL-2. Thus, this cytokine production profile also reflects the highly differentiated nature of the residual cell population. Overall, these studies provide new information on the trafficking of naive/mature and Th1/Th2 T cell populations in this disease model.

Oxazolone colitis: A murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4.

In this study we describe oxazolone colitis, a new form of experimental colitis. This model is induced in SJL/J mice by the rectal instillation of the haptenating agent, oxazolone, and is characterized by a rapidly developing colitis confined to the distal half of the colon; it consists of a mixed neutrophil/lymphocyte infiltration limited to the superficial layer of the mucosa which is associated with ulceration. Oxazolone colitis is a T helper cell type 2 (Th2)-mediated process since stimulated T cells from lesional tissue produce markedly increased amounts of interleukin (IL)-4 and IL-5; in addition, anti-IL-4 administration leads to a striking amelioration of disease, whereas anti-IL-12 administration either has no effect or exacerbates disease. Finally, this proinflammatory Th2 cytokine response is counterbalanced by a massive transforming growth factor-beta (TGF-beta) response which limits both the extent and duration of disease: lesional (distal) T cells manifest a 20-30-fold increase in TGF-beta production, whereas nonlesional (proximal) T cells manifest an even greater 40-50-fold increase. In addition, anti-TGF-beta administration leads to more severe inflammation which now involves the entire colon. The histologic features and distribution of oxazolone colitis have characteristics that resemble ulcerative colitis (UC) and thus sharply distinguish this model from most other models, which usually resemble Crohn's disease. This feature of oxazolone colitis as well as its cytokine profile have important implications to the pathogenesis and treatment of UC.

The pathogenesis of mucosal inflammation in murine models of inflammatory bowel disease and Crohn disease.

In recent years, it has become apparent that overproduction of the Th1 cytokines interleukin-12 and interferon-gamma is the probable driving force behind murine models of intestinal inflammation resembling Crohn disease and intestinal inflammation in humans with Crohn disease. In addition, studies of murine models strongly suggest that this overproduction is associated with inadequate secretion of the counter-regulatory and anti-inflammatory cytokine transforming growth factor-beta. Thus, mucosal inflammation in models (and possibly in humans) may result from an imbalance between normally occurring positive (immunogenic or inflammatory) responses and negative (tolerogenic or anti-inflammatory) mucosal immune responses. These new findings and the hypotheses that arise from them are being used to construct new approaches to the treatment of Crohn disease that are based on the administration of anti-inflammatory cytokines and anti-cytokine antibodies.

Characteristic T helper 2 T cell cytokine abnormalities in autoimmune lymphoproliferative syndrome, a syndrome marked by defective apoptosis and humoral autoimmunity.

Autoimmune lymphoproliferative syndrome (ALPS) is marked by massive lymphadenopathy, hepatosplenomegaly, autoimmunity and the presence of increased numbers of circulating and tissue TCR-alpha beta, CD4- CD8- T cells. The underlying defect is that of decreased T cell and B cell apoptosis, due in most, but not all, cases to heterozygous mutations of the Fas gene and corresponding defective Fas signaling function. Here we measure in vivo and in vitro cytokine secretion in ALPS to shed light on the relation of apoptosis defects to the development of autoimmunity. In in vivo studies, ALPS patients manifested greatly increased circulating levels of IL-10 (> 100-fold), compared with both healthy individuals and various disease controls; in contrast, their levels of IL-1 beta, IL-4, and IFN-gamma were normal and their levels of IL-2 and TNF-alpha were marginally increased. In parallel in vitro studies, ALPS patients CD4+ DR+ T cells stimulated either with anti-CD3/CD28 or anti-CD2/CD28 produced increased amounts of IL-4 and IL-5 (10 to 20-fold) and decreased amounts of IFN-gamma (4-fold) as compared with those of control CD4+ DR+ T cells. In contrast, ALPS patients' CD4-/CD8- T cells produced very low amounts of cytokines. Finally, ALPS patients' peripheral monocytes/macrophages produced decreased amounts of IL-12 (30-fold) and increased amounts of IL-10 (5-fold). In conclusion, ALPS is marked by the presence of DR+ T cells that exhibit a skewed Th2 cytokine response upon various forms of stimulation. This cytokine response, in the presence of increased circulating IL-10 levels, is likely to define the cytokine milieu that accounts for the humoral autoimmune features of ALPS and, perhaps, of other humoral autoimmune states.

Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.

In this study, we investigate whether human inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) is associated with altered lymphokine secretion profiles, as recently found in various animal models of chronic intestinal inflammation. In initial studies, we determined the proliferative responses of purified lamina propria (LP) CD4+ T cells from patients with IBD under defined conditions of T cell stimulation. We found that IBD LP CD4+ T cells in comparison with control LP CD4+ T cells have diminished TCR/CD3 pathway proliferative responses, whereas CD2/CD28 accessory pathway proliferative responses are relatively preserved. In further studies centering on lymphokine production, we showed that LP T cells from inflamed Crohn's disease mucosa manifest increased IFN-gamma secretion compared with control LP T cells, particularly when stimulated via the CD2/CD28 pathway. Subsequent ELISPOT analysis indicated that this was due to an increased number of IFN-gamma-secreting CD4+ T cells. In contrast, IL-4 and IL-5 production by Crohn's disease LP T cells was decreased compared with that of control LP T cells. Of interest, IL-2 production by Crohn's disease LP T cells was also reduced, as was IL-2 production by peripheral blood T cells. In parallel studies, LP T cells from inflamed ulcerative colitis mucosa stimulated via either the TCR/CD3/CD28 or CD2/CD28 produced increased amounts of IL-5, again when measured either as secreted IL-5 or by ELISPOT analysis. Such increased IL-5 production was not associated with increased IL-4 secretion and, in contrast to Crohn's disease, ulcerative colitis LP T cell production of IL-2 and IFN-gamma was normal. Taken together, these studies provide strong evidence that the immunopathologic process characteristic of the two major forms of IBD is associated with very different cytokine secretion patterns. These different patterns may determine the type of inflammatory process present.