Ivan J. Fuss

The fundamental basis of inflammatory bowel disease

Two broad hypotheses have arisen regarding the fundamental nature of the pathogenesis of inflammatory bowel diseases (IBDs, which include ulcerative colitis and Crohn disease). The first contends that primary dysregulation of the mucosal immune system leads to excessive immunologic responses to normal microflora. The second suggests that changes in the composition of gut microflora and/or deranged epithelial barrier function elicits pathologic responses from the normal mucosal immune system. Here we examine these hypotheses and conclude that IBD is indeed characterized by an abnormal mucosal immune response but that microbial factors and epithelial cell abnormalities can facilitate this response.

TGF-β1 Plays an Important Role in the Mechanism of CD4 + CD25 + Regulatory T Cell Activity in Both Humans and Mice

In previous studies, we have shown that murine CD4+CD25+ regulatory T cells produce high levels of TGF-β1 in a cell surface and/or secreted form, and blockade of such TGF-β1 by anti-TGF-β curtails the ability of these cells to suppress CD25− T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-β1 in suppression by CD4+CD25+ T cells, we show in this study that another TGF-β1-blocking molecule, recombinant latency-associated peptide of TGF-β1 (rLAP), also reverses suppression by mouse CD4+CD25+ T cells as well as their human counterparts, CD4+CD25high T cells. In addition, we show that CD25− T cells exposed to CD4+CD25+ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF-β-inducible surface integrin. In further studies, we show that while CD4+CD25+ T cells from TGF-β1-deficient mice can suppress CD25− T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4+LAP+, but not CD4+LAP− T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-β1 produced by CD4+CD25+ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.

Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis

While Crohn disease (CD) has been clearly identified as a Th1 inflammation, the immunopathogenesis of its counterpart inflammatory bowel disease, ulcerative colitis (UC), remains enigmatic. Here we show that lamina propria T (LPT) cells from UC patients produce significantly greater amounts of IL-13 (and IL-5) than control cells and little IFN-gamma, whereas comparable cells from CD patients produce large amounts of IFN-gamma and small amounts of IL-13. We then show that stimulation of UC LPT cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing transfected CD1d induces substantial IL-13 production. While this provided firm evidence that the IL-13-producing cell is an NK T (NKT) cell, it became clear that this cell does not express invariant NKT cell receptors characteristic of most NKT cells since there was no increase in cells binding alpha-galactosylceramide-loaded tetramers, and alpha-galactosylceramide did not induce IL-13 secretion. Finally, we show that both human NKT cell lines as well as UC CD161(+) LPT cells are cytotoxic for HT-29 epithelial cells and that this cytotoxicity is augmented by IL-13. These studies show that UC is associated with an atypical Th2 response mediated by nonclassical NKT cells producing IL-13 and having cytotoxic potential for epithelial cells.

Cutting Edge: Regulatory T Cells Induce CD4+CD25−Foxp3− T Cells or Are Self-Induced to Become Th17 Cells in the Absence of Exogenous TGF-β

Recent studies have shown that TGF-β together with IL-6 induce the differentiation of IL-17-producing T cells (Th17) T cells. We therefore examined whether CD4+CD25+Foxp3+ regulatory T cells, i.e., cells previously shown to produce TGF-β, serve as Th17 inducers. We found that upon activation purified CD25+ T cells (or sorted GFP+ T cells obtained from Foxp3-GFP knockin mice) produce high amounts of soluble TGF-β and when cultured with CD4+CD25−Foxp3− T cells in the presence of IL-6 induce the latter to differentiate into Th17 cells. Perhaps more importantly, upon activation, CD4+CD25+Foxp3+(GFP+) T cells themselves differentiate into Th17 cells in the presence of IL-6 (and in the absence of exogenous TGF-β). These results indicate that CD4+CD25+Foxp3+ regulatory T cells can function as inducers of Th17 cells and can differentiate into Th17 cells. They thus have important implications to our understanding of regulatory T cell function and their possible therapeutic use.

Proinflammatory Cytokines in the Pathogenesis of Inflammatory Bowel Diseases

The cytokine responses characterizing the inflammatory bowel diseases are the key pathophysiologic elements that govern the initiation, evolution, and, ultimately, the resolution of these forms of inflammation. Studies during the last 2 decades now provide a detailed (but not yet complete) picture of the nature of these responses. The first tier of cytokine responses are governed by the T-cell differentiation patterns dominating the disease. In Crohns disease, the major cytokines arise from T-helper cell (Th) 1 and Th17 CD4(+) T-cell differentiation and consist of interferon-γ and interleukin (IL)-17/IL-22 generated by these types of differentiation. The relative importance of these cytokines to Crohns inflammation is still unclear, although evidence is mounting that interferon-γ is primus inter pare (first among equals). In contrast, in ulcerative colitis, a Th2-like differentiation process is paramount, which results in expansion of natural killer T cells producing IL-13 (and perhaps IL-5). These disease-specific cytokine patterns give rise to a second tier of cytokines that span the Th1/Th17-Th2 divide and act as upstream facilitators and downstream mediators of inflammation. These cytokines include the well-known tumor necrosis factor-α, IL-1β, IL-6 triumphirate, as well as a more recently studied cytokine known as TL1A (tumor necrosis factor-like ligand). In this review, we will explore this cytokine landscape with the view of providing an understanding of how recent and future anticytokine therapies actually function.

Oxazolone Colitis, a Th2 Colitis Model Resembling Ulcerative Colitis, Is Mediated by IL-13-Producing NK-T Cells

Oxazolone colitis (OC) is an experimental colitis that has a histologic resemblance to human ulcerative colitis. Here we show that IL-13 production is a significant pathologic factor in OC since its neutralization by IL-13Ralpha2-Fc administration prevents colitis. We further show that OC is mediated by NK-T cells since it can be induced neither in mice depleted of NK-T cells nor in mice that cannot present antigen to NK-T cells and mice lacking an NK-T cell-associated TCR. Finally, we show that NK-T cells are the source of the IL-13, since they produce IL-13 upon stimulation by alpha-galactosylceramide, an NK-T cell-specific antigen. These data thus describe a cellular mechanism underlying an experimental colitis that may explain the pathogenesis of ulcerative colitis.

Reciprocal IFN-γ and TGF-β responses regulate the occurrence of mucosal inflammation

Abstract Recent studies of oral tolerance and experimental colitis indicate that the occurrence of gastrointestinal inflammation is determined by a balance between proinflammatory interferon γ responses and anti-inflammatory transforming growth factor β responses. Here, Warren Strober and colleagues discuss the new findings.

Both IL-12p70 and IL-23 Are Synthesized During Active Crohn's Disease and Are Down-regulated by Treatment with Anti-IL-12 p40 Monoclonal Antibody

Background: Interleukin (IL)‐12p70 and IL‐23 are key T helper‐1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL‐12 and a p19 chain in IL‐23, making both potentially susceptible to modulation by an anti‐IL‐12p40 monoclonal antibody (mAb). Methods: In the present study, we sought to determine whether active inflammation in Crohns disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti‐IL‐12p40 mAb down‐regulate IL‐23 as well as IL‐12p70 as previous reported. Results: To this end we initially determined that IL‐12p70 secretion by control and CD antigen‐presenting cells (macrophages) in lamina propria mononuclear populations is optimized by stimulation with CD40L and interferon‐&ggr;. In subsequent studies using these stimulation conditions we found that patients with CD manifested both increased IL‐12p70 and IL‐23 secretion before anti‐IL‐12p40 mAb treatment and normal levels of secretion of these cytokines following cessation of treatment. Antigen‐presenting cells in lamina propria mononuclear cells from ulcerative colitis patients, in contrast, produced only baseline levels of IL‐23. Finally, we found that IL‐23‐induced T cell production of IL‐17 and IL‐6 are also greatly reduced after antibody treatment. The latter data are parallel to those from previous studies showing that anti‐IL‐12p40 down‐regulates IFN‐&ggr; and tumor necrosis factor‐&agr; secretion. Conclusions: We conclude that CD but not ulcerative colitis is associated with high levels of both IL‐12p70 and IL‐23 secretion as well as the secretion of downstream effector cytokines, and that this cytokine production is down‐regulated following administration of IL‐12p40 mAb.

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease.

The Interrelated Roles of TGF-β and IL-10 in the Regulation of Experimental Colitis

In the present study, we define the relation between TGF-β and IL-10 in the regulation of the Th1-mediated inflammation occurring in trinitrobenzene sulfonic acid (TNBS)-colitis. In initial studies, we showed that the feeding of trinitrophenol-haptenated colonic protein to SJL/J mice induces CD4+ regulatory T cells that transfer protection from induction of TNBS-colitis, and that such protection correlates with cells producing TGF-β, not IL-10. Further studies in which SJL/J mice were fed haptenated colonic protein, and then administered either anti-TGF-β or anti-IL-10 at the time of subsequent TNBS administration per rectum, showed that while both Abs abolished protection, anti-TGF-β administration prevented TGF-β secretion, but left IL-10 secretion intact; whereas anti-IL-10 administration prevented both TGF-β secretion and IL-10 secretion. Thus, it appeared that the protective effect of IL-10 was an indirect consequence of its effect on TGF-β secretion. To establish this point further, we conducted adoptive transfer studies and showed that anti-IL-10 administration had no effect on induction of TGF-β producing T cells in donor mice. However, it did inhibit their subsequent expansion in recipient mice, probably by regulating the magnitude of the Th1 T cell response which would otherwise inhibit the TGF-β response. Therefore, these studies suggest that TGF-β production is a primary mechanism of counter-regulation of Th1 T cell-mediated mucosal inflammation, and that IL-10 is necessary as a secondary factor that facilitates TGF-β production.