Zhiqiong Yang

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.

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.

Suppression of inflammation in ulcerative colitis by interferon-β-1a is accompanied by inhibition of IL-13 production

Objective Ulcerative colitis is associated with increased interleukin 13 (IL-13) production by natural killer T cells. Taking advantage of the inhibitory actions of interferon β on IL-13 expression, this proof-of-concept study aimed to show that decreasing IL-13 production is associated with clinical improvement of ulcerative colitis symptoms. Design Open-label interventional drug trial. Setting Outpatient clinical research hospital. Patients Adult patients with active ulcerative colitis (Short Clinical Colitis Activity Index (SCCAI)≥5). Interventions Treatment with 30 μg IM interferon-β-1a (Avonex) weekly for 12 weeks with 6 month follow-up. Main outcome measures Clinical response was defined as ≥3 point drop in the SCCAI for at least two consecutive monitoring visits, and cytokine production was measured in cultured peripheral blood and lamina propria mononuclear cells (LPMC) before and after treatment. Results 11 of 16 patients were clinical responders, and 4 were in remission (SCCAI ≤ 2) at the end of treatment. Rectal bleeding subscores improved dramatically by week 4 (38% with frank bleeding vs 87% pretreatment). Increased IL-13 production by LPMC T cells fell significantly in clinical responders (690±99 vs 297±58 pg/ml p=0.015) but was unchanged in non-responders (542±83 vs 510±39 pg/ml). In addition, non-responders had significantly higher production of IL-17 and IL-6 pre-treatment compared to responders. Conclusions Interferon-β-1a induces clinical response and remission in a large subset of patients with ulcerative colitis that is associated with significant inhibition of IL-13 production. In addition, increased IL-17 and IL-6 production is associated with no response to interferon-β. These data provide a proof-of-concept that IL-13 is an effector cytokine in ulcerative colitis and should be a target for novel therapies.

IL-13Rα2-bearing, type II NKT cells reactive to sulfatide self-antigen populate the mucosa of ulcerative colitis

Objective Previous studies have shown that ulcerative colitis (UC) is associated with the presence of lamina propria non-invariant (Type II) NKT cells producing IL-13 and mediating epithelial cell cytotoxicity. Here we sought to define the antigen(s) stimulating the NKT cells and to quantitate these cells in the UC lamina propria. Design Detection of Type II NKT cells in UC lamina propria mononuclear cells (LPMC) with lyso-sulfatide loaded tetramer and quantum dot-based flow cytometry and staining. Culture of UC LPMCs with lyso-sulfatide glycolipid to determine sulfatide induction of epithelial cell cytotoxicity, IL-13 production and IL-13Rα2 expression. Blinded quantum dot-based phenotypic analysis to assess UC LPMC expression of IL-13Rα2, CD161 and IL-13. Results Approximately 36% of UC LPMC were lyso-sulfatide tetramer positive, whereas few, if any, control LPMCs were positive. When tested, the positive cells were also CD3 and IL-13Rα2 positive. Culture of UC LPMC with lyso-sulfatide glycolipid showed that sulfatide stimulates UC LPMC production of IL-13 and induces UC CD161 LPMC-mediated cytotoxicity of activated epithelial cells; additionally, lyso-sulfatide induces enhanced expression of IL-13Rα2. Finally, blinded phenotypic analysis of UC LP MC using multicolour quantum dot-staining technology showed that approximately 60% of the LPMC bear both IL-13Rα2 and CD161 and most of these cells also produce IL-13. Conclusions These studies show that UC lamina propria is replete with Type II NKT cells responsive to lyso-sulfatide glycolipid and bearing IL-13Rα2. Since lyso-sulfatide is a self-antigen, these data suggest that an autoimmune response is involved in UC pathogenesis.

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 Crohns 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 Crohns disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohns patients were treated with G‐CSF (5 µg/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. Crohns patients who achieved a clinical response or remission based on the decrease in the Crohns 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‐γ production capacity was not changed significantly except in non‐responders, where it increased. These data show that clinical benefit from G‐CSF treatment in Crohns 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.

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-β, and IL-6, implying that these transcription factors are critical regulators of Th17 induction.

Dynamic changes in E-protein activity regulate T reg cell development

Gao et al. show that E-box proteins dampen the generation and function of Foxp3+ regulatory T cells in part by inhibiting IL-2Rα expression and IL-2 responsiveness.

860 Natural Killer T Cells Bearing IL-13rα2, Producing IL-13 and Having Cytotoxicity for Epithelial Cells Populate the Mucosa in Ulcerative Colitis

exposition in patients after pouch surgery (pouch patients versus IBD patients, p < 0.02). Conclusions:Increased cell shedding and transient epithelial cell destruction with subsequent bacterial translocation are newly discovered features of IBD pathophysiology. These changes can be identified and quantified in humans during ongoing colonoscopy using CLE. This might further support the notion that intrinsic micro-flora can trigger flares in patients with IBD. Thus, CLE might play a crucial role in the future for monitoring mucosal healing in patients with IBD. Epithelial changes of the terminal ileum before and after the exposition of rectal micro-flora