Monica Boirivant

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.

Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-β-Bearing Regulatory Cells

Recent studies of murine models of mucosal inflammation suggest that, whereas some kinds of bacterial microflora are inducers of disease, others, known as probiotics, prevent disease. In the present study, we analyzed the regulatory cytokine and cell response to probiotic (VSL#3) administration in the context of the Th1 T cell colitis induced by trinitrobenzene sulfonic acid treatment of SJL/J mice. Daily administration of probiotics for 3 wk to mice during a remission period between a first and second course of colitis induced by trinitrobenzene sulfonic acid, resulted in a milder form of recurrent colitis than observed in mice administered PBS during this same period. This protective effect was attributable to effects on the lamina propria mononuclear cell (LPMC) population, because it could be transferred by LPMC from probiotic-treated mice to naive mice. Probiotic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4+ T cells bearing surface TGF-β in the form of latency-associated protein (LAP) (LAP+ T cells). The latter were dependent on the IL-10 production because administration of anti-IL-10R mAb blocked their appearance. Finally, the LAP+ T cells were essential to the protective effect of probiotics because administration of anti-IL-10R or anti-TGF-β at the initiation of recurrent colitis induction or depletion of LAP+ T cells from LPMC abolished the latter’s capacity to transfer protection to naive recipients. These studies show that probiotic (VSL#3) administration during a remission period ameliorates the severity of recurrent colitis by inducing an immunoregulatory response involving TGF-β-bearing regulatory cells.

Lamina propria T cells in Crohn's disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis

BACKGROUND & AIMS Normal human lamina propria lymphocytes manifest increased unstimulated apoptosis compared with peripheral lymphocytes, which are enhanced after stimulation via the CD2 activation pathway. This activation-induced apoptosis down-regulates cell expansion and cytokine production. In previous studies, it was shown that lamina propria T cells from patients with Crohns disease and ulcerative colitis manifest abnormal proliferation and cytokine production. It was therefore of interest to determine if such cells also showed abnormal patterns of apoptosis. METHODS Apoptosis was evaluated by propidium iodide staining of cells followed by flow cytometric analysis. Fas expression and Bcl-2 levels in cells were evaluated by immunofluorescence. RESULTS Lamina propria lymphocytes from patients with Crohns disease and ulcerative colitis as well as from 2 patients with diverticulitis showed defective CD2 pathway-induced apoptosis. Studies of the mechanisms of this defect focusing on cells from patients with Crohns disease showed that Crohns disease lamina propria lymphocytes from inflamed tissues express the same amount of cell surface Fas but are less sensitive to Fas-mediated apoptosis than control cells. In addition, lamina propria lymphocytes from inflamed Crohns disease tissues manifest increased expression of Bcl-2 after CD2 pathway stimulation and elevated Bcl-2 levels in cultures of unstimulated T cells. CONCLUSIONS T cells isolated from areas of inflammation in Crohns disease, ulcerative colitis, and other inflammatory states manifest decreased CD2 pathway-induced apoptosis. Studies of cells from inflamed Crohns disease tissue indicate that this defect is accompanied by elevated Bcl-2 levels. These changes are probably caused by the chronic inflammation and may aggravate the underlying disease processes that are present.

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.

The mechanism of action of probiotics.

Purpose of review Probiotics are a heterogeneous group of nonpathologic bacteria that are functionally defined by their ability to allay inflammation when introduced into the inflamed intestine. The purpose of this review is to discuss recent data bearing on the possible mechanisms of action of these bacteria, with a particular focus on the relation of these mechanisms to the pathogenesis of inflammatory bowel disease, their main arena of use. Recent findings Studies of probiotic activity in recent years provide evidence that probiotics counter experimental and human gastrointestinal inflammation (human inflammatory bowel disease) by their effects on epithelial cell function, including epithelial cell barrier function, epithelial cytokine secretion, and their antibacterial effects relating to colonization of the epithelial layer. In addition, there is emerging evidence that probiotics induce regulatory T cells that act as a break on the effector T cells that would otherwise cause inflammation. Summary This review of probiotics and inflammatory bowel disease marshals support for the concept that administration of probiotics ameliorates inflammation by exerting positive effects on the epithelial cell dysfunction and mucosal immune system dysfunction that forms the basis of the inflammation.

Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse

Aims/hypothesisRecent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice.MethodsVSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-γ and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer’s patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies.ResultsEarly oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer’s patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice.Conclusions/interpretationOrally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

IL-21 Counteracts the Regulatory T Cell-Mediated Suppression of Human CD4+ T Lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4+CD25+ regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4+CD25+ T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4+CD25− T cells and counteracts the suppressive activities of CD4+CD25+ T cells on CD4+CD25− T cells without affecting the percentage of Foxp3+ cells or survival of Treg. Additionally, CD4+CD25+ T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8+CD25− T cells but does not revert the CD4+CD25+ T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4+ T cells resistant to suppression rather than inhibiting CD4+CD25+ T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4+CD25+ T cells. Data indicate that IL-21 renders human CD4+CD25− T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

BACKGROUND & AIMS Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

The clinical significance of serum C reactive protein levels in Crohn's disease. Results of a prospective longitudinal study.

We measured the serum C reactive protein (CRP) levels by a sensitive immunochemistry method in 101 outpatients with Crohns disease enrolled in a prospective longitudinal study. At entry, 51 of the 101 patients had raised CRP levels, which showed good correlation with the clinical score. However, one-third of the patients with clinically active disease had normal CRP while one-third of the patients in clinical remission had a raised CRP. The longitudinal study showed that (a) in patients with active disease, CRP levels tended to decrease as the disease went into remission; (b) in patients who achieved remission, the likelihood of clinical relapse after 2 years of entry was higher in those with persistently raised CRP than in those with persistently normal CRP; and (c) in patients with quiescent disease at entry who remained in remission throughout the first year of the study, the likelihood of clinical relapse during the second year of the study was higher for those with persistently raised CRP than for the others. This study confirms that serum CRP is a useful laboratory index for supplementing clinical scores in patients with Crohns disease, in monitoring the response to treatment, in helping to predict the course of the disease, and in contributing to defining subgroups of patients.

Probiotic administration in patients with ileal pouch–anal anastomosis for ulcerative colitis is associated with expansion of mucosal regulatory cells

Background: Probiotics have anti‐inflammatory effects in patients with inflammatory bowel disease and appear to regulate mucosal immune response through reductions in proinflammatory cytokines. The probiotic VSL#3 prevents pouchitis if started within a week of ileostomy closure and maintains remission following antibacterial treatment in patients with refractory or recurrent pouchitis. However, the efficacy of probiotics and their effects on regulatory cells if started at a greater time after surgery in patients undergoing ileal pouch anal anastomosis (IPAA) for ulcerative colitis are unknown. Methods: We conducted an open‐label study in which 31 patients at different periods from surgery without signs and symptoms of pouchitis were randomized to 2 sachets of VSL#3 once daily or no treatment for 12 months. Pouchitis disease activity index (PDAI) was evaluated at baseline and after 3, 6, and 12 months. The percentage of CD4+ T lymphocytes expressing CD25 and the inactive form of transforming growth factor‐&bgr; [latency‐associated peptide (LAP)] were evaluated at baseline and after 3 and 6 months in peripheral‐blood mononuclear cells and mucosal biopsies. Variation in tissue interleukin‐1&bgr; and Foxp3 mRNA expression was also evaluated. Results: During the study period, VSL#3‐treated patients showed a significant reduction in PDAI score and a significant increase in the percentage of mucosal CD4+CD25high and CD4+ LAP‐positive cells compared with baseline values. Tissue samples at different points showed a significant reduction in IL‐1&bgr; mRNA expression, and a significant increase in Foxp3 mRNA expression. Conclusions: We conclude that VSL#3 administration in patients with IPAA modulates the PDAI and expands the number of mucosal regulatory T cells.