T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Recent advances in gut immunology.

In recent years, there have been significant advances in our understanding of the mucosal immune system. In addition to unravelling some of the complexities of this system, including the discovery of completely new cells types, further insights into the three-way interactions between mucosal immune cells, the intestinal epithelium and the microbial communities colonizing the GI tract promise to redefine our understanding of how intestinal homeostasis is maintained, but also how dysregulation of these highly integrated interactions conspires to cause disease. In this review, we will discuss major recent advances in the role of key immune players in the gut, including innate lymphoid cells (ILCs), mucosa-associated invariant T cells (MAIT cells) and cells of the mononuclear phagocyte system (MPS), including how these cells interact with the intestinal epithelial and their crosstalk with components of the intestinal microbiota, and how these interactions shape host health.

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease.

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNß in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNß selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNß in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.

Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

Defective expression of SIRT1 contributes to sustain inflammatory pathways in the gut.

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.

A functional role for Smad7 in sustaining colon cancer cell growth and survival.

Initially identified as an inhibitor of transforming growth factor (TGF)-ß mainly owing to its ability to bind TGF-ß receptor type I and abrogate TGF-ß-driven signaling, Smad7 can interact with additional intracellular proteins and regulate TGF-ß-independent pathways, thus having a key role in the control of neoplastic processes in various organs. Genome-wide association studies have shown that common alleles of Smad7 influence the risk of colorectal cancer (CRC), even though the contribution of Smad7 in colon carcinogenesis is not fully understood. In this study, we assessed the expression and role of Smad7 in human and mouse models of sporadic CRC. We document a significant increase of Smad7 in human CRC relative to the surrounding nontumor tissues and show that silencing of Smad7 inhibits the growth of CRC cell lines both in vitro and in vivo after transplantation into immunodeficient mice. Knockdown of Smad7 results in enhanced phosphorylation of the cyclin-dependent kinase (CDK)2, accumulation of CRC cells in S phase and enhanced cell death. Smad7-deficient CRC cells have lower levels of CDC25A, a phosphatase that dephosphorylates CDK2, and hyperphosphorylated eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, knockdown of Smad7 associates with inactivation of eIF2α, lower CDC25A expression and diminished fraction of proliferating cells in human CRC explants, and reduces the number of intestinal tumors in Apc(min/+) mice. Altogether, these data support a role for Smad7 in sustaining colon tumorigenesis.

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease.

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Interleukin-25 production is differently regulated by TNF-α and TGF-ß1 in the human gut.

An altered balance between effector and regulatory factors is supposed to sustain the tissue-damaging immune response in inflammatory bowel disease (IBD). We have recently shown that in IBD, there is a defective synthesis of the counter-regulatory cytokine, interleukin (IL)-25. In this study we investigated factors that control IL-25 production in the gut. IBD patients produced less IL-25 when compared with normal controls. Stimulation of normal intestinal explants with tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ) or IL-21, reduced IL-25 synthesis. Consistently, IL-25 production was enhanced by anti-TNF-α both in vitro and in vivo. Upregulation of IL-25 was also seen in normal colonic explants stimulated with transforming growth factor-ß1 (TGF-ß1). As in IBD, TGF-ß1 activity is abrogated by Smad7, we next assessed whether inhibition of Smad7 with an antisense oligonucleotide enhanced IL-25 expression. Knockdown of Smad7 was accompanied by an increase in IL-25 production. Data show that IL-25 production is differently regulated by TNF-α and TGF-ß1 in the human gut.

Transcriptional regulation of the mucosal immune system mediated by T-bet.

The immune system faces the arduous task of defending the mucosal surfaces from invading pathogens, but must simultaneously repress responses against commensal organisms and other inert antigens that are abundant in the external environment, as inappropriate immune activation might expose the host to increased risk of autoimmunity. The behavior of individual immune cells is governed by the expression of transcription factors that are responsible for switching immune response genes on and off. T-bet (T-box expressed in T cells) has emerged as one of the key transcription factors responsible for controlling the fate of both innate and adaptive immune cells, and its expression in different immune cells found at mucosal surfaces is capable of dictating the critical balance between permitting robust host immunity and limiting susceptibility to autoimmunity and allergy.

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1ß and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1ß and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

Differential regulation of interleukin 17 and interferon gamma production in inflammatory bowel disease.

BACKGROUND AND AIMS: Interleukin 17 (IL17) is now known to be involved in a number of chronic inflammatory disorders. However, the mechanisms regulating its production in inflammatory bowel disease (IBD) are still unclear. METHODS: Endoscopic biopsies or surgical specimens were taken from inflamed and uninflamed colonic mucosa of 72 patients with IBD (38 with Crohn's disease and 34 with ulcerative colitis), and normal colon of 38 control subjects. IL17 and interferon gamma (IFNgamma) were detected by ELISA in the supernatants of biopsies cultured ex vivo, and anti-CD3/CD28-stimulated lamina propria mononuclear cells (LPMCs) incubated with IL12, IL23, IL1beta plus IL6, transforming growth factor beta1 (TGFbeta1), or anti-IL21 neutralising antibody. Intracellular flow cytometry was performed to analyse mucosal Th17 and Th1/Th17 cells. RESULTS: IL17 production by organ culture biopsies was higher in IBD inflamed mucosa than IBD uninflamed mucosa and controls, and was equivalent in amount to IFNgamma. Anti-CD3/CD28-stimulated IBD LPMCs produced higher IL17 amounts compared to controls. The percentages of Th17 and Th1/Th17 cells were increased in patients with IBD. IL23 and IL1beta plus IL6 had no effect on IBD LPMC production of IL17; however, IL12 markedly increased IFNgamma production and decreased IL17 production. TGFbeta1 dose-dependently decreased IFNgamma, but had no significant inhibitory effect on IL17 production. Blocking IL21 significantly downregulated IL17 production. CONCLUSIONS: Our findings support a role for IL12, TGFbeta and IL21 in modulating IL17/IFNgamma production in IBD. The abundant IL17 in inflamed IBD mucosa may help explain the relative lack of efficacy of anti-IFNgamma antibodies in clinical trials of Crohn's disease.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Cow's milk protein-specific T-helper type I/II cytokine responses in infants with necrotizing enterocolitis.

Enteral feeding, in particular with formula feeds, is associated with necrotizing enterocolitis (NEC). In this study, we have examined, in the systemic and mucosal immune compartments, for evidence of bovine milk antigen sensitization in infants with NEC. Eleven newborns with Bell's staging 2-3 NEC [median post-conceptional age 31 wk (range 27-41 wk)], 21 neonatal controls [33 (28-40) wk] and 15 infants undergoing intestinal resection or mucosal biopsy for non-inflammatory conditions [39 (34-42) wk] were studied. Spontaneous and antigen or mitogen elicited interferon-gamma (IFN-gamma) [T-helper type I (Th1)], interleukin (IL)-4 and IL-5 [T-helper type II (Th2)] responses were enumerated using single-cell enzyme-linked immunospot (ELISPOT) assay in peripheral blood (PBMC) or lamina propria mononuclear cells. NEC infants, compared with controls, showed a significant elevation in baseline PBMC cytokine secreting cells, vigorous mitogen responses (20- to 120-fold increase) for IFN-gamma, IL-4 and IL-5 (p < 0.001), strong responses to beta-lactoglobulin (betalg) (IFN-gamma > IL-4/IL-5, p < or = 0.001), and somewhat smaller casein responses. Similarly, in the lamina propria, a small but significant increase in spontaneous cytokine-secreting cells was detected in NEC infants (p < 0.01), with an IFN-gamma/IL-4 predominant phytohemagglutinin (PHA)/concanavalin-A (ConA) response. Three of nine NEC infants (but no controls) also showed a positive ELISPOT response to betalg (IFN-gamma only) but none to casein. We have thus demonstrated significant cow's milk protein (CMP) sensitization in NEC, at least in the systemic compartment (mixed Th1/Th2), with minimal mucosal activation in some cases. These novel findings provide a potential mechanism for a direct contributory role of CMP in the pathogenesis of NEC.

CC-10004 but not thalidomide or lenalidomide inhibits lamina propria mononuclear cell TNF-α and MMP-3 production in patients with inflammatory bowel disease.

BACKGROUND: Thalidomide, one of whose activities is to inhibit Tumour Necrosis Factor (TNF)-α production, has been reported to be an effective treatment for refractory inflammatory bowel disease (IBD). TNF-α driven production of matrix metalloproteinase (MMP)-3 by gut lamina propria mononuclear cells (LPMCs) is a major pathway of tissue injury in IBD; however the effect of thalidomide and newer more potent immunomodulatory derivatives on this pathway has not been studied. AIM: To investigate the effect of thalidomide, CC-4047 (pomalidomide), CC-5013 (lenalidomide), and CC-10004 (apremilast) on gut LPMC TNFα and MMP-3 production in patients with IBD. METHODS: Gut LPMCs and myofibroblasts were isolated from patients with IBD, and cultured with thalidomide, CC-4047, CC-5013, and CC-10004. MMP-3 and TIMP-1 levels were determined by western blotting and real-time PCR, and TNF-α levels by ELISA. RESULTS: CC-10004 significantly reduced both TNF-α production and MMP-3 production by cultured LPMCs. Thalidomide and CC-4047 and CC-5013 had no significant effect on the production of TNF-α or MMP-3 by LPMCs. CONCLUSION: These results provides a mechanistic rationale for both the failure of lenalidomide (CC-5013) in a recent randomised controlled trial in Crohn's disease, and for the evaluation of CC-10004 as a novel oral therapy in the treatment of CD and UC.

TGF-beta1 and Smad7 in the regulation of IBD.

Inflammatory bowel diseases (IBDs) are caused by an aberrant and excessive local immune response to components of the bacterial microflora that are: poorly controlled by endogenous counter regulatory mechanisms such as the immunosuppressive cytokine transforming growth factor-beta1 (TGF-beta1). Studies in human IBD tissues have documented a disruption of TGF-beta1 signaling marked by a block in the phosphorylation of the activated TGF-beta receptor-associated signaling molecule, Smad3, caused by the upregulation of the intracellular inhibitor of Smad signaling, Smad7. Inhibition of Smad7 with a specific antisense oligonucleotide restores TGF-beta1/Smad3 signaling, resulting in a marked suppression of inflammatory cytokine production. The functional relevance of Smad7 in gut inflammation was confirmed by studies in murine models of IBD. In inflamed tissues of mice with colitis induced by either the trinitrobenzene sulfonic acid or oxazolone, p-Smad3 was low despite active TGF-beta1 being produced in excess. In vivo administration of Smad7 antisense oligonucleotides to mice with colitis restored TGF-beta1 signaling and decreased the synthesis of inflammatory molecules and the extent of gut damage. These data support a role for Smad7 in maintaining intestinal inflammation, and suggest that blocking Smad7 could be a promising way to dampen the ongoing inflammation in IBD.

IL-21 comes of age as a regulator of effector T cells in the gut.

In healthy individuals, antigens from the gut lumen are tolerated by the mucosal immune system. However, a loss of tolerance toward the bacterial microflora probably causes inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis. The abnormal activation of the immune system in the gut of IBD patients is characterized by a cascade of cellular events orchestrated by cytokine cross talk between immune and non-immune cells. Interleukin (IL)-21, the newest member of the common gamma-chain-dependent cytokine family, is a key component of the inflammatory cascade in the gut. It is highly expressed in CD and sustains the ongoing T helper type 1 (Th1)-mediated immune response. IL-21 is essential for the differentiation of Th17 cells. IL-21 is also involved in recruiting T cells to the inflamed gut and eliciting the secretion of matrix-degrading enzymes by gut fibroblasts. Overall, there is now sufficient evidence to suggest that targeting IL-21 will be of therapeutic benefit in IBD.

Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa.

BACKGROUND AND AIMS: The role of transforming growth factor beta (TGFbeta) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFbeta inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFbeta blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon gamma (IFN gamma) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS: TGFbeta transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFbeta neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN gamma, tumour necrosis factor alpha (TNFalpha), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS: A higher number of TGFbeta transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFbeta antibody than in those cultured with control antibody. TGFbeta blockade downregulated T cell apoptosis, and induced a significant increase in IFN gamma, TNFalpha, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFbeta antibody. CONCLUSIONS: The findings support a crucial role for TGFbeta in dampening T cell-mediated tissue-damaging responses in the human gut.

Interleukin 21 contributes to the mucosal T helper cell type 1 response in coeliac disease.

BACKGROUND: In coeliac disease (CD), the upper bowel lesion is associated with a marked infiltration of the mucosa with Th1 cells secreting interferon gamma (IFNgamma) and expressing the Th1-associated transcription factor, T-bet. However, the molecular mechanisms which regulate T-bet and promote the Th1 cell response are unknown. OBJECTIVE: To examine whether interleukin 21 (IL21), a cytokine that regulates T cell activation, has a role in CD. METHODS: Duodenal mucosal samples were taken from CD patients and normal controls. IL21 and T-bet were examined by real-time PCR and western blotting, and IFNgamma was assessed by real-time PCR and ELISA. The effect of blockade of endogenous IL21 on the expression of T-bet was examined in an ex vivo culture of biopsies taken from untreated CD patients. Finally, the role of IL21 in controlling T-bet and IFNgamma was also evaluated in cultures of biopsies taken from treated CD patients and cultured with a peptic-tryptic digest of gliadin (PT) in the presence or absence of a neutralising IL21 antibody. RESULTS: Enhanced IL21 RNA and protein expression was seen in duodenal samples from untreated CD patients. Blockade of IL21 activity in biopsies of untreated CD patients reduced T-bet and IFNgamma secretion. Stimulation of treated CD biopsies with PT enhanced IL21 expression, and neutralisation of IL21 largely prevented PT-driven T-bet and IFNgamma induction. CONCLUSIONS: IL21 is overproduced in the mucosa of CD patients, where it helps sustain T-bet expression and IFNgamma production.

The role of matrix metalloproteinases in stromal/epithelial interactions in the gut.

The gastrointestinal mucosa is an extremely soft, highly vascularised tissue, with a single layer of epithelium separating the gut lumen from the host. Epithelial cells adhere to a thin basement membrane that is produced by both epithelial cells and the underlying stromal cells. Signals passing between epithelial cells and stromal cells are needed for normal gut structure. In gut diseases, however, epithelial cells and stromal cells produce large amounts of matrix degrading enzymes (matrix metalloproteinases), the function of which is only beginning to be elucidated. Here, we review the role of matrix metalloproteonases (MMPs) in the gut in health, in gut inflammation, and in cancer.