Zhanju Liu

IL-15 Is Highly Expressed in Inflammatory Bowel Disease and Regulates Local T Cell-Dependent Cytokine Production

IL-15 shares biological activities but no significant sequence homology with IL-2. It induces T cell recruitment to sites of inflammation, T cell proliferation, and cytokine production and rescue from apoptosis. The aim of this study was to investigate expression of IL-15 and its effects on proinflammatory cytokine production in inflammatory bowel disease (IBD). Immunohistochemistry demonstrated local IL-15 production by macrophages in inflamed mucosa from IBD patients. Isolated lamina propria mononuclear cells from these patients but not from controls produced IL-15 when stimulated with LPS or IFN-γ. Moreover, lamina propria T cells (LP-T) from IBD patients were more responsive to IL-15 as compared with controls, and IL-15 alone without a primary T cell stimulus induced IFN-γ and TNF production by isolated IBD LP-T cells, especially by LP-T cells from patients with Crohn’s disease. LP-T cells from IBD patients could induce CD40-CD40 ligand (CD40L) interaction-dependent TNF and IL-12 production by monocytes in a coculture system. This capacity of LP-T cells was strongly enhanced by preincubation in IL-15 and was the result of higher CD40L expression after culture in IL-15. These data indicate that IL-15 is overexpressed in the inflamed mucosa in IBD and that IL-15 enhances local T cell activation, proliferation, and proinflammatory cytokine production by both T cells and macrophages, the latter via a CD40-CD40L interaction-dependent mechanism. Treatment directed against IL-15 may have therapeutic potential in IBD.

A global consensus on the classification, diagnosis and multidisciplinary treatment of perianal fistulising Crohn's disease

Objective To develop a consensus on the classification, diagnosis and multidisciplinary treatment of perianal fistulising Crohns disease (pCD), based on best available evidence. Methods Based on a systematic literature review, statements were formed, discussed and approved in multiple rounds by the 20 working group participants. Consensus was defined as at least 80% agreement among voters. Evidence was assessed using the modified GRADE (Grading of Recommendations Assessment, Development, and Evaluation) criteria. Results Highest diagnostic accuracy can only be established if a combination of modalities is used. Drainage of sepsis is always first line therapy before initiating immunosuppressive treatment. Mucosal healing is the goal in the presence of proctitis. Whereas antibiotics and thiopurines have a role as adjunctive treatments in pCD, anti-tumour necrosis factor (anti-TNF) is the current gold standard. The efficacy of infliximab is best documented although adalimumab and certolizumab pegol are moderately effective. Oral tacrolimus could be used in patients failing anti-TNF therapy. Definite surgical repair is only of consideration in the absence of luminal inflammation. Conclusions Based on a multidisciplinary approach, items relevant for fistula management were identified and algorithms on diagnosis and treatment of pCD were developed.

Prevention of Experimental Colitis in SCID Mice Reconstituted with CD45RBhigh CD4+ T Cells by Blocking the CD40-CD154 Interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-γ, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis.

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RBhighCD4+ T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RBhighCD4+ cells from CD28−/− mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RBhighCD4+ cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-γ by lamina propria CD4+ cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25+CD4+ cells from CD28−/− mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RBhigh cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.

The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity

This study analyzed IL‐23p19 expression in inflamed mucosa of IBD and the role in the induction of IEL and NK cell activation as well as Th17 cell differentiation. Expression of IL‐23p19 was performed by immunohistochemistry and quantitative real‐time PCR. Expression of IL‐23R was assessed by flow cytometry. Cytolytic activities of IEL and NK cells by IL‐23 were determined by a standard 51Cr‐release assay. Cytokine levels were analyzed by ELISA and quantitative real‐time PCR. Expression of IL‐23p19 was increased significantly in inflamed mucosa of CD compared with that in UC and healthy controls. Double‐staining confirmed that IL‐23p19+ cells were mainly CD68+ macrophages/DCs. IL‐23R+ cells were increased significantly in PB‐ and LP‐CD4+ and ‐CD8+ T and NK cells. IL‐23 markedly promoted IBD IEL and NK cell activation and cytotoxicity and triggered IBD PB‐ and LP‐T cells to secrete significantly higher levels of IFN‐γ, TNF, IL‐2, and IL‐17A compared with controls. Importantly, IL‐23 promoted IBD PB‐ or LP‐CD4+ T cells to differentiate into Th17 cells, characterized by increased expression of IL‐17A and RORC. Anti‐TNF treatment could markedly reduce IL‐23 expression and Th17 cell infiltration in inflamed mucosa of CD patients. These data indicate that IL‐23 is highly expressed in inflamed mucosa of IBD and plays an important role in the induction of IEL, NK, and T cell activation, proinflammatory cytokine secretion, and Th17 cell differentiation. Targeted therapy directed against IL‐23p19 may have a therapeutic role in treatment of IBD.

Il‐21 enhances NK cell activation and cytolytic activity and induces Th17 cell differentiation in inflammatory bowel disease

Background: Interleukin‐21 (IL‐21) is involved in T and NK cell activation and effector response and promotes Th17 cell differentiation. Here we investigated IL‐21 receptor (IL‐21R) expression in inflamed mucosa of inflammatory bowel disease (IBD) and evaluated its role in the induction of NK cell cytotoxicity and activation as well as Th17 differentiation. Methods: Expression of IL‐21R was performed by immunohistochemistry and flow cytometry. NK cell cytotoxicity was detected by a standard 51Cr‐release assay. Cytokine levels were analyzed by enzyme‐linked immunosorbent assay (ELISA) and quantitative real‐time polymerase chain reaction (PCR). Results: IL‐21R‐positive cells were significantly increased in inflamed mucosa of IBD compared with controls, and mainly expressed in freshly isolated peripheral blood (PB)‐ and lamina propria (LP)‐CD4+, CD8+ T, B, and NK cells. PB‐NK cells from IBD patients produced higher levels of interferon gamma (IFN‐γ) and tumor necrosis factor (TNF) than controls when stimulated with immobilized human IgG and IL‐21. IL‐21‐primed IBD NK cells showed a more potent antitumor cytotoxicity to NK‐sensitive K562 cells than controls. Moreover, PB‐T and LP‐T cells from IBD patients produced large amounts of proinflammatory cytokines (e.g., TNF, IFN‐γ) than controls when stimulated with IL‐21 and anti‐CD3. Importantly, IL‐21 facilitated IBD CD4+ T cell to differentiate into Th17 cells, characterized by increased expression of IL‐17A and RORγt. Conclusions: IL‐21 enhances IBD NK cell cytotoxic response, triggers T cells to produce proinflammatory cytokines, and induces IBD CD4+ T cells to differentiate into Th17 cells, suggesting that IL‐21 is involved in the pathogenesis of IBD and that blocking IL‐21R signaling may have a therapeutic potential in IBD. (Inflamm Bowel Dis 2009)

Dysregulation of mucosal immune response in pathogenesis of inflammatory bowel disease

Inflammatory bowel disease (IBD) includes Crohns disease and ulcerative colitis. The exact etiology and pathology of IBD remain unknown. Available evidence suggests that an abnormal immune response against the microorganisms in the intestine is responsible for the disease in genetically susceptible individuals. Dysregulation of immune response in the intestine plays a critical role in the pathogenesis of IBD, involving a wide range of molecules including cytokines. On the other hand, besides T helper (Th) 1 and Th2 cell immune responses, other subsets of T cells, namely Th17 and regulatory T cells, are likely associated with disease progression. Studying the interactions between various constituents of the innate and adaptive immune systems will certainly open new horizons of the knowledge about the immunologic mechanisms in IBD.

Role of interleukin‐12 in the induction of mucosal inflammation and abrogation of regulatory T cell function in chronic experimental colitis

IL‐12 promotes Th1 cell differentiation and cell‐mediated immunity. In the present study, the potential role of IL‐12 was analyzed in an experimental colitis model in scid mice reconstituted with syngeneic CD45RBhighCD4+ T cells. Real‐time reverse transcription‐PCR studies demonstrated that IL‐12 p40 mRNA in inflamed colon is induced shortly after T cell transfer and maintained at a stable level after week 4, at the time when wasting disease starts. Administration of anti‐IL‐12 on days 0, 14, and 28 (early treatment) or on days 28, 42, and 56 (delayed treatment) after T cell transfer, effectively prevented or, respectively cured wasting disease and colitis in scid recipients. Anti‐IL‐12 treatment abrogated mucosal inflammation with significantly diminished leukocyte infiltration (CD4 cells, macrophages) and CD54 expression, and down‐regulated proinflammatory cytokines IFN‐γ and IL‐2. Of note, although splenic CD4+ T cells are unable to induce disease as a result of the presence of regulatory CD45RBlow cells, splenic CD4+ T cells, preactivated by IL‐12 and anti‐CD3 in vitro, were highly pathogenic in inducing severe mucosal inflammation, suggesting that IL‐12 and anti‐CD3 abrogated regulatory T cell function. These findings indicate that IL‐12 is important for the induction of experimental colitis through effects on proinflammatory cytokine production and on regulatory T cell function.

Long Non-coding RNA Growth Arrest-specific Transcript 5 (GAS5) Inhibits Liver Fibrogenesis through a Mechanism of Competing Endogenous RNA

Background: Long non-coding RNAs function as competing endogenous RNAs (ceRNAs). Whether growth arrest-specific transcript 5 (GAS5) acts as a ceRNA for microRNA-222 in liver fibrosis remains undefined. Results: GAS5 increases p27 expression as a ceRNA for microRNA-222, thereby inhibiting liver fibrosis progression. Conclusion: The GAS5/microRNA-222/p27 axis underlies the pathogenesis of liver fibrosis. Significance: The ceRNA network helps to understand liver fibrogenesis. Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.

miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD

Objective Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD. Methods miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells. Results The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon-γ inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses. Conclusions Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.