The Immunorequtation Mechanisms Of IL-17/IL-17R Pathway In The Inflammatoy Bowel Disease

Backgroup: Inflammatory bowel diseases (IBD), including ulcerative colitis(UC) and Crohn’s disease (CD), are associated with chronic relapsing inflammationof the intestinal tract of unknown etiology. IBD is fairly common in western countries,the prevalence rate of100-200/100000. In the past10years, the incidence of IBD hasan increase of about10-20times in China. Given the serious harm and highermalignant transformation probability in IBD, more people are concerned about thisdisease.Currently, it is believed that IBD is caused by the multifactorial interaction ofenvironment, gene, infection and immune disorders. The abnormalities of intestinalmucosal immune system that lead to the inflammatory response play an important rolein the pathogenesis of IBD. Initially, the imbalance of CD4+Th1/Th2subsets isclosely related to the incidence of CD and UC. However, recent reports havedemonstrated that Th17cells and Th17cells cytokines play an important role in IBD.IL-17expression and IL-17-secreting cells in the mucosa and serum were increased inIBD patients, suggesting a pathogenic role of IL-17in the progression of IBD. Manysubsequent studies have investigated the feasibility of IL-17as IBD interventiontargets.Unfortunately, IL-17neutralizing antibody failed to show an effect on theclinical trials in IBD. These studies indicate that the role of IL-17in IBD may be morecomplex. Roles of IL-17in the pathophysiology of IBD remian to be determined.It is known that IL-17can induce neutrophil aggregation and inflammatoryresponse by the activation of MAPK p38pathway. Act1(activator of NF-B), animportant adapter protein in IL-17signaling pathway, is involved in IL-17-inducedactivation of the MAPK pathway. However, it is not very clear whether and how Act1molecules participate in the IL-17-mediated activation of colonic epithelial cells inIBD. On the other hand, the data show that IL-17can play a protective role in IBD.Our previous data showed that Th1cell function is inhibited and IL-17expressionincreased in both a DSS-induced colitis model and UC patients. Whether the immuneprotection role of IL-17is mediated by inhibiting the Th1activity is not yet clear.The mechanism is well worth exploring. Therefore, understanding the mechanism ofIL-17-mediated inflammatory response will provide theoretical and experimentalevidence for the intervention strategies in inflammatory bowel disease.Objective: As colonic epithelial cell (CEC) is the main target cell of IBD and isalso the important mediator of local immune response, here CECs were used toinvestigate the effect and signal transduction mechanisms of IL-17in IBD (UC). Byexploring the molecular mechanism of the IL-17-mediated dual role in CECs, hopethis study will shed new light on therapeutic strategies against IL-17-related clinicaldiseases.Methods and Results:1. Using colonic epithelial cell line HT-29cells, weexplored the molecular mechanism that IL-17mediates the pro-inflammatory signalmechanisms by real-time PCR and Western Blot. Our results indicated that IL-17alone had no significant effect, but can synergistically promote TNF-a-mediated cellactivation in HT-29cells. The results are as follows.1) IL-17significantly promoted TNF-.-induced neutrophil chemokines(CXCL1/2/5/6/IL-8) and Th17cells chemokine (CCL20) gene expression. WesternBlot results showed that IL-17obviously enhanced TNF-. induced P38, ERK andAKT phosphorylation.2) Pathway specific inhibition assay showed that P38pathways play essentialroles in IL-17induced inflammatory response.3) Act1stable knock down HT-29cell line were established by shRNA, asmarked by decreased Act1expression at both mRNA and protein level. Act1knockdown decreased IL-17A mediated P38phosphorylation, which confirmed thatAct1-dependent P38pathway is important molecular mechanism for IL-17mediatedpro-inflammatory response.2. Relative to the pro-inflammatory role of IL-17, the immunoregulationmechanism of IL-17in autoimmune diseases is far from clear. Our previous studiessuggested that IL-17may play a protective immune function by inhibiting the Th1activity. Therefore, in this study we first focused on whether and how IL-17siganlingin CECs inhibited the expression of Th1-associated cytokines. The results include:1) Acute DSS colitis model UC was established by fedding DSS water in male C57BL/6mice. The gene expression levels of IL-17and IFN-in mouse spleen,mesenteric lymph nodes and colonic lamina propria lymphoid were detected byreal-time PCR. The results showed that, expression of IL-17was significantlyincreased while expression of IFN-significant decreases in DSS mice, which isconsistent with our previously published data (Dysregulated of Tim-3expression and itscorrelation with imbalanced CD4helper T cell in ulcerative colitis). Those data indicate thatIL-17may inhibite Th1cell activity in IBD. However, the underlying mechanismsremain largely unclear.2) In this study we also examined the expression of CXCL11and IL-12P35which is related to Th1cell chemotaxis and differentiation. The results showed thatthe average expression levels of the two cytokines were significantly decreased inDSS mice. These results suggest that high level of IL-17may inhibit the function ofTh1cells by suppressing the differentiation and chemotaxis of Th1cells.3) Using colonic epithelial cell line HT-29cells, we then explored the effects ofIL-17signaling on the expression of CXCL11and IL-12P35which are closely relatedto Th1cell chemotaxis and differentiation. The data showed that IL-17inhibited theTNF-. induced expression of CXCL11and IL-12p35at mRNAs level. The resultindicated that IL-17may play an anti-inflammatory effect by negatively regulatingTh1cell chemotaxis and differentiation. Subsequently, we explored the intracellularsignaling mechanisms of IL-17-mediated immune regulation (inhibiting Th1activity)by Western Blot, shRNA and expression microarray in HT-29cells. The resultsinclude:7IL-17synergistically enhanced TNF-. induced ERK-C/EBP and AKTphosphorylation. The data with specific inhibitors of ERK (U0126) or PI3K(wortmannin) showed that the ERK and PI3K-AKT pathways play essential roles inIL-17-mediated immune regulation.8In Act1knockdown cells, IL-17signalingfailed to enhance TNF-induced phosphorylation of ERK and AKT. Finally, the effectsof Act1knockdown on IL-17-mediated negative regulation were examined and thedata showed that Act1knockdown attenuated IL-17-induced inhibition ofTNF D-induced increase in CXCL11and IL-12P35expression. These data show thatAct1is essential for IL-17-induced enhancement of TNF-D-induced phosphorylationof ERK and PI3K-AKT and for IL-17-mediated negative regulation.9Toinvestigate the mechanisms by which IL-17induced negative regulation, microarrayanalysis was carried out. We were intrigued by the unexpected finding that PI3K-cat gamma (one subunit of PI3K-IB) expression was more than two-fold lower in Act1knockdown HT-29cells and this was confirmed by real-time PCR and Westernblotting. Notably, we found that IL-17signaling in the absence of TNF-DincreasedPI3K-CG expression in control HT-29cells, but not in Act1knockdown cells. Thesedata suggest that IL-17signaling might induce activation of PI3K B-AKT byincreasing PI3K-CG expression, a process dependent on Act1.4) Finally, to further explore the possible negative effects of IL-17, we used anHT-29cell and human PBMC co-culture system. These data demonstrate that IL-17can negatively regulate TNF-D-induced Th1cell function by targeting CECs andinhibiting the expression of IL-12P35by HT-29cells.5) To further examine the axis by which IL-17mediates negative regulationthrough CECs, we performed the following experiments in vivo.7IL-17neutralization was performed by injection of anti-IL-17antibody on days1,3,5, and7during induction of DSS-induced colitis and the effects on the activity of CEC wereexamined. Physical and histopathological examination of colon tissue revealedmarked tissue injury and infiltration of inflammatory cells in DSS mice receivinganti-IL-17antibody. It is worth noting that CECs isolated from mice receivinganti-IL-17antibody showed increased expression of CXCL-11, IL-12P35, and IFN-Jcompared to those from mice that received control IgG. The result suggested thatneutralization of IL-17in vivo affected the activity of CECs and enhanced the activityof Th1cells. As a result, the severity of DSS colitis was aggravated.8To furthervalidate the key role of the CECs in IL-17-mediated immune regulation, CECsisolated from mice on day8of DSS-induced colitis were transferred alone or togetherwith recombinant IL-17into previously untreated mice on days1and4of inductionof DSS-induced colitis. Accompanied by elevated expression of CXCL11, IL-12P35and IFN-J, adoptively transferred CECs from DSS-induced colitis mice exacerbatedcolitis in DSS-colitis mice. Interestingly, our data clearly showed that administrationof recombinant IL-17attenuated the ability of CECs from DSS-induced colitis mice toinduce colitis. These data further demonstrate the existence of an IL-17-CECs-Th1inhibition axis in IL-17-mediated negative regulation in UC.Conclusion: The above study systematically explored the immunoregulatorymechanisms of IL-17in IBD. On the one hand, IL-17promotes local neutrophilinflammatory response through Act1-P38pathway.On the other hand, we demonstrated that IL-17signaling in CECs can also trigger anti-inflammatorymechanisms by activating the PI3K B-AKT and ERK-CEBP/pathways in anAct1-dependent manner, finally leading to inhibition of TNF-D-induced expressionof IL-12P35and of a Th1cell chemokine, CXCL11, and of Th1cell function. In thisstudy, we explored the bidirectional regulation mechanisms of IL-17, which furtherexplained why IL-17antibody failed to prevent IBD in clinical experiments.Meanwhile, this study also provided valuable information for the other moleculeswith similar immune regulation. The function of inflammatory mediator(s) can beinfluenced by time, space and other micro-environmental factors. Therefore, furtherelucidating the molecular mechanisms of IL-17-mediated immune regulation isexpected to provide a more accurate basis for the specific intervention toinflammatory bowel disease by targeting IL-17.