以DSS誘發急性腸炎為模型，探討介白素-22對貼附行性侵襲性大腸桿菌感染之影響

Abstract

摘要 克隆氏症患者的發炎迴腸黏膜組織中有相當高的機率能夠分離出貼附性侵襲性大腸桿菌(AIEC)。而細菌感染和過度的免疫反應皆為導致發炎性腸道疾病(IBD)的因素，若能釐清與IBD具有高度相關的病原菌感染宿主時會引起的免疫反應能幫助我們更詳細地瞭解IBD的致病過程。一提到協助維持腸道第一道免疫防線的細胞激素就會想到介白素-22(IL-22)，表現IL-22受體的腸道上皮細胞在接收到該細胞激素後會促進上皮細胞增生以及誘發抗菌物質的產生。在本篇研究計畫中，我們揭露在飲用DSS水引發腸道發炎的小鼠身上進行AIEC感染時，IL-22會扮演重要的保護角色，B6野生型小鼠感染後可以見到IL-22表現量上升，且IL-22的基因剔除小鼠上會觀察到較高的AIEC含量和較嚴重的腸道發炎情形。然而，透過分析IL-22基因剔除小鼠在DSS-AIEC感染情況下的上皮細胞發現其修復能力較為低落，並伴隨 IL-18、Reg3和CXCL1的表現量下降，且配合我們利用體外organoid培養證實IL-22的刺激能夠促進上皮細胞IL-18、Reg3和CXCL1 mRNA的生成和隱窩毒殺實驗中發現IL-22能夠使抗菌物質從上皮細胞釋放毒殺AIEC來釐清IL-22在DSS-AIEC感染時確切地防禦功能。除此之外，當B6野生型小鼠感染AIEC時除了見到IL-22表現量上升以外上皮細胞IL-18的表現量也會上升，證實IL-22刺激上皮細胞表現IL-18亦為宿主在DSS-AIEC感染防禦的一環，進一步探究其作用機轉和IL-18的防禦功能發現，IL-22作用到腸道上皮細胞後會刺激轉錄因子STAT3的活化使其結合到IL-18 promoter區域促進IL-18基因進行轉錄，而IL-18雖然無法提升抗菌物質mRNA的表現量，但卻能夠促使上皮細胞進行抗菌物質的釋放從上皮細胞釋放殺菌。另外，因著在DSS-AIEC感染的IL-22基因剔除小鼠身上見到固有層淋巴球IFNg和IL-17A的生成降低，我們也透過分別對IFNg和IL-17A的基因剔除小鼠進行相同的感染模型證實，缺乏IFNg或IL-17A的小鼠會有較為嚴重的腸道發炎現象、明顯較高的疾病活動指數和較多的AIEC病原菌的存在，以此說明這兩種細胞激素對於AIEC亦參與在AIEC感染時得宿主防禦。

Inflamed ileal mucosa of patient with Crohn’s disease have high prevalence of AIEC isolation. Believed that enteric pathogens targeting to epithelial cells and out of control immune response also can trigger inflammatory bowel disease (IBD). So If studies fully understand the host immune response to enteric pathogen, it will become useful for better understanding of IBD pathogenesis. When it comes to immune barrier maintenance in the gut, we will remind interleukin-22 (IL-22), the cytokine exclusively targets IL-22 receptor-expressing epithelial cells to promote cell regeneration and anti-microbial peptide (AMP) production. Here, we showed that IL-22 is crucial for controlling AIEC bacterial load in the context of inflammation induced by dextran sodium sulfate (DSS). After DSS-AIEC treatment, IL-22 expression level is upregulated on WT mice and mice without il-22, pathogenic AIEC load and inflammatory condition in the gut were more serious. In DSS-AIEC, we found Ki67+ proliferating crypts and the production of interleukin-18 (IL-18), Reg3g (an AMP) and CXCL1 (a chemokine) is significantly reduced in IL-22-deficient mice. In addition, we used ex vivo organoid culture and crypt killing assay respectively to showed that IL-22 is able to induce mRNA levels of IL-18, Reg3g, and CXCL1 and also promote AMP releasing from epithelial cell to kill AIEC. Not only IL-22 but also IL-18 were increased in WT mice during DSS-AIEC, indicate that IL-22 stimulates epithelial cell IL-18 expression in AIEC infection. It is likely via IL-22-induced phospho-STAT3 to bind the promoters of IL-18. As for investigating the the role of IL-18 in DSS-AIEC, we also found that IL-18 cannot induce mRNA level of AMP and chemokine, but it can stimulate AMP from epithelial cell leading to in vitro AIEC killing. Furthermore, we showed that lamina propria IFNg-producing T cells and IL-17A-producing T cells were reduced in IL-22-deficient mice and that IFNg-deficient or IL-17A-deficient mice were more susceptible to DSS-AIEC, suggesting that these cytokines also participate in host defense to control AIEC infection during intestine inflammatory.