探討GEF-H1在小腸中調控第三型干擾素表現與抗病毒反應

Abstract

諾羅病毒、輪狀病毒等腸胃道病毒常見的腸胃炎病因，尤其對免疫系統尚未發育完全的嬰幼兒是足以致命的。近年來有越來越多證據顯示第三型干擾素是黏膜組織中主要的抗病毒分子，然而目前仍不清楚第三型干擾素在腸道黏膜組織的抗病毒反應中是如何被調控的。過去的研究已經證實GEF-H1 (guanine nucleotide exchange factor H1) 能夠調控第一型干擾素在抗病毒反應中的表現，因此我們想要探討GEF-H1對於第三型干擾素是否也有調控作用，並且在腸道抗病毒反應中是否扮演著調控的角色。 我們發現在呼腸孤病毒感染模型中，GEF-H1缺陷會造成小鼠較容易被病毒感染，而且小腸組織的病變程度會比較嚴重。此外，GEF-H1缺陷小鼠小腸中的第一型和第三型干擾素表現量也是較低的。我們進一步探討GEF-H1在上皮細胞以及固有層的白血球中是否有不同的影響，結果發現在聚肌胞苷酸 (Poly I:C) 刺激下，GEF-H1剔除的上皮細胞中第三型干擾素的表現明顯下降，顯示GEF-H1在腸道上皮細胞中扮演著重要的角色。為了探討GEF-H1在腸道上皮細胞中如何調控第三型干擾素的生成，我們利用CRISPR/Cas9技術剔除掉大鼠小腸上皮細胞株 IEC-6中的GEF-H1。如同小鼠體內實驗的結果，第一型和第三型干擾素的表現在GEF-H1剔除的IEC-6中顯著降低。我們也檢驗了典型的和第三型干擾素活化的干擾素激活基因(ISGs)的表現，兩者在剔除的IEC-6中也是降低的。另外我們也使用螢光素酶報告分析方法研究GEF-H1在類RIG-I受體信號通路中所調控的途徑。我們發現GEF-H1主要是參與過氧化體上的線粒體抗病毒信號蛋白的下游反應。活化的線粒體抗病毒信號蛋白會透過信號通路啟動干擾素調控因子1和3，進而引起干擾素的表現。我們也發現干擾素調控因子1和3的活化需要GEF-H1的調控。 總結我們的發現，我們揭露了GEF-H1在小腸上皮組織中扮演著調控干擾素生成的角色，進而影響宿主的抗病毒感染能力。

Enteric viruses, such as rotaviruses and noroviruses, are the most common causes of gastroenteritis in infants and young children. Growing evidence have established that type III interferon (IFN) is the major cytokine which mediates antiviral immune responses at mucosal barriers. However, the key regulator of type III IFN expression in the gut during enteric virus infection is still unclear. Here we showed that a microtubule-associated protein, guanine nucleotide exchange factor H1 (GEF-H1), which has been identified as the essential molecule for RIG-I-like receptor (RLR)-mediated type I IFN responses, is critical for antiviral immunity in the gut. Reovirus infection model showed that GEF-H1-deficient mice were more susceptible to reovirus infection, which was accompanied by impaired type I and type III IFN production in the small intestine. Furthermore, the expressions of Ifnb and Ifnl were profoundly reduced in the epithelial cells isolated from poly(I:C)-administrated GEF-H1-deficient mice, revealing the role of GEF-H1 in the intestinal epithelial cells. To characterize the role of GEF-H1 in the induction of IFN in the intestinal epithelial cells, we generated GEF-H1 knockout intestinal epithelial cell line IEC-6 by CRISPR/Cas9 system. The GEF-H1 knockout IEC-6 exhibited diminished Ifnb and Ifnl expressions as well as interferon-stimulated genes (ISGs). Moreover, ISGs that were specifically induced by IFN-λ were also reduced. In addition, we also used luciferase reporter assays to examine type I and type III IFN promoter activation in GEF-H1 overexpressed HEK293T cells. Expression of GEF-H1 significantly amplified MAVS-induced activation of type I and type III IFN promoter. Moreover, the activation of IRF1 and IRF3 that were the transcription factors of type I and type III IFNs was dependent on the expression of GEF-H1. In summary, our results revealed that GEF-H1 is the critical factor that controls both type I and III IFN expressions for the enteric virus restriction in the intestine.