Pattern Recognition Receptor-initiated Innate Antiviral Responses In The Mouse Testis And Epididymis

Background and objectives:Viral infections of the testis and epididymis may cause orchitis and epididymitis, which impair male fertility. The mechanisms underlying innate antiviral responses merit clarification. The present study investigated pattern recognition receptor-initiated innate antiviral responses in the tissue-specific cells.Materials and methods:Primary testicular cells and epididymal epithelial cells (EECs) were isolated from C57/BL6 and TLR3 knockout (TLR3-/-) mice. Gene expression at mRNA level was analyzed by real-time qRT-PCR. Proteins were determined by Western blot and immunohistochemistry. The concentration of cytokines and testosterone was measured using ELISA. TLR3//- cells and specific siRNA for target genes were used to distinguish the functions of PRRs in testicular and epididymal innate antiviral responses.Results:Mouse Leydig cells and spermatids display innate antiviral capacities through MDA5/RIG-I signaling in response to viral RNA analog poly(I:C). Moreover, Leydig cells have innate antiviral activities through p204 activation in response to viral DNA analog HSV60. EECs constitutively express several viral sensors, including TLR3, RIG-I, and DAI. P204 and cGAS can be induced by HSV60 and activated in EECs. TLR3 and RIG-I in EECs can be activated by poly(I:C), while DAI can be initiated by HSV60. Both poly(I:C) and HSV60 induced the expression of type Ⅰ interferons and various antiviral proteins, including ISG15, OAS1 and MX1. Poly(I:C) but not HSV60 dramatically induced the expression of major proinflammatory cytokines TNF-a and MCP-1. The antiviral responses were significantly reduced by TLR3-deficience and knockdown of individual RIG-I, DAI, p204 and cGAS. The results suggest that these viral sensors cooperatively initiate the innate antiviral responses in the mouse testis and epididymis. Conclusions:Various viral sensors are expressed and mediate innate antiviral responses in Leydig, male germ cells and EECs. Results provided novel insights into the mechanisms underlying the testicular and epididymal innate antiviral responses.