Virus-triggered Nucleic Acid Sensor Signaling Pathways And Their Effects On Testicular Functions

Background and objectives:A large spectrum of viruses can infect testis.Only some RNA viruses induce orchitis and impair testicular functions,and the underlying mechanisms remain unclear.This thesis focuses on the innate immune signaling pathways triggered by viral RNA and DNA in testicular Sertoli and Leydig cells and their impacts on testicular functions.The viral RNA and DNA-induced innate immune responses in other cells,including HeLa,mouse peritoneal macrophages,and 15P-1,are also evaluated.Materials and methods:Primary testicular Sertoli and Leydig cells and peritoneal macrophages were isolated from C57BL/6J,Toll-like receptor 3(Tlr3)gene knockout(Tlr3-/-),IFN-? promoter stimulator-1(Ips-1)gene knockout(Ips-1-/-)and Sting mutant(Stinggt/gt)mice.The mice and primary cells were stimulated by viral RNA and DNA analogues:polyinosinic-polycytidylic acid[poly(I:C)]and 60 bp oligonucleotide containing herpes simplex virus(HSV)genome DNA motif(HSV60).Hematoxylin-eosin(HE)staining was used for histopathological analysis.Real-time qRT-PCR was used for quantitative analysis of gene expression at mRNA level.ELISA was used to measure the levels of cytokines and testosterone in the culture medium.Western blot and immunofluorescence staining were used for the determination of protein expression and distribution.RNA interference was used to inhibit gene expression.Results:Poly(I:C)injection into the testis damaged spermatogenesis in mice,whereas the injection of HSV60 did not affect spermatogenesis.Poly(I:C)transfection in vitro induced the production of pro-inflammatory cytokines TNF-? and IL-6 in Sertoli and Leydig cells through TLR3 and IPS-1 signaling pathways,which significantly impaired the blood-testicular barrier(BTB)and testosterone synthesis.The poly(I:C)-induced TNF-? plays a critical role in impairing the cellular functions.By contrast,HSV60 predominantly induced the expression of type I interferons and antiviral proteins via the stimulator of interferon gene(STING)signaling pathway,which did not apparently affect the cell functions.We further demonstrated that Zika virus(ZIKV)induced higher levels of TNF-? and IL-6 than herpes simplex virus-2(HSV-2)in Sertoli and Leydig cells and impaired the cell functions.Moreover,poly(I:C)also induced a relatively high levels of TNF-? and IL-6 in HeLa,mouse peritoneal macrophages and 15P-1.Conclusions:Poly(I:C)-induced innate immune responses through TLR3 and IPS-1 signaling pathways,while HSV60 predominantly induces innate antiviral responses through STING pathway.The activation of TLR3 and IPS-1 signaling pathways damage spermatogenesis,disrupt BTB formation and inhibit testosterone synthesis,whereas STING pathway does not apparently impair testicular functions.Poly(I:C)also induces the high levels of pro-inflammatory cytokines in HeLa,mouse peritoneal macrophages and 15P-1,which may be one of the mechanisms by which RNA viruses predominantly induce inflammatory conditions.