Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis.

No abstract

Summary Activation of Toll‐like receptors (TLRs) triggers rapid inflammatory cytokine production in various cell types. The exogenous product of growth‐arrest‐specific gene 6 (Gas6) and Protein S (ProS) inhibit the TLR‐triggered inflammatory responses through the activation of Tyro3, Axl and Mer (TAM) receptors. However, regulation of the Gas6/ProS‐TAM system remains largely unknown. In the current study, mouse macrophages are shown to constitutively express Gas6 and ProS, which synergistically suppress the basal and TLR‐triggered production of inflammatory cytokines, including those of tumour necrosis factor‐α, interleukin‐6 and interleukin‐1β, by the macrophages in an autocrine manner. Notably, TLR signalling markedly decreases Gas6 and ProS expression in macrophages through the activation of the nuclear factor‐κB. Further, the down‐regulation of Gas6 and ProS by TLR signalling facilitates the TLR‐mediated inflammatory cytokine production in mouse macrophages. These results describe a self‐regulatory mechanism of TLR signalling through the suppression of Gas6 and ProS expression.

Tyro3, Axl and Mer (TAM) receptor tyrosine kinases triple knockout (TAM(-/-)) mice are male infertile due to impaired spermatogenesis. However, the mechanism by which TAM receptors regulate spermatogenesis remains unclear. In this study, we demonstrate that the testicular immune homeostasis was impaired in TAM(-/-) mice. As development after the onset of sexual maturity, germ cells were progressively degenerated. Macrophages and lymphocytes infiltrated into the testis as TAM(-/-) mice aged. Moreover, the integrity of blood-testis barrier was impaired, and the autoantibodies against germ cell antigens were produced. Major inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1 were upregulated in the testis of TAM(-/-) mice, and predominantly located in Sertoli cells (SCs). In vitro assays showed that TAM(-/-) SCs secrete significantly high levels of inflammatory cytokines compared with wild-type SCs after coculture with apoptotic germ cells. These results suggest that TAM receptors are important in the maintenance of the immune homeostasis in the testis.

The testis is an immune privileged organ in which the tissue-specific cells have adopted effective innate immune functions against microbial pathogens. Toll-like receptors (TLRs) mediate innate immune response in the testis. The current study demonstrates that melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) initiate the testicular innate antiviral response. Both MDA5 and RIG-I are expressed in Leydig cells, and MDA5 is also expressed in spermatids. Polyinosinic-polycytidylic acid [poly(I:C)], a common agonist of MDA5 and RIG-I, significantly induces the expression of type I interferons (IFN-α/β) and antiviral proteins, including IFN-stimulated gene 15, 2'5'-oligoadenylate synthetase 1, and Mx GTPase 1, in primary TLR3-deficient (TLR3(-/-)) Leydig and germ cells. Moreover, major proinflammatory cytokines, including TNF-α and IL-6, are significantly up-regulated by poly(I:C) in these testicular cells. The poly(I:C)-induced innate antiviral response in the testicular cells is significantly reduced by knockdown of individual MDA5 and RIG-I using specific small interfering RNA. We also provide evidence that local injection of poly(I:C) induces antiviral response in the testis of TLR3(-/-) mice. These data provide novel insights into the mechanisms underlying testicular antiviral response.

The testis is an immunoprivileged site where local cell-initiated innate immunity plays a crucial role in antimicrobial responses. Toll-like receptors (TLRs) mediate innate immune responses in testicular somatic cells. Although several TLRs are expressed in some stages of male germ cells, the potential role of TLRs in triggering antimicrobial responses in the germ cells has yet to be exclusively studied. The current study demonstrates that TLR3 is constitutively expressed in spermatogonia and spermatocytes and can be activated by a synthetic double-strained RNA analog, polyinosinic-polycytidylic acid. TLR3 activation in these male germ cells up-regulates the expression of proinflammatory cytokines, such as interleukin IL1B, IL6, and tumor necrosis factor alpha, through activation of nuclear factor kappa B; it also induces production of type 1 interferons (IFNA and IFNB) through the activation of IFN regulatory factor 3. In addition, TLR3 activation increases the production of two major antiviral proteins, namely, double-stranded RNA-activated protein kinase and MX1 protein, by germ cells. Data in this article describe an antiviral response of male germ cells through the activation of TLR3 in vitro.

Viral infections of the epididymis may impair male fertility and spread sexually transmitted pathogens. The innate antiviral immune responses in the epididymis have yet to be intensively investigated. This study found that mouse epididymal epithelial cells (EECs) constitutively express several viral sensors, including TLR3, retinoic acid–inducible gene I, and DNA-dependent activator of IFN regulatory factors. Other DNA sensors, including p204 and cGMP-AMP synthase, can be induced by transfection of synthetic HSV genomic DNA (HSV60). TLR3 and retinoic acid–inducible gene I in EECs can be activated by their common agonist, polyinosinic-polycytidylic acid [poly(I:C)]. The signaling pathway of DNA sensors can be initiated by HSV60. Both poly(I:C) and HSV60 induced the expression of type 1 IFNs and various antiviral proteins, including IFN-stimulated gene 15, 2′,5′-oligoadenylate synthetase, and myxovirus resistance 1. Poly(I:C), but not HSV60, also dramatically induced the expression of major proinflammatory cytokines, including TNF-α and MCP-1, in EECs. In vivo assay confirmed that the local injection of poly(I:C) or HSV60 induced the innate antiviral responses in EECs. This study provided novel insights into the mechanisms underlying the innate antiviral responses in the mouse epididymis.

No abstract