Qiaoyuan Chen

Toll-like receptor-mediated inhibition of Gas6 and ProS expression facilitates inflammatory cytokine production in mouse macrophages

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.

Toll-Like Receptor 3-Initiated Antiviral Responses in Mouse Male Germ Cells In Vitro

ABSTRACT 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.

Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells

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.

Breakdown of immune homeostasis in the testis of mice lacking Tyro3, Axl and Mer receptor tyrosine kinases

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.

Toll-Like Receptor 11-Initiated Innate Immune Response in Male Mouse Germ Cells

ABSTRACT Toxoplasma gondii and uropathogenic Escherichia coli (UPEC) may infect the testis and impair testicular function. Mechanisms underlying testicular innate immune response to these two pathogens remain to be clarified. The present study examined the function of TLR11, which can be recognized by T. gondii-derived profilin and UPEC, in initiating innate immune response in male mouse germ cells. TLR11 is predominantly expressed in spermatids. Profilin and UPEC induced the expressions of different inflammatory cytokine profiles in the germ cells. In particular, profilin induced the expressions of macrophage chemotactic protein 1 (MCP1), interleukin 12 (IL12), and interferon gamma (IFNG) through nuclear factor KB (NFKB) activation. UPEC induced the expressions of MCP1, IL12, and IFNG, as well as tumor necrosis factor alpha (TNFA), IL6, and IFNB, through the activation of NFKB, IFN regulatory factor 3, and mitogen-activated protein kinases. Evidence showed that profilin induced the innate response in male germ cells through TLR11 signaling, and UPEC triggered the response through TLR11 and other TLR-signaling pathways. We also provided evidence that local injection of profilin or UPEC induces the innate immune response in the germ cells. Data describe TLR11-mediated innate immune function of male germ cells in response to T. gondii profilin and UPEC stimulations. This system may play a role in testicular defense against T. gondii and UPEC infections in mice.

Testicular immunoregulation and spermatogenesis.

The mammalian testis possesses a unique immune environment that is essential for testicular function. The testis is a remarkable immunoprivileged site that protects immunogenic germ cells from the detrimental effects of immune responses. However, the testis can be infected by various microbial pathogens. To overcome the immune privilege and enable testicular defense against microbes, the testis adopts local effective innate immune responses to microbial infections. The mechanisms underlying the testicular immune privilege have been investigated for several decades and the innate defense system in the testis is being revealed based on the identification of pattern recognition receptor-initiated innate immune responses in testicular cells. The coordination between immune privilege and local innate immune responses is critical in the maintenance of testicular immune homeostasis. Disruption of the testicular immune homeostasis may lead to orchitis and impair spermatogenesis, an etiological factor of male infertility. Dissection of the immunoregulatory mechanisms in the testis can aid in establishing preventive and therapeutic approaches for orchitis. This review discusses current understanding of the mechanisms which underlie the testicular immunoregulation and its effect on spermatogenesis.

Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double‐knockout (Axl−/−Mer−/−) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freunds adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro‐inflammatory cytokine upregulation in the testes, impaired permeability of the blood–testis barrier and generation of autoantibodies against germ cell antigens in Axl−/−Mer−/− mice. Immunization also induced mild EAO in Axl or Mer single‐gene‐knockout mice. By contrast, a single immunization failed to induce EAO in wild‐type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.

Pattern Recognition Receptor–Initiated Innate Antiviral Responses in Mouse Epididymal Epithelial Cells

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.

p204-mediated innate antiviral responses in mouse adipose cells and their effects on cell functions

Viruses can infect adipose tissues. However, innate antiviral responses in adipose cells and their effects on adipocyte function have not yet been intensively investigated. In this study, p204‐initiated innate antiviral responses in mouse adipose cells were examined. Cytosolic DNA sensor p204 and its signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in primary preadipocytes. Synthetic herpes simplex viral DNA (HSV60), a p204 ligand, induced type I IFN expression by activating IFN regulatory factor 3. Major antiviral proteins, including IFN‐stimulating gene 15, 2′,5′‐oligoadenylate synthetase and Mx GTPase 1, in preadipocytes were upregulated by HSV60. HSV60‐triggered innate antiviral responses were significantly reduced by inhibition of p204 signaling with specific small interfering RNA targeting p204 or STING. HSV60 inhibited the differentiation of preadipocytes to mature adipocytes and enhanced the proliferation of adipose cells. Moreover, HSV60 induced innate antiviral responses in mature adipocytes and inhibited expressions of several adipokines, including leptin, adiponectin and resistin. These results indicated that p204 initiated innate antiviral responses in adipose cells, thereby modulating adipocyte function.

p204-Initiated Innate Antiviral Response in Mouse Leydig Cells

ABSTRACT The mammalian testis is an immunoprivileged organ where local tissue-specific cells acquire an effective innate immune function against invading microbial pathogens. The present study demonstrated that mouse Leydig cells had innate antiviral activities in response to viral DNA challenge through p204 activation. The DNA sensor p204 and its signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in Leydig cells. Synthetic herpes simplex virus DNA analog (HSV60), a p204 agonist, induced the expression of type I IFNs and various antiviral proteins, including IFN-stimulating gene 15, 2′5′-oligoadenylate synthetase, and Mx glutamyl transpeptidase 1, in Leydig cells. The HSV60-induced innate antiviral response in Leydig cells was significantly reduced by inhibiting p204 signaling using specific small interfering RNAs targeting p204 and Sting. The antiviral response did not affect steroidogenesis in Leydig cells. These results indicated a novel mechanism underlying the testicular innate antiviral response.