Spermatozoa-induced seminal vesiculitis in mice.

BACKGROUND: Seminal vesiculitis is a common inflammation in the male genital tract. Etiologically, microbial infection and non-infectious factors can be responsible for seminal vesiculitis. The pathogenic triggers and mechanisms underlying non-infectious seminal vesiculitis remain unclear. OBJECTIVES: To demonstrate that spermatozoa can induce seminal vesiculitis in mice, which could be attributable to spermatozoa-induced innate immune responses in seminal vesicular epithelial cells. MATERIAL AND METHODS: Spermatozoa from epididymis were injected into seminal vesicles at the tail of the gland. Histopathology of seminal vesicles were examined by hematoxylin-eosin staining. Infiltration of leukocytes were identified by immunohistochemistry. Seminal vesicular epithelial cells were isolated from 5-week-old mice and cell types were detected by immunofluoresence. Western blot and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect protein and gene expression levels. RESULTS: In vivo, local injection of epididymal spermatozoa into seminal vesicles resulted in seminal vesiculitis characterized by tissue swelling and leukocyte infiltration. In vitro, spermatozoa induced the expression of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, CXCL10, and MCP1, and the activation of NF-κB in seminal vesicular epithelial cells. DISCUSSION AND CONCLUSION: Spermatozoa may induce seminal vesiculitis through the activation of innate immune responses in seminal vesicular epithelial cells, which provide novel insights into the mechanisms underlying non-infectious seminal vesiculitis.

Viral tropism for the testis and sexual transmission.

The mammalian testis adopts an immune privileged environment to protect male germ cells from adverse autoimmune reaction. The testicular immune privileged status can be also hijacked by various microbial pathogens as a sanctuary to escape systemic immune surveillance. In particular, several viruses have a tropism for the testis. To overcome the immune privileged status and mount an effective local defense against invading viruses, testicular cells are well equipped with innate antiviral machinery. However, several viruses may persist an elongated duration in the testis and disrupt the local immune homeostasis, thereby impairing testicular functions and male fertility. Moreover, the viruses in the testis, as well as other organs of the male reproductive system, can shed to the semen, thus allowing sexual transmission to partners. Viral infection in the testis, which can impair male fertility and lead to sexual transmission, is a serious concern in research on known and on new emerging viruses. To provide references for our scientific peers, this article reviews research achievements and suggests future research focuses in the field.

Immune homeostasis and disorder in the testis - Roles of Sertoli cells.

The mammalian testis requires a highly organized microenvironment for the execution of its main functions, sperm production and testosterone synthesis. The testis possesses an immunoprivileged status essential for the protection of immunogenic male germ cells from detrimental immune responses. To counteract microbial infections, the tissue-specific cells are well equipped with innate defense machineries, termed pattern recognition receptors (PRRs), that can initiate innate immune responses. However, PRR-initiated innate immune responses in testicular cells may disrupt the immunoprivileged status and result in orchitis, thereby impairing spermatogenesis and testosterone synthesis. The mechanisms underlying the innate defense system and immunological pathophysiology in the testis have been intensively studied. Testicular immune homeostasis and disorders are regulated by a coordination of the tissue structure and cellular functions. This article focuses on the pivotal roles that Sertoli cells play in regulating testicular immune homeostasis by facilitating the blood-testis barrier, phagocytosis, paracrine, and innate immune responses.

Differential Effects of Viral Nucleic Acid Sensor Signaling Pathways on Testicular Sertoli and Leydig Cells.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon ß promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.

Immunologic Environment of the Testis.

Mammalian spermatogenesis is a carefully orchestrated male germ cell differentiation process by which spermatogonia differentiate to spermatozoa in the testis. A highly organized testicular microenvironment is therefore necessary to support spermatogenesis. Regarding immunologic aspects, the testis adapts a specialized immune environment for the protection of male germ cells and testicular functions. The mammalian testis possesses two immunologic features: (1) it is an immunoprivileged organ where immunogenic germ cells do not induce deleterious immune responses under physiologic conditions; and (2) it creates its own effective innate defense system against microbial infection. Various pathologic conditions may disrupt testicular immune homeostasis, thereby resulting in a detrimental immune response and perturbing testicular functions, one of the etiologic factors of male infertility. Understanding the mechanisms underlying immunoregulation in the testis can aid in establishing strategies for the prevention and therapy of immunologic testicular dysfunction and male infertility. This chapter focuses on the mechanisms underlying immune privilege, local innate immunity, and immunologic diseases of the testis.

Pattern recognition receptor-initiated innate immune responses in mouse prostatic epithelial cells‡.

Three major pathogenic states of the prostate, including benign prostatic hyperplasia, prostate cancer, and prostatitis, are related to the local inflammation. However, the mechanisms underlying the initiation of prostate inflammation remain largely unknown. Given that the innate immune responses of the tissue-specific cells to microbial infection or autoantigens contribute to local inflammation, this study focused on pattern recognition receptor (PRR)-initiated innate immune responses in mouse prostatic epithelial cells (PECs). Primary mouse PECs abundantly expressed Toll-like receptor 3 (TLR3), TLR4, TLR5, melanoma differentiation-associated protein 5 (MDA5), and IFN-inducible protein 16 (p204 in mouse). These PRRs can be activated by their respective ligands: lipopolysaccharide (LPS) and flagellin of Gram-negative bacteria for TLR4 and TLR5, polyinosinic-polycytidylic acid (poly(I:C)) for TLR3 and MDA5, and herpes simplex virus DNA analog (HSV60) for p204. LPS and flagellin predominantly induced the expression of inflammatory cytokines, including tumor necrosis factor alpha (TNFA), interleukin 6 (IL6), chemokines monocyte chemoattractant protein-1 (MCP1), and C-X-C motif chemokine 10 (CXCL10). Poly(I:C) and HSV60 predominantly induced the expression of type 1 interferons (IFNA and IFNB) and antiviral proteins: Mx GTPase 1, 2',5'-oligoadenylate synthetase 1, and IFN-stimulated gene 15. The replication of mumps virus in PECs was inhibited by type 1 IFN signaling. These findings provide insights into the mechanisms underlying innate immune response in the prostate.

Mumps Orchitis: Clinical Aspects and Mechanisms.

The causative agent of mumps is a single-stranded, non-segmented, negative sense RNA virus belonging to the Paramyxoviridae family. Besides the classic symptom of painfully swollen parotid salivary glands (parotitis) in mumps virus (MuV)-infected men, orchitis is the most common form of extra-salivary gland inflammation. Mumps orchitis frequently occurs in young adult men, and leads to pain and swelling of the testis. The administration of MuV vaccines in children has been proven highly effective in reducing the incidence of mumps. However, a recent global outbreak of mumps and the high rate of orchitis have recently been considered as threats to male fertility. The pathogenesis of mumps orchitis remains largely unclear due to lack of systematic clinical data analysis and animal models studies. The alarming increase in the incidence of mumps orchitis and the high risk of the male fertility have thus become a major health concern. Recent studies have revealed the mechanisms by which MuV-host cells interact and MuV infection induces inflammatory responses in testicular cells. In this mini-review, we highlight advances in our knowledge of the clinical aspects and possible mechanisms of mumps orchitis.

Characterization of an Antiviral Component in Human Seminal Plasma.

Numerous types of viruses have been found in human semen, which raises concerns about the sexual transmission of these viruses. The overall effect of semen on viral infection and transmission have yet to be fully investigated. In the present study, we aimed at the effect of seminal plasma (SP) on viral infection by focusing on the mumps viral (MuV) infection of HeLa cells. MuV efficiently infected HeLa cells in vitro. MuV infection was strongly inhibited by the pre-treatment of viruses with SP. SP inhibited MuV infection through the impairment of the virus's attachment to cells. The antiviral activity of SP was resistant to the treatment of SP with boiling water, Proteinase K, RNase A, and DNase I, suggesting that the antiviral factor would not be proteins and nucleic acids. PNGase or PLA2 treatments did not abrogate the antiviral effect of SP against MuV. Further, we showed that the prostatic fluid (PF) showed similar inhibition as SP, whereas the epididymal fluid and seminal vesicle extract did not inhibit MuV infection. Both SP and PF also inhibited MuV infection of other cell types, including another human cervical carcinoma cell line C33a, mouse primary epididymal epithelial cells, and Sertoli cell line 15P1. Moreover, this inhibitory effect was not specific to MuV, as the herpes simplex virus 1, dengue virus 2, and adenovirus 5 infections were also inhibited by SP and PF. Our findings suggest that SP contains a prostate-derived pan-antiviral factor that may limit the sexual transmission of various viruses.

Zika virus disrupts the barrier structure and Absorption/Secretion functions of the epididymis in mice.

Several studies have demonstrated that Zika virus (ZIKV) damages testis and leads to infertility in mice; however, the infection in the epididymis, another important organ of male reproductive health, has gained less attention. Previously, we detected lesions in the epididymis in interferon type I and II receptor knockout male mice during ZIKV infection. Herein, the pathogenesis of ZIKV in the epididymis was further assessed in the infected mice after footpad inoculation. ZIKV efficiently replicated in the epididymis, and principal cells were susceptible to ZIKV. ZIKV infection disrupted the histomorphology of the epididymis, and the effects were characterized by a decrease in the thickness of the epithelial layer and an increase in the luminal diameter, especially at the proximal end. Significant inflammatory cell infiltration was observed in the epididymis accompanied by an increase in the levels of interleukin (IL)-6 and IL-28. The expression of tight junction proteins was downregulated and associated with disordered arrangement of the junctions. Importantly, the expression levels of aquaporin 1 and lipocalin 8, indicators of the absorption and secretion functions of the epididymis, were markedly reduced, and the proteins were redistributed. These events synergistically altered the microenvironment for sperm maturation, disturbed sperm transport downstream, and may impact male reproductive health. Overall, these results provide new insights into the pathogenesis of the male reproductive damage caused by ZIKV infection and the possible contribution of epididymal injury into this process. Therefore, male fertility of the population in areas of ZIKV epidemic requires additional attention.

Axl Deficiency Promotes the Neuroinvasion of Japanese Encephalitis Virus by Enhancing IL-1α Production from Pyroptotic Macrophages.

Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis (JE), which has an unclear pathogenesis. Despite vaccination, thousands of deaths attributed to JE are reported annually. In this study, we report that mice deficient for Axl, a receptor tyrosine kinase that plays multiple roles in flaviviral infection, displayed greater mortality upon JEV infection. The effect of Axl deficiency on JEV infection was mediated by markedly elevated serum interleukin-1α (IL-1α) levels, which devastated the blood-brain-barrier and promoted viral neuroinvasion within 24 h postinfection. Using an in situ infection model, we showed that dead macrophages were the primary source of observed increased serum IL-1α levels. Axl deficiency enhanced cell death and caused pyroptosis in 80% of JEV-infected macrophages by disrupting phosphatidylinositol 3-kinase (PI3K)-Akt signaling. Intriguingly, the primary effector released by pyroptotic macrophages in our model was IL-1α rather than IL-1ß. Finally, we assessed the effect of an IL-1α antagonist and demonstrated that it effectively prevented the incidence of JE. Our results indicate that Axl plays a protective role in JEV infection, identify IL-1α released by pyroptotic macrophages as a crucial factor promoting JEV neuroinvasion, and suggest that an IL-1α antagonist may be a candidate for JE therapy.IMPORTANCE Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese encephalitis (JE), the most commonly diagnosed viral encephalitis worldwide. The fatality rate of JE is 20%, and nearly half of the surviving patients develop neuropsychiatric sequelae. Axl is a receptor tyrosine kinase that plays multiple roles in flaviviral infections. Currently, the involvement of Axl in JEV infection remains enigmatic. In this study, we demonstrate that Axl impedes the pathogenesis of severe JE in mice by maintaining blood-brain-barrier (BBB) integrity and restricting viral neuroinvasion. Furthermore, serum IL-1α is a key mediator of this process and is primarily released by JEV-infected pyroptotic macrophages to elicit BBB breakdown, while an IL-1α antagonist can effectively reduce the incidence of severe JE. Our work uncovers the protective role of Axl in antagonizing severe JE and shows that the use of an IL-1α antagonist may be a promising tactic to prevent severe JE.

Roles of Sialic Acid, AXL, and MER Receptor Tyrosine Kinases in Mumps Virus Infection of Mouse Sertoli and Leydig Cells.

The mumps virus (MuV) causes epidemic parotitis. MuV also frequently infects the testis and induces orchitis, an important etiological factor contributing to male infertility. However, mechanisms underlying MuV infection of the testis remain unknown. Here, we describe that sialic acid, AXL, and MER receptor tyrosine kinases regulate MuV entry and replication in mouse major testicular cells, including Sertoli and Leydig cells. Sialic acid, AXL, and MER were present in Sertoli and Leydig cells. Sialic acid specifically mediated MuV entry into Sertoli and Leydig cells, whereas both AXL and MER facilitated MuV replication within cells through the inhibition of cellular innate antiviral responses. Mechanistically, the inhibition of type 1 interferon signaling by AXL and MER is essential for MuV replication in Sertoli and Leydig cells. Our findings provide novel insights into the mechanisms behind MuV infection and replication in the testis.

Damaged male germ cells induce epididymitis in mice.

Epididymitis can be caused by infectious and noninfectious etiological factors. While microbial infections are responsible for infectious epididymitis, the etiological factors contributing to noninfectious epididymitis remain to be defined. The present study demonstrated that damaged male germ cells (DMGCs) induce epididymitis in mice. Intraperitoneal injection of the alkylating agent busulfan damaged murine male germ cells. Epididymitis was observed in mice 4 weeks after the injection of busulfan and was characterized by massive macrophage infiltration. Epididymitis was coincident with an accumulation of DMGCs in the epididymis. In contrast, busulfan injection into mice lacking male germ cells did not induce epididymitis. DMGCs induced innate immune responses in epididymal epithelial cells (EECs), thereby upregulating the pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), as well as the chemokines such as monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-5 (MCP-5), and chemokine ligand-10 (CXCL10). These results suggest that male germ cell damage may induce noninfectious epididymitis through the induction of innate immune responses in EECs. These findings provide novel insights into the mechanisms underlying noninfectious epididymitis, which might aid in the diagnosis and treatment of the disease.

Mumps virus infection disrupts blood-testis barrier through the induction of TNF-α in Sertoli cells.

Mumps virus (MuV) has high tropism to the testis and may lead to male infertility. Sertoli cells are the major targets of MuV infection. However, the mechanisms by which MuV infection impairs male fertility and Sertoli cell function remain unclear. The present study elucidated the effect of MuV infection on the blood-testis barrier (BTB). The transepithelial electrical resistance of MuV-infected mouse Sertoli cells was monitored, and the expression of major proteins of the BTB was examined. We demonstrated that MuV infection disrupted the BTB by reducing the levels of occludin and zonula occludens 1. Sertoli cells derived from Tlr2-/- and Tnfa-/- mice were analyzed for mediating MuV-induced impairment. TLR2-mediated TNF-α production by Sertoli cells in response to MuV infection impaired BTB integrity. MuV-impaired BTB was not observed in Tlr2-/- and Tnfa-/- Sertoli cells. Moreover, an inhibitor of TNF-α, pomalidomide, prevents the disruption of BTB in response to MuV infection. FITC-labeled biotin tracing assay confirmed that BTB permeability and spermatogenesis were transiently impaired by MuV infection in vivo. These findings suggest that the disruption of the BTB could be one of the mechanisms underlying MuV-impaired male fertility, in which TNF-α could play a critical role.-Wu, H., Jiang, X., Gao, Y., Liu, W., Wang, F., Gong, M., Chen, R., Yu, X., Zhang, W., Gao, B., Song, C., Han, D. Mumps virus infection disrupts blood-testis barrier through the induction of TNF-α in Sertoli cells.

Pattern recognition receptor-mediated innate immune responses in seminal vesicle epithelial cell and their impacts on cellular function†.

The seminal vesicles can be infected by microorganisms, thereby resulting in vesiculitis and impairment in male fertility. Innate immune responses in seminal vesicles cells to microbial infections, which facilitate vesiculitis, have yet to be investigated. The present study aims to elucidate pattern recognition receptor-mediated innate immune responses in seminal vesicles epithelial cells. Various pattern recognition receptors, including Toll-like receptor 3, Toll-like receptor 4, cytosolic ribonucleic acid, and deoxyribonucleic acid sensors, are abundantly expressed in seminal vesicles epithelial cells. These pattern recognition receptors can recognize their respective ligands, thus activating nuclear factor kappa B and interferon regulatory factor 3. The pattern recognition receptor signaling induces expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (Tnfa) and interleukin 6 (Il6), chemokines monocyte chemoattractant protein-1 (Mcp1) and C-X-C motif chemokine 10 (Cxcl10), and type 1 interferons Ifna and Ifnb. Moreover, pattern recognition receptor-mediated innate immune responses up-regulated the expression of microsomal prostaglandin E synthase and cyclooxygenase 2, but they down-regulated semenogelin-1 expression. These results provide novel insights into the mechanism underlying vesiculitis and its impact on the functions of the seminal vesicles.

Lipopolysaccharide-induced testicular dysfunction and epididymitis in mice: a critical role of tumor necrosis factor alpha†.

Systemic inflammation may impair male fertility, and its underlying mechanisms remain poorly understood. The present study investigates the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the testis and epididymis in mice. Intraperitoneal injection of LPS significantly impaired testicular functions, including testosterone production, spermatogenesis, and blood-testis barrier permeability. The epididymitis characterized by leukocyte infiltration and fibrosis was observed in the cauda epididymis after LPS injection. LPS-induced testicular dysfunction and epididymitis were abolished in tumor necrosis factor alpha (Tnfa) knockout mice. Pomalidomide, a TNFA inhibitor, blocked the detrimental effects of LPS on the testis and epididymis. The results indicate that LPS-induced systemic inflammation impairs male fertility through TNFA production, suggesting that the intervention on TNFA production would be considered for the prevention and treatment of inflammatory impairment of male fertility.

Viral threat to male fertility.

The detrimental effects of Zika virus (ZIKV) infection on mouse testicular functions have reminded a viral threat to male fertility. A broad range of virus families has tropism for male reproductive system, particularly the testes. Certain virus types of these viruses, such as mumps virus and human immunodeficiency virus (HIV), may severely damage the testes and consequently lead to male infertility. ZIKV has been recently found to damage testicular functions and lead to male infertility in mice. Many other viruses also have detrimental effects on host reproduction. Public attention has been paid to sexually transmitted viruses, such as HIV and hepatitis B and C viruses in humans and likewise in economically important farm animals. This article provides an overview on main viruses affecting the male reproductive system and their detrimental effects on fertility, and outlines some important issues for future study.

Correction: C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis.

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C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis.

Mumps virus (MuV) infection usually results in germ cell degeneration in the testis, which is an etiological factor for male infertility. However, the mechanisms by which MuV infection damages male germ cells remain unclear. The present study showed that C-X-C motif chemokine ligand 10 (CXCL10) is produced by mouse Sertoli cells in response to MuV infection, which induces germ cell apoptosis through the activation of caspase-3. CXC chemokine receptor 3 (CXCR3), a functional receptor of CXCL10, is constitutively expressed in male germ cells. Neutralizing antibodies against CXCR3 and an inhibitor of caspase-3 activation significantly inhibited CXCL10-induced male germ cell apoptosis. Furthermore, the tumor necrosis factor-α (TNF-α) upregulated CXCL10 production in Sertoli cells after MuV infection. The knockout of either CXCL10 or TNF-α reduced germ cell apoptosis in the co-cultures of germ cells and Sertoli cells in response to MuV infection. Local injection of MuV into the testes of mice confirmed the involvement of CXCL10 in germ cell apoptosis in vivo. These results provide novel insights into MuV-induced germ cell apoptosis in the testis.