Mumps virus infection triggers early pro-inflammatory responses and impairs Leydig and Sertoli cell function in an ex vivo human testis model.

study questionWhat is the direct effect of mumps virus (MuV) replication within the human testis on the tissue innate immune responses and testicular cell functions? SUMMARY ANSWER: MuV induces an early pro-inflammatory response in the human testis ex vivo and infects both Leydig cells and Sertoli cells, which drastically alters testosterone and inhibin B production. WHAT IS KNOWN ALREADY: Despite widespread vaccination efforts, orchitis remains a significant complication of MuV infection, especially in young men, which potentially results in infertility in up to 87% of patients with bilateral orchitis. Our understanding of MuV pathogenesis in the human testis has been limited by the lack of relevant animal models, impairing the development of effective treatments. STUDY DESIGN, SIZE, DURATION: Normal testes were collected from seven uninfected post-mortem donors (median age of 55 years, range 29-79). Organotypic cultures of human testis explants exposed or not to MuV ex vivo were undertaken for 10 days. Utilizing this original ex vivo model, we investigated the replication kinetics of MuV, identified its target cells, characterized the innate immune responses of the testis to the virus, and assessed the impact of the infection on testicular cell functions. PARTICIPANTS/MATERIALS, SETTING,

methodsHuman testis explants were exposed overnight to MuV at a multiplicity of infection of 1 and cultured on polyethylene terephthalate inserts at the air/medium interface for 10 days. MuV replication in human testis explants was evidenced by measuring the release of infectious viral particles in plaque-forming assay and viral RNA in RT-qPCR, as well as by in situ detection of replicative viral RNA in testicular cells all along the 10-day culture period. Infected cells were characterized by microscopy using specific cell markers and a probe against viral RNA. The innate immune response was assessed using RT-qPCR, in situ hybridization, and LegendPlex. Testosterone and its precursors were measured in the supernatants of MuV and mock-infected explants by mass spectrometry, while inhibin B was measured by ELISA. The impact of MuV infection on testis tissue and cells was further explored by lactate dehydrogenase viability assay, RT-qPCR, immunohistochemistry, and western blot. MAIN RESULTS AND THE ROLE OF CHANCE: MuV robustly replicated in human testicular explants all along the 10-day culture, progressing from the interstitial tissue, where it infected Leydig cells, macrophages, and peritubular cells, to the seminiferous tubules, where it targeted Sertoli cells. Unlike Zika virus, another testis-tropic virus, MuV triggered a pro-inflammatory response within 4 h in exposed human testis explants, characterized by transcriptional upregulation of interleukin 1 beta (IL1B) in sentinel cells. This was followed by the tissue release of inflammatory mediators (P = 0.02 for IL1B at 72 h and Day 7) and the dynamic regulation of interleukin 10 (IL10) upon viral replication. MuV replication inhibited testosterone production from Day 7 onwards (P < 0.03) by disrupting the steroidogenic activity of Leydig cells at the level of cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and decreased inhibin B secretion from Sertoli cells from Day 4 onwards (P < 0.03), which exhibited features of pyroptosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This ex vivo study, which demonstrates the direct impact of MuV replication in the human testis, does not assess the additional role of infiltrating peripheral immune cells in testicular lesions. WIDER IMPLICATIONS OF THE

findingsThese findings demonstrate that MuV infection of the human testis elicits a distinct early innate immune response in contrast to Zika virus, known for its silent persistence. This difference offers a potential explanation for the development of MuV-induced testis inflammation. Furthermore, our study provides evidence that MuV directly disrupts crucial testicular functions in the absence of leukocytic infiltrates. These data advance our understanding of the early events of MuV pathogenesis in the testis and provide a basis for further investigation into the mechanisms of orchitis versus silent infection. The ex vivo model of MuV-infected human testis developed in this study will serve as a valuable tool for evaluating antiviral strategies aimed at preserving testicular function in MuV-infected men. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the French National Research Agency (grant number ANR-21-CE15-0021-01) and from the Fondation pour la Recherche Médicale (FRM EQU202203014611), as well as by Institut National de la Santé et de la Recherche Médicale and the University of Rennes. The authors have no competing interests. TRIAL REGISTRATION NUMBER: N/A.