Franklin Lina, Kuassivi Ohiniba-Nadège, Satie Anne-Pascale, Abiven Hervé, Mathieu Romain, Miaadi Naoufel, Plotton Ingrid, Le Tortorec Anna, Dejucq-Rainsford Nathalie
Institut National de la Santé et de la Recherche Médicale, Ecole des Hautes Etudes en Santé Publique, Institut de recherche en santé, environnement et travail, Université de Rennes-UMR_S1085, Rennes, France.
Service d'Urologie, Centre Hospitalier Universitaire de Rennes, Rennes, France.
Hum Reprod. 2025 Sep 9. doi: 10.1093/humrep/deaf155.
What is the direct effect of mumps virus (MuV) replication within the human testis on the tissue innate immune responses and testicular cell functions?
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.
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, METHODS: Human 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.
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.
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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.
These 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.
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腮腺炎病毒（MuV）在人类睾丸内复制对组织固有免疫反应和睾丸细胞功能有何直接影响？
MuV在体外人睾丸中诱导早期促炎反应，并感染睾丸间质细胞和支持细胞，这会显著改变睾酮和抑制素B的产生。
尽管进行了广泛的疫苗接种，但睾丸炎仍是MuV感染的一个重要并发症，尤其是在年轻男性中，这可能导致高达87%的双侧睾丸炎患者不育。由于缺乏相关动物模型，我们对MuV在人类睾丸中的发病机制的理解受到限制，这阻碍了有效治疗方法的开发。
研究设计、规模、持续时间：从7名未感染的尸体供体（中位年龄55岁，范围29 - 79岁）收集正常睾丸。对体外暴露或未暴露于MuV的人睾丸外植体进行器官型培养10天。利用这个原始的体外模型，我们研究了MuV的复制动力学，确定了其靶细胞，表征了睾丸对病毒的固有免疫反应，并评估了感染对睾丸细胞功能的影响。
参与者/材料、设置、方法：将人睾丸外植体以感染复数1过夜暴露于MuV，并在空气/培养基界面的聚对苯二甲酸乙二醇酯插入物上培养10天。通过在空斑形成试验中测量感染性病毒颗粒的释放以及在RT - qPCR中测量病毒RNA，以及在整个10天培养期内对睾丸细胞中复制性病毒RNA进行原位检测，来证明MuV在人睾丸外植体中的复制。使用特异性细胞标志物和针对病毒RNA的探针通过显微镜对感染细胞进行表征。使用RT - qPCR、原位杂交和LegendPlex评估固有免疫反应。通过质谱法测量MuV感染和模拟感染外植体上清液中的睾酮及其前体，而通过ELISA测量抑制素B。通过乳酸脱氢酶活力测定、RT - qPCR、免疫组织化学和蛋白质免疫印迹进一步探索MuV感染对睾丸组织和细胞的影响。
在整个10天的培养过程中，MuV在人睾丸外植体中大量复制，从感染睾丸间质细胞、巨噬细胞和管周细胞的间质组织发展到感染支持细胞的生精小管。与另一种嗜睾丸病毒寨卡病毒不同，MuV在暴露的人睾丸外植体中4小时内就引发了促炎反应，其特征是前哨细胞中白细胞介素1β（IL1B）的转录上调。随后是炎症介质的组织释放（72小时和第7天时IL1B的P = 0.02）以及病毒复制时白细胞介素10（IL10）的动态调节。从第7天起，MuV复制通过在细胞色素P450家族17亚家族A成员1（CYP17A1）水平破坏睾丸间质细胞的类固醇生成活性而抑制睾酮产生（P < 0.03），并且从第4天起降低支持细胞中抑制素B的分泌（P < 0.03），支持细胞表现出细胞焦亡的特征。
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局限性、谨慎原因：这项体外研究证明了MuV在人睾丸中复制的直接影响，但未评估浸润的外周免疫细胞在睾丸病变中的额外作用。
这些发现表明，与以沉默持续存在而闻名的寨卡病毒相比，MuV感染人睾丸会引发独特的早期固有免疫反应。这种差异为MuV诱导的睾丸炎的发展提供了一个潜在的解释。此外，我们的研究提供了证据表明，在没有白细胞浸润的情况下，MuV直接破坏关键的睾丸功能。这些数据推进了我们对MuV在睾丸中发病机制早期事件的理解，并为进一步研究睾丸炎与无症状感染的机制提供了基础。本研究中开发的MuV感染人睾丸的体外模型将作为评估旨在保护MuV感染男性睾丸功能的抗病毒策略的宝贵工具。
研究资金/利益冲突：本研究得到了法国国家研究机构（资助编号ANR - 21 - CE15 - 0021 - 01）和医学研究基金会（FRM EQU202203014611）的资助，以及法国国家健康与医学研究院和雷恩大学的支持。作者没有利益冲突。
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