Proton-secreting epithelial cells as key modulators of epididymal mucosal immunity.

项目总结/摘要 男性生殖生理学中最有趣和研究不足的方面之一是附睾的能力， 阻止针对自身抗原精子的免疫应答的发展，同时启动非常有效的免疫应答， 对病原体的反应。这项申请的长期目标是开发新的战略疗法， 疾病，如男性不育症和附睾炎，并确定男性避孕的新目标。整体 目的是阐明附睾上皮透明细胞（CC）如何与免疫细胞通讯，以促进附睾上皮细胞的免疫功能。 “免疫特权”环境是精子成熟和储存的最佳环境。我们最近展示了一个 CC在免疫激活和精子耐受中的全新作用。重要的是，这些细胞建立亲密的 与附睾中单核吞噬细胞（MP）的区域特异性异质亚群接触。我们的中央 一种假设是，CC被战略性地定位成与免疫细胞以协调一致的方式工作，以调查免疫细胞的免疫功能。 附睾屏障，调节睾丸后炎症和免疫耐受之间的平衡 环境基本原理是基于我们的初步数据显示，CC和MP通信在稳定状态 在感染或损伤的情况下，这两种细胞类型表达不同的致耐受性介质 允许调节精子耐受性。因此，这些细胞在附睾粘膜中起着中心作用。在 第一个具体目标，我们将确定在附睾炎期间启动免疫激活的CC-MP通信网络。 为此，将使用两种小鼠附睾炎模型：向尾部管腔注射细菌抗原， 附睾-睾丸扭转导致不育性炎症。在第二个具体目标中，我们将评估CC-MP 用白喉耗竭调节性T细胞诱导附睾精子耐受消除后的串扰 毒素转基因小鼠本申请中提出的研究将采用创新的多学科方法 它采用了很少用于研究生殖生理学的强大免疫学技术，3D共聚焦显微镜， 转录组学分析、流式细胞术、细胞分选和生物信息学分析，以及使用小鼠模型， 描述了特化上皮细胞和免疫细胞如何在附睾中交流。特别是，我们将提供一个- 三种特定附睾细胞类型的深度表征：CC和MP的两个子集。我们的研究计划是 重要的是，它有望为附睾免疫调节的主要机制提供新的见解。 CC与MP一起保护精子免受有害抗原和自身免疫的侵害。最终，这些知识将 通过解决粘膜免疫学和细胞免疫学的关键概念，填补了与男性生殖生物学相关的重要空白， 细胞相互作用，所有这些都是人类男性生殖健康的关键方面，但尚未得到充分研究。

PROJECT SUMMARY/ABSTRACT One of the most intriguing and understudied aspects of male reproductive physiology is the ability of the epididymis to prevent the development of immune responses against autoantigenic spermatozoa, while initiating very efficient immune responses against pathogens. The long-term goal of this application is to develop new strategic therapies for common disorders such as male infertility and epididymitis, and to identify novel targets for male-contraception. The overall objective is to elucidate how epididymal epithelial clear cells (CCs) communicate with immunocytes to contribute to the “immune privileged” environment that is optimal for sperm maturation and storage. We have recently showed a completely novel role for CCs in immune activation and sperm tolerance. Importantly, these cells establish intimate contact with region-specific heterogeneous subsets of mononuclear phagocytes (MPs) in the epididymis. Our central hypothesis is that CCs are strategically positioned to work in a concerted manner with immune cells to survey the epididymis barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment. The rationale is based on our preliminary data showing that CCs and MPs communicate in the steady state epididymis and also in the presence of infection or injury, and that both cell types express different tolerogenic mediators allowing the modulation of sperm tolerance. Therefore, these cells play a central role in the epididymal mucosa. In the first specific aim, we will determine CC – MP communication networks that initiate immune activation during epididymitis. In this aim, two mouse models of epididymitis will be used: injection of bacterial antigens into the cauda lumen, and epididymal-testicular torsion that induces sterile inflammation. In the second specific aim, we will evaluate CC - MP crosstalk after ablation of sperm tolerance in the epididymis, induced by depletion of regulatory T cells using diphtheria toxin-based transgenic mice. The research proposed in this application will use an innovative multidisciplinary approach that employs powerful immunology techniques rarely used to study reproductive physiology, 3D confocal microscopy, transcriptomics analysis, flow cytometry, cell sorting and bioinformatic analysis, as well as the use of mouse models to describe how specialized epithelial cells and immunocytes communicate in the epididymis. Notably, we will provide an in- depth characterization of three specific epididymal cell types: CCs and two subsets of MPs. Our proposed research is significant because it is expected to provide new insights into major immunoregulatory mechanisms by which epididymal CCs together with MPs protect spermatozoa against harmful antigens and autoimmunity. Ultimately, such knowledge will fill important gaps related to male reproductive biology by addressing crucial concepts of mucosal immunology and cell– cell interactions, all of which are critical but understudied facets of human male reproductive health.