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

项目摘要/摘要 男性生殖生理学最耐人寻味和最未被研究的一个方面是附睾会 防止针对自身抗原精子的免疫反应的发展，同时启动非常有效的免疫 对病原体的反应。该应用的长期目标是开发新的治疗常见病的策略疗法。 这项工作的目的是解决诸如男性不育症和附睾炎等疾病，并确定新的男性避孕目标。整体而言 目的：阐明附睾腺上皮透明细胞(CCS)与免疫细胞之间的通讯机制，以促进附睾上皮细胞与免疫细胞的相互作用。 “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 CCS在免疫激活和精子耐受性方面的全新作用。重要的是，这些细胞建立了亲密的 与附睾单核巨噬细胞(MPS)的区域特异性异质性亚群接触。我们的中央 假设CCS在战略上处于与免疫细胞协同工作的位置，以调查 附睾屏障和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CCS和MPS在稳定状态下通信 在感染或损伤存在的情况下，两种细胞类型表达不同的耐受介质 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 第一个具体目标是，我们将确定在附睾炎期间启动免疫激活的CC-MP通信网络。 为了达到这个目的，将使用两种附睾炎的小鼠模型：将细菌抗原注射到尾部腔内，以及 导致不育性炎症的附睾-睾丸扭转。在第二个具体目标中，我们将评估CC-MP 用白喉去除调节性T细胞引起的精子耐受性消融后的串扰 基于毒素的转基因小鼠。本申请中提出的研究将使用创新的多学科方法 使用了很少用来研究生殖生理学的强大免疫学技术，3D共聚焦显微镜， 转录组分析、流式细胞术、细胞分类和生物信息学分析，以及使用小鼠模型 描述特化的上皮细胞和免疫细胞在附睾体中是如何沟通的。值得注意的是，我们将提供一个- 三种特定的附睾细胞类型：CCS和MPS的两个亚集的深度特征。我们建议的研究是 这一研究意义重大，因为它有望为附睾腺的主要免疫调节机制提供新的见解。 CCS和MPS一起保护精子免受有害抗原和自身免疫的影响。最终，这样的知识将 通过解决粘膜免疫学和细胞生物学的关键概念，填补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但未被研究的方面。

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.