Day-3 SYMPOSIUM II: ENVIRONMENT AND MALE INFERTILITYChairs: Maciej Kurpisz | Bettina Toth | Guy Cassuto (replaced Satish)9:00-9:20 How immune cell phenotypes determine a region-specific immune response in the epididymis

The epididymis is a tightly coiled single tubule that connects to the testis via the efferent ducts. The epididymis comprises four distinct regions in mouse: the initial segment which receives the spermatozoa from the efferent ducts, the caput (head), the corpus (body) and the cauda (tail), where sperm are stored and pass to the vas deferens. The epididymis faces opposing immunological challenges. In the initial segment and caput, local tolerance is required to avoid autoimmune reactions against immunogenic spermatozoa. Conversely, the distal region/cauda is a port of entry for bacterial microbes ascending from the environment via the urethra. In both mice and human, the magnitude of immune response in the cauda following bacterial infection is much higher than in the proximal parts of the epididymis, often leading to male infertility due to fibrosis and duct obstruction. We assume that immune cell populations are strategically positioned along the epididymis to maintain the immunological equilibrium across the organ. Epididymitis in mice was elicited injection of uropathogenic Escherichia coli (UPEC CFT073) in the lumen of the vas deferens close to the epididymis. Morphological alterations were assessed by histological staining and related to quantity and localization of infiltrating leukocyte populations by flow cytometry and immunofluorescence. Epididymal regions from wt mice were simultaneously exposed to an inflammatory stimulus ex vivo (LPS, 50ng/ml, 6h). Levels of inflammatory cytokines (e.g. TNFα, MCP-1, IL-1α, IL-1β, IL-6) were quantified using RT-qPCR and bead-based immunoassay. Multipanel flow cytometry and immunofluorescence were performed to characterize resident immune cells under physiological conditions. At steady-state, immune cell populations exhibited striking region-specific properties. The proximal regions were densely populated by immunoregulatory immune cells, i.e. macrophage subsets involved in tissue homeostasis (identified by alternating expression of CX3CR1, CCR2, MHC-II, CD206). Corpus and cauda were characterized by a smaller but more diverse network of immune cells (T cells, NK cells, DC subsets, CCR2+ monocyte-derived macrophages) suggesting an immune-sensitive environment within the distal regions predestined to sense invading pathogens. UPEC infection resulted in severe immune responses within the cauda associated with epithelial detachment and fibrotic remodeling, while the caput remained mostly unresponsive. Tissue-damage was positively correlated with an increase of CD45+ leukocytes, i.e. infiltrating Ly6G+ neutrophils followed by Ly6ChiCD11bhiF4/80lo monocytes that appear to differentiate into Ly6Clo-intCD11bhiF4/80intMHC-IIhi macrophages. Immune cell infiltration was associated with upregulation of inflammatory cytokines within the cauda in ascending bacterial infection in vivo, similar to the ex vivo model where LPS challenge occurred simultaneously. In summation, our data imply that strategically positioned leukocyte populations could be a key factor in maintaining the immunological equilibrium within the epididymis and are responsible for the differences in immune responsivenesses of epididymal regions.