CX3CR1+ mononuclear phagocytes gene-expression profile based on their location in the epididymis using RNA-sequencing

In the epididymis, prevention of autoimmune responses against spermatozoa, while providing protection against pathogens, is important for male fertility. We previously showed that immune cells known as mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these “intraepithelial” MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterization of MPs isolated from IS, caput-corpus, and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in ‘dendritic cells’ or ‘macrophages’. RNA sequencing identified distinct transcriptomic signatures in MPs from each region, and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity, and antigen processing and presentation. Functional fluorescent in vivo labeling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS and corpus, circulatory antigens were internalized and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalized and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-born pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for common disorders such as male infertility and epididymitis, and identify potential targets for immuno-contraception. EGFP+ CX3CR1+ cells were isolated by double cell sorting from the initial segments (IS), caput/corpus and cauda epididymal regions of CX3CR1 EGFP +/- transgenic mice that specifically express EGFP in mononuclear phagocytes.