Etebar, F.; Whatmore, P.; Harkin, D. G.; Dando, S. J.

Background: Bordering the central nervous system (CNS) parenchyma are the pia mater (the innermost layer of the meninges enveloping the brain) and the choroid (underlying the retina). While near the neural parenchyma, the pia mater and choroid are external to the immune privileged environment of the brain and retina and thus are distinct immune compartments. This study aimed to characterise the transcriptomic signatures of immune cells within the pia mater and choroid bordering the healthy adult mouse CNS. Methods: Brains and eyes were obtained from 7-week-old female C57Bl/6J mice. Pia mater-enriched tissue and choroid were dissected and processed for fluorescence activated cell sorting of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ. Results: A total of 3,606 cells were sequenced from mouse tissues, including 1,481 CD45+ cells from pia mater-enriched tissue and 2,125 CD45+ cells from choroid. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells and B cells. While some clusters were common to both pia mater and choroid, others exhibited tissue-specific gene expression profiles and potential functional specialisations. Analysis of 6,501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid. Conclusions: This study provides a detailed characterisation of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy brain and retina, and their potential roles in immune protection.