CD163+ macrophages monitor enhanced permeability at the blood–dorsal root ganglion barrier

In dorsal root ganglia (DRG), macrophages reside in close proximity to sensory neurons, and their functions have largely been explored in the context of pain, nerve injury and repair. In this study, however, we discovered that the majority of macrophages in DRGs are in direct contact with the vasculature where they constantly monitor the circulation, efficiently phagocytosing proteins and macromolecules from the blood. Characterization of the DRG endothelium revealed a specialized vascular network spanning the arteriovenous axis, which gradually transformed from a barrier type endothelium in arteries to a highly permeable endothelium in veins. Macrophage phagocytosis spatially aligned with peak endothelial permeability and we identified caveolar transcytosis as a mechanism regulating endothelial permeability. Profiling of the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover and function. CD163 expressing macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation and were conserved in mouse and Man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit. DOI: 10.1084/jem.20230675 Mice were perfused with ice- cold PBS and neural tissues dissected into cold PBS. On average 40 pooled cervical, thoracic and lumbar DRGs were used. The tissues were digested in 2mg/ml Collagenase I (Gibco 17100-017), 5mg/ml Dispase II (Sigma, D4693) and 0.5mg/ml DNAse I (Roche, 11284932001) at 37C for 30min (Brain) or 40min DRGs. Myelin was removed using 38% Percoll (Sigma, GE17-0891-02). Single cell suspensions of DRG cells from 3 male and 3 female BALB/cAnNRj mice were blocked with FcR blocking solution (Miltenyi Biotec l No.130-092-575) and stained with anti-CD45:PE (BioLegend 103105). Additionally, TotalSeq™ anti-mouse Hashtag antibodies were used to label individual samples The samples were strained through a 35-micron filter. DAPI was added to the cell solution in order to exclude dead cells. CD45+DAPI− cells were sorted on a BD Influx sorter. The sorted cells were pooled into a single lane on the 10x Genomics Chromium Single Cell 3 ′ v3 system. Library preparation and sequencing were performed at the SciLifeLab sequencing facility, Solna using an Illumina HiSeq at 61,006 reads/cell.