Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury

Abstract

While central nervous system (CNS) microglia have been studied extensively, surprisingly little is known about macrophages populating the peripheral nervous system (PNS Macs).We performed ontogenic, transcriptomic and spatial characterization of sciatic nerve Macs (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and get replaced by bone marrow-derived Macs over time. Using single-cell profiling, we identified a tissue-specific core signature of snMacs and found two spatially-separated snMacs: Relmα + Mgl1 + snMacs in the epineurium and Relmα -Mgl1-snMacs in the endoneurium. Globally, snMacs lack most core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. Single-cell transcriptomics revealed that in response to injury both snMacs respond differently and that the PNS, in contrast to the CNS, is permissive to prolonged engraftment of monocytederived Macs recruited upon injury.

Single cell analysis of the cellular heterogeneity and interactions in the injured mouse spinal cord

“…Our finding that Macrophage-A and B subtypes do not correspond to the M1/M2 nomenclature (Extended Data Fig. 5) is consistent with findings from other sc-RNAseq studies [29][30][31] . However, there are parallels with the generally accepted paradigm that macrophages shift from an anti-inflammatory to a more pro-inflammatory state over time after SCI [32][33][34] .…”

Imaging resident and recruited macrophage contribution to Wallerian degeneration

“…In contralateral nerves representative of noninjured nerves (control nerves), only a few EGFP + cells and no ECFP + cells were detected. These cells, located at the epi-and perineurium, displayed a stellar and elongated shape when located at the endoneurium in close contact with nerve fibers as reported by Ydens et al (2020) and in other tissue-associated nerves (Kolter et al, 2020). Flow cytometry analysis confirmed that resident MPs express EGFP, but not ECFP, arguing for the recent recruitment of ECFP + cells (Fig.…”

“…We chose to simplify the nomenclature and defined the ECFP + EGFP + subset as ECFP + cells and the ECFP − EGFP + subsets as EGFP + cells. ECFP + cells are composed of Ly6C high -Mo's as well as Ly6C low CD64 + MPs (previously named "Mono-Mac waterfall"; Ydens et al, 2020), while EGFP + cells represent exclusively Ly6C low CD64 + MPs and accumulate by 10 d after injury ( Fig. 1 e, right panels).…”

“…Lyve1 is associated with vascular-(Lyve1 high MHCII low ) and nerve-associated (Lyve1 low MHCII high ) interstitial MPs in the lungs (Chakarov et al, 2019), and Mgl2 is associated with endoneurial MPs after injury (Ydens et al, 2020). Out of these 124 genes, in accordance with the resident epineurial MPs signature (Ydens et al, 2020), Retnla and Clec10a, coding for Relmα and Mgl1, respectively, were both significantly enriched in the EGFP + -sorted MPs compared with ECFP + MPs at a log fold change of 1.4 and 1.3, respectively. No inflammatory pathway-associated gene cluster was uncovered among these 124 genes, suggesting that either the two MP subsets evenly up-regulated inflammatory genes or that inflammation might already be dampened by day 14 in favor of the regenerative process.…”

“…This subset has been demonstrated to proliferate upon nerve injury and participate in the process of WD along with MPs of hematogenous origin (Mueller et al, 2003). Ydens et al (2020) have recently investigated this question and identified that MPs in the sciatic nerve arise from late embryonic precursors progressively replaced by MPs of adult Mo origin. Based on single-cell RNA sequencing (RNA-seq), they uncovered that endoneurial and epineurial MPs harbor distinct transcriptomic signatures specific for the sciatic nerve.…”