Post-natal meningeal macrophages protect against viral neuroinfection

Due to the vital importance of the Central Nervous System (CNS), its potential infection and inflammation have to be tightly controlled. The surface of the CNS is connected to the periphery by a rich and complex tissue, the meninges. They contain a vast network of macrophages subdivided in at least two subpopulations endowed with elusive functions: a neonatal, MHC-II negative macrophage population, and an post-natal population expressing MHC-II. Using in situ-histocytometry, flow cytometry, and single-cell RNA sequencing approaches, we showed that those populations have opposite dynamic behaviors in response to in vivo peripheral challenges such as LPS, SARS-CoV2 and lymphocytic choriomeningitis virus (LCMV), with an apparent contraction of the MHC-II+ population. Focusing on LCMV infection in experimental mouse models and using innovative pharmacological and genetic depletion strategies, we show that meningeal macrophages (MM) represent an early line of protection against this neuroinvasive pathogen. In their absence, specific areas in the meninges became highly infected, leading to fatal brain disease. While their intrinsic sensing of viral replication through the Mitochondrial antiviral-signaling protein (MAVS) was dispensable, sensing of IFNs through the STAT1 pathway played an important role in controlling viral spread. Unexpectedly, the post-natal MHC-II+ macrophage population had an important role in controlling neuroinfection, by shutting down biosynthesis pathways and efficiently blocking viral replication. This work helps understanding the spatial organization of the brain defense system and the cellular and molecular mechanisms involved in CNS protection. Overall design: Meningeal cells were extracted as described in the Mononuclear cell isolation and Flow cytometry sections above (apart from sacrifice, which was performed using an overdose of Ketamine/Xylasine) and stained with Hoescht 33342 (10µg/ml, Sigma) and Draq7 (0.3µM, ThermoFischer). The 4 samples were differentially labelled using 4 Hashtags (TotalSeq-A, clone M1/42; 30-F11, Biolegend) at 0.1mg/ml for 15min at 4°C. After a wash, nucleated cells were isolated from 2 PBS or 2 LPS-injected mice (7 weeks-old males) at day 1 post-injection using a FACS Aria III SORP (BD) using a 100 µm nozzle. 52 000 cells of each sample were pooled, centrifuged at 350g for 3 minutes, and 35 000 cells of the pool were loaded on a Chromium Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEM and scRNA-libraries were prepared using the Single Cell 3'v3 Gel Bead and Library Kit (10X Genomics, No. 1000092) and the Chromium i7 Multiplex kit (10x Genomics, No 120262) according to the manufacturer's instructions.