Temporal tracking of microglial and monocyte single-cell transcriptomics in lethal flavivirus infection

Abstract

As the resident parenchymal myeloid population in the central nervous system (CNS), microglia are strategically positioned to respond to neurotropic virus invasion and have been implicated in promoting both disease resolution and progression in the acute and post-infectious phase of virus encephalitis. In a mouse model of West Nile virus encephalitis (WNE), infection of the CNS results in recruitment of large numbers of peripheral immune cells into the brain, the majority being nitric oxide (NO)-producing Ly6Chi inflammatory monocyte-derived cells (MCs). In this model, these cells enhance immunopathology and mortality. However, the contribution of microglia to this response is currently undefined. Here we used a combination of experimental tools, including single-cell RNA sequencing (scRNA-seq), microglia and MC depletion reagents, high-dimensional spectral cytometry and computational algorithms to dissect the differential contribution of microglia and MCs to the anti-viral immune response in severe neuroinflammation seen in WNE. Intriguingly, analysis of scRNA-seq data revealed 6 unique microglia and 3 unique MC clusters that were predominantly timepoint-specific, demonstrating substantial transcriptional adaptation with disease progression over the course of WNE. While microglia and MC adopted unique gene expression profiles, gene ontology enrichment analysis, coupled with microglia and MC depletion studies, demonstrated a role for both of these cells in the trafficking of peripheral immune cells into the CNS, T cell responses and viral clearance. Over the course of infection, microglia transitioned from a homeostatic to an anti-viral and then into an immune cell-recruiting phenotype. Conversely, MC adopted antigen-presenting, immune cell-recruiting and NO-producing phenotypes, which all had anti-viral function. Overall, this study defines for the first time the single-cell transcriptomic responses of microglia and MCs over the course of WNE, demonstrating both protective and pathological roles of these cells that could potentially be targeted for differential therapeutic intervention to dampen immune-mediated pathology, while maintaining viral clearance functions.

High‐Dimensional Methods of Single‐Cell Microglial Profiling to Enhance Understanding of Neuropathological Disease

“…Although multiple RNA studies, including our own, have shown that microglia downregulate the expression of the nominal homeostatic microglia-specific markers P2ry12 and Tmem119 during neurodegeneration and neuroinflammation (Jordão et al, 2019;Keren-Shaul et al, 2017;Krasemann et al, 2017;Spiteri, Wishart et al, 2023;Wishart et al, 2022), on a protein level, microglia do not downregulate P2RY12 expression in WNE (Spiteri et al, 2021) or alpha-herpesvirus CNS infection (Fekete et al, 2018). As TMEM119 expression is downregulated by microglia in certain inflammatory diseases, such as WNE, P2RY12 enables the separation of microglia from infiltrating myeloid populations when used in combination with CD45.…”

“…Proceed with manufacturer's instructions for cell capture. Details of sample processing for sequencing can be found in Spiteri, Wishart et al (2023).…”

“…Advances in cytometry and computational analysis platforms have better elucidated the identities of these cell types (Ashhurst et al., 2021). This has enabled the development of more accurate gating strategies to phenotype and sort microglia and infiltrating myeloid cells (Spiteri, Wishart et al., 2023).…”

“…These include microglia‐specific identification markers such as P2RY12 and TMEM119 (Bennett et al., 2016; Butovsky et al., 2014) and the CSF1R inhibitor and microglia‐depletion drug PLX5622 (Spangenberg et al., 2019). Although these represent significant advances in the field, subsequent work has shown that microglia‐specific markers are often downregulated during inflammation, while PLX5622 can also target other cells in the periphery (Depaula‐Silva et al., 2019; Greter et al., 2015; Lei et al., 2020; Lewis et al., 2014; Spiteri & King, 2023; Spiteri, Ni et al., 2022; Spiteri et al., 2021; Spiteri, Wishart et al., 2023; Spiteri et al., 2020; Vankriekelsvenne et al., 2022). Moreover, isolated and cultured microglia undergo considerable change from their in vivo state (Bennett et al., 2018; Bohlen et al., 2017; Gosselin et al., 2017), significantly limiting the translatability of in vitro studies.…”

“…a Cerebral interferonopathy modelled in transgenic mice with CNS-targeted chronic production of IFN-α (GFAP-IFN; West et al, 2022) or demyelination modeled in mice treated with cuprizone (Song et al, 2021). b West Nile virus encephalitis (Spiteri, Ni et al, 2022;Spiteri et al, 2021;Spiteri, Wishart et al, 2023;Spiteri, Van Vreden et al, 2023;Wishart et al, 2022).…”