Single-Cell Analysis of Neuroinflammatory Responses Following Intracranial Injection of G-Deleted Rabies Viruses

Abstract

Viral vectors are essential tools for the study of neural circuits, with glycoprotein-deleted rabies viruses being widely used for monosynaptic retrograde tracing to map connectivity between specific cell types in the nervous system. However, the use of rabies virus is limited by the cytotoxicity and the inflammatory responses these viruses trigger. While components of the rabies virus genome contribute to its cytotoxic effects, the function of other neuronal and non-neuronal cells within the vicinity of the infected host neurons in either effecting or mitigating virally-induced tissue damage are still being elucidated. Here, we analyzed 60,212 single-cell RNA profiles to assess both global and cell-typespecific transcriptional responses in the mouse dorsal raphe nucleus (DRN) following intracranial injection of glycoprotein-deleted rabies viruses and axonal infection of dorsal raphe serotonergic neurons. Gene pathway analyses revealed a down-regulation of genes involved in metabolic processes and neurotransmission following infection. We also identified several transcriptionally diverse leukocyte populations that infiltrate the brain and are distinct from resident immune cells. Cell type-specific patterns of cytokine expression showed that antiviral responses were likely orchestrated by Type I and Type II interferon signaling from microglia and infiltrating CD4 + T cells, respectively. Additionally, we uncovered transcriptionally distinct states of microglia along an activation trajectory that may serve different functions, which range from surveillance to antigen presentation and cytokine secretion. Intercellular interactions inferred from transcriptional data suggest that CD4 + T cells facilitate microglial state transitions during the inflammatory response. Our study uncovers the heterogeneity of immune cells mediating neuroinflammatory responses and provides a critical evaluation of the compatibility between rabies-mediated connectivity mapping and single-cell transcriptional profiling. These findings provide additional insights into the distinct contributions of various cell types in mediating different facets of antiviral responses in the brain and will facilitate the design of strategies to circumvent immune responses to improve the efficacy of viral gene delivery.

Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

“…Collecting the entire transcriptome of injected and non-injected animals offers an opportunity to study the effects of AAV transduction on the host cell transcriptome. A similar investigation has been conducted with G-deleted rabies virus (Huang and Sabatini, 2020). This study demonstrated that virus infection led to the downregulation of genes involved in metabolic processes and 90 neurotransmission in host cells, whereas genes related to cytokine signaling and the adaptive immune system were upregulated.…”

A combination of two human monoclonal antibodies cures symptomatic rabies

“…In a comparative manner, in neurodegenerative diseases, the antibody‐mediated clearance of tau protein by microglia in vitro is dependent on Fc‐gamma receptor binding (Andersson et al , 2019); Fc‐gamma receptor is key to antibody uptake by neurons (Congdon et al , 2013). Consequently, the effective rabies virus clearance by the RVC20/RVC58 mAbs may be reinforced by Fc‐gamma receptor binding, by permitting the access of the mAbs inside infected neurons, and by modulating microglial activity and inflammatory mediators’ production, or even by promoting the infiltration of leukocytes (Quan et al , 2009; Chauhan et al , 2017; Huang & Sabatini, 2020).…”

Tissue-Resident NK Cells: Development, Maturation, and Clinical Relevance

“…Vento et al analyzed the transcriptome of about 70,000 single-cells from human first-trimester placentas, and they found three subsets decidual NK cells (dNK1. dNK2, and dNK3) [ 205 ]. These subsets co-express the tissue-resident markers CD49a (ITGA1) and CD9.…”

“…These subsets co-express the tissue-resident markers CD49a (ITGA1) and CD9. dNK1 cells express CD39 (ENTPD1), CYP26A1, and B4GALNT1, whereas the defining markers of dNK2 cells are ANXA1 and ITGB2; dNK3 cells express CD160, KLRB1, and CD103 (ITGAE), but not the innate lymphocyte cell marker CD127 [ 205 ].…”