RESUMEN
Microglia serve as a front-line defense against neuroinvasive viral infection, however, determination of their actual transcriptional profiles under conditions of health and disease is challenging. Here, we used various experimental approaches to delineate the transcriptional landscape of microglia during viral infection. Intriguingly, multiple activation genes were found to be artificially induced in sorted microglia and we demonstrated that shear stress encountered during cell sorting was one of the key inducers. Post-hoc analysis revealed that publicly available large-scale single-cell RNA sequencing datasets were significantly tainted by aberrant signatures that are associated with cell sorting. By exploiting the ribosomal tagging approach, we developed a strategy to enrich microglia-specific transcripts by comparing immunoprecipitated RNA with total RNA. Such enriched transcripts were instrumental in defining bona fide signatures of microglia under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia are involved in shaping antiviral T-cell responses in the brain. Collectively, our study defines the transcriptional landscape of microglia under steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination.
Asunto(s)
Encefalitis Viral , Perfilación de la Expresión Génica , Homeostasis , Microglía , Microglía/metabolismo , Microglía/virología , Animales , Encefalitis Viral/genética , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Homeostasis/fisiología , Homeostasis/genética , Ratones , Perfilación de la Expresión Génica/métodos , Transcriptoma , Ratones Endogámicos C57BLRESUMEN
Sindbis virus (SINV) infection of mice provides a model system for studying the pathogenesis of alphaviruses that infect the central nervous system (CNS) to cause encephalomyelitis. While studies of human viral infections typically focus on accessible cells from the blood, this compartment is rarely evaluated in mice. To bridge this gap, single-cell RNA sequencing (scRNAseq) was combined with flow cytometry to characterize the transcriptional and phenotypic changes of peripheral blood mononuclear cells (PBMCs) from SINV-infected mice. Twenty-one clusters were identified by scRNAseq at 7 days after infection, with a unique cluster and overall increase in naive B cells for infected mice. Uninfected mice had fewer immature T cells and CCR9+ CD4 T cells and a unique immature T cell cluster. Gene expression was most altered in the Ki67+ CD8 T cell cluster, with chemotaxis and proliferation-related genes upregulated. Global analysis indicated metabolic changes in myeloid cells and increased expression of Ccl5 by NK cells. Phenotypes of PBMCs and cells infiltrating the CNS were analyzed by flow cytometry over 14 days after infection. In PBMCs, CD8 and Th1 CD4 T cells increased in representation, while B cells showed a transient decrease at day 5 in total, Ly6a+, and naive cells, and an increase in activated B cells. In the brain, CD8 T cells increased for the first 7 days, while Th1 CD4 T cells and naive and Ly6a+ B cells continued to accumulate for 14 days. Therefore, dynamic immune cell changes can be identified in the blood as well as the CNS during viral encephalomyelitis. IMPORTANCE: The outcome of viral encephalomyelitis is dependent on the host immune response, with clearance and resolution of infection mediated by the adaptive immune response. These processes are frequently studied in mouse models of infection, where infected tissues are examined to understand the mechanisms of clearance and recovery. However, studies of human infection typically focus on the analysis of cells from the blood, a compartment rarely examined in mice, rather than inaccessible tissue. To close this gap, we used single-cell RNA sequencing and flow cytometry to profile the transcriptomic and phenotypic changes of peripheral blood mononuclear cells (PBMCs) before and after central nervous system (CNS) infection in mice. Changes to T and B cell gene expression and cell composition occurred in PBMC and during entry into the CNS, with CCL5 being a differentially expressed chemokine. Therefore, dynamic changes occur in the blood as well as the CNS during the response of mice to virus infection, which will inform the analysis of human studies.
Asunto(s)
Infecciones por Alphavirus , Leucocitos Mononucleares , Animales , Ratones , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Infecciones por Alphavirus/virología , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/genética , Virus Sindbis/genética , Virus Sindbis/inmunología , Ratones Endogámicos C57BL , Fenotipo , Femenino , Modelos Animales de Enfermedad , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Encefalitis Viral/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Análisis de la Célula IndividualRESUMEN
Inherited deficiency of the RNA lariat-debranching enzyme 1 (DBR1) is a rare etiology of brainstem viral encephalitis. The cellular basis of disease and the range of viral predisposition are unclear. We report inherited DBR1 deficiency in a 14-year-old boy who suffered from isolated SARS-CoV-2 brainstem encephalitis. The patient is homozygous for a previously reported hypomorphic and pathogenic DBR1 variant (I120T). Consistently, DBR1 I120T/I120T fibroblasts from affected individuals from this and another unrelated kindred have similarly low levels of DBR1 protein and high levels of RNA lariats. DBR1 I120T/I120T human pluripotent stem cell (hPSC)-derived hindbrain neurons are highly susceptible to SARS-CoV-2 infection. Exogenous WT DBR1 expression in DBR1 I120T/I120T fibroblasts and hindbrain neurons rescued the RNA lariat accumulation phenotype. Moreover, expression of exogenous RNA lariats, mimicking DBR1 deficiency, increased the susceptibility of WT hindbrain neurons to SARS-CoV-2 infection. Inborn errors of DBR1 impair hindbrain neuron-intrinsic antiviral immunity, predisposing to viral infections of the brainstem, including that by SARS-CoV-2.