Sustained infiltration of neutrophils into the CNS results in increased demyelination in a viral-induced model of multiple sclerosis. Sustained infiltration of neutrophils into the CNS results in increased demyelination in a viral-induced model of multiple sclerosis

Intracranial inoculation of the neuroadapted JHM strain of mouse hepatitis virus (JHMV) into susceptible strains of mice results in acute encephalomyelitis and chronic immune-mediated demyelination similar to the human demyelinating disease multiple sclerosis (MS). JHMV infection of transgenic mice in which expression of the neutrophil chemoattractant chemokine CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells results in sustained neutrophil infiltration in the central nervous system (CNS) that correlates with an increase in spinal cord demyelination. We used single cell RNA sequencing (scRNAseq) and flow cytometry to characterize molecular and cellular changes within the CNS associated with increased demyelination in transgenic mice compared to control animals. These approaches revealed the presence of activated neutrophils as determined by increased expression of mRNA transcripts associated with increased neutrophil effector functions, such as CD63, MMP9, S100a8, S100a9, and ASPRV1, as well as by altered neutrophil morphology and protein expression. Collectively, these findings reveal insight into changes in the profile of neutrophils associated with increased white matter damage in mice persistently infected with a neurotropic coronavirus. Overall design: Double-tg mice and single-tg (male and female, 6-8 weeks) were injected intracranially (i.c.) in the right brain hemisphere with 250 plaque forming units (PFU) of JHMV and CD45-positive (CD45+) cells were flow sorted from the brains of JHMV-infected mice at day post-infection (p.i.). FACS sorted CD45+ cells then underwent single-cell RNA-sequencing analysis

将神经适应型小鼠肝炎病毒JHM株（neuroadapted JHM strain of mouse hepatitis virus, JHMV）颅内接种至易感小鼠品系，可引发急性脑脊髓炎与慢性免疫介导性脱髓鞘病变，其病理表型与人类脱髓鞘疾病多发性硬化（multiple sclerosis, MS）高度相似。在星形胶质细胞纤维酸性蛋白（glial fibrillary acidic protein, GFAP）阳性细胞中受四环素诱导型启动子（tetracycline-inducible promoter）调控表达中性粒细胞趋化因子CXCL1的转基因小鼠，经JHMV感染后，中枢神经系统（central nervous system, CNS）会出现持续的中性粒细胞浸润，该现象与脊髓脱髓鞘程度加重呈显著正相关。本研究采用单细胞RNA测序（single cell RNA sequencing, scRNAseq）与流式细胞术（flow cytometry），对比转基因小鼠与对照动物，对中枢神经系统内与脱髓鞘程度加重相关的分子与细胞变化进行了系统表征。通过上述分析，研究人员鉴定到活化的中性粒细胞：其分子特征为与中性粒细胞效应功能增强相关的mRNA转录本表达上调，涵盖CD63、MMP9、S100a8、S100a9及ASPRV1，同时伴随中性粒细胞形态改变与蛋白表达异常。综上，本研究揭示了嗜神经性冠状病毒持续感染小鼠中，与白质损伤加重相关的中性粒细胞谱变化。实验设计：将双转基因小鼠与单转基因小鼠（雌雄各半，6~8周龄）经颅内注射（intracranial, i.c.）至右侧大脑半球，接种250噬斑形成单位（plaque forming units, PFU）的JHMV；于感染后天（day post-infection, p.i.）从JHMV感染小鼠的脑组织中流式分选CD45阳性（CD45+）细胞，随后对分选得到的CD45+细胞开展单细胞RNA测序分析。