PDF(Pubmed)
Abstract：
BACKGROUND: Cystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination.
METHODS: Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3\' single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.
RESULTS: JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.
CONCLUSIONS: Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.
METHODS: Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3\' single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.
RESULTS: JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.
CONCLUSIONS: Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.
摘要：
背景：胱抑素F是一种分泌型溶酶体半胱氨酸蛋白酶抑制剂，在免疫介导的脱髓鞘的临床前模型中，其与影响脱髓鞘的严重程度和增强髓鞘再生有关。胱抑素F如何影响中枢神经系统（CNS）病毒感染后神经系统疾病的严重程度尚未得到很好的表征，并且是本研究的重点。我们使用胱抑素F无效突变小鼠（Cst7-/-）与建立良好的小鼠冠状病毒诱导的神经系统疾病模型来评估胱抑素F在宿主防御中的贡献，脱髓鞘和髓鞘再生。
方法：野生型对照和Cst7-/-小鼠颅内（i.c.）感染亚致死剂量的嗜神经JHM小鼠肝炎病毒（JHMV），每天监测疾病进展和生存。病毒噬斑测定和qPCR用于评估CNS中的病毒水平。通过流式细胞术和10X基因组学铬3'单细胞RNA测序（scRNA-seq）确定免疫细胞向CNS的浸润和免疫细胞活化。脊髓脱髓鞘通过卢克索耐蓝(LFB)和苏木精/伊红(H&E)染色来确定,并且通过SMI-32的免疫组织化学染色来评估轴突损伤。通过电子显微镜（EM）和g比的计算来评估髓鞘再生。
结果：JHMV感染的Cst7-/-小鼠能够控制CNS内的病毒复制，表明胱抑素F对于有效的Th1抗病毒免疫应答不是必需的。与感染对照相比，感染JHMV的Cst7-/-小鼠的T细胞向脊髓的浸润增加，这与轴突损伤增加和髓鞘再生受损相关的脱髓鞘有关。从感染的Cst7-/-和对照小鼠的脊髓中富集的CD45细胞的单细胞RNA-seq显示编码T细胞化学引诱物的转录本的表达增强，Cxcl9和Cxcl10与来自Cst7-/-小鼠的CD8+T细胞中的干扰素-g(Ifng)和穿孔素(Prf1)转录物的升高表达相组合,与对照相比。
结论：胱抑素F不是CNS内JHMV复制的免疫介导控制所必需的。然而,JHMV感染的Cst7-/-小鼠表现出与脱髓鞘增多和髓鞘再生受损相关的更严重的临床疾病。疾病严重程度的增加与T细胞趋化因子的表达升高有关，同时神经炎症增加。这些发现支持胱抑素F影响影响神经炎症的促炎基因表达的观点。T细胞活化和/或神经胶质细胞反应最终影响神经炎症和神经系统疾病。
方法：野生型对照和Cst7-/-小鼠颅内（i.c.）感染亚致死剂量的嗜神经JHM小鼠肝炎病毒（JHMV），每天监测疾病进展和生存。病毒噬斑测定和qPCR用于评估CNS中的病毒水平。通过流式细胞术和10X基因组学铬3'单细胞RNA测序（scRNA-seq）确定免疫细胞向CNS的浸润和免疫细胞活化。脊髓脱髓鞘通过卢克索耐蓝(LFB)和苏木精/伊红(H&E)染色来确定,并且通过SMI-32的免疫组织化学染色来评估轴突损伤。通过电子显微镜（EM）和g比的计算来评估髓鞘再生。
结果：JHMV感染的Cst7-/-小鼠能够控制CNS内的病毒复制，表明胱抑素F对于有效的Th1抗病毒免疫应答不是必需的。与感染对照相比，感染JHMV的Cst7-/-小鼠的T细胞向脊髓的浸润增加，这与轴突损伤增加和髓鞘再生受损相关的脱髓鞘有关。从感染的Cst7-/-和对照小鼠的脊髓中富集的CD45细胞的单细胞RNA-seq显示编码T细胞化学引诱物的转录本的表达增强，Cxcl9和Cxcl10与来自Cst7-/-小鼠的CD8+T细胞中的干扰素-g(Ifng)和穿孔素(Prf1)转录物的升高表达相组合,与对照相比。
结论：胱抑素F不是CNS内JHMV复制的免疫介导控制所必需的。然而,JHMV感染的Cst7-/-小鼠表现出与脱髓鞘增多和髓鞘再生受损相关的更严重的临床疾病。疾病严重程度的增加与T细胞趋化因子的表达升高有关，同时神经炎症增加。这些发现支持胱抑素F影响影响神经炎症的促炎基因表达的观点。T细胞活化和/或神经胶质细胞反应最终影响神经炎症和神经系统疾病。
