Department of Neurobiology & Behavior, School of Biological Sciences, University of California, Irvine, 92697, USA.
Department of Molecular Biology & Biochemistry, School of Biological Sciences, University of California, Irvine, 92697, USA.
J Neuroinflammation. 2024 Jun 15;21(1):157. doi: 10.1186/s12974-024-03153-0.
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.
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.
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.
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.
半胱氨酸蛋白酶抑制剂 F 是一种分泌性溶酶体胱氨酸蛋白酶抑制剂，先前的研究表明它可影响脱髓鞘的严重程度，并增强免疫介导的脱髓鞘动物模型中的髓鞘再生。然而，关于半胱氨酸蛋白酶抑制剂 F 如何影响中枢神经系统（CNS）病毒感染后的神经疾病严重程度，目前仍知之甚少，这也是本研究的重点。我们使用了半胱氨酸蛋白酶抑制剂 F 缺失突变小鼠（Cst7-/-），该动物模型已被广泛用于研究鼠冠状病毒诱导的神经疾病，以此来评估半胱氨酸蛋白酶抑制剂 F 在宿主防御、脱髓鞘和髓鞘再生中的作用。
野生型对照和 Cst7-/- 小鼠通过脑内（i.c.）感染神经嗜性 JHM 株鼠肝炎病毒（JHMV）的亚致死剂量，每天监测疾病进展和存活率。通过病毒斑形成试验和 qPCR 检测 CNS 中的病毒水平。通过流式细胞术和 10X 基因组学 Chromium 3' 单细胞 RNA 测序（scRNA-seq）评估免疫细胞浸润和免疫细胞激活。通过卢索快速蓝（LFB）和苏木精/伊红（H&E）染色评估脊髓脱髓鞘，通过 SMI-32 免疫组化染色评估轴突损伤。通过电子显微镜（EM）评估髓鞘再生并计算 g-比值。
感染 JHMV 的 Cst7-/- 小鼠能够控制 CNS 内的病毒复制，这表明半胱氨酸蛋白酶抑制剂 F 对于有效的 Th1 抗病毒免疫反应并非必需。与感染对照相比，感染 JHMV 的 Cst7-/- 小鼠脊髓中 T 细胞的浸润增加，这与轴突损伤和脱髓鞘有关，同时也与髓鞘再生受损有关。从感染 Cst7-/- 和对照小鼠的脊髓中分离出 CD45+细胞进行单细胞 RNA-seq 分析，揭示了趋化因子 Cxcl9 和 Cxcl10 编码转录物的表达增强，同时 Cst7-/- 小鼠 CD8+T 细胞中干扰素-g（Ifng）和穿孔素（Prf1）转录物的表达升高。
半胱氨酸蛋白酶抑制剂 F 对于 JHMV 在 CNS 内的免疫介导复制并非必需。然而，感染 JHMV 的 Cst7-/- 小鼠表现出更严重的临床疾病，与脱髓鞘增加和髓鞘再生受损有关。疾病严重程度的增加与 T 细胞趋化因子的表达增加有关，同时伴随着神经炎症的增加。这些发现支持这样一种观点，即半胱氨酸蛋白酶抑制剂 F 影响促炎基因表达，从而影响神经炎症、T 细胞激活和/或胶质细胞反应，最终影响神经炎症和神经疾病。
