Cystatin F attentuates neuroinflammation and demyelination following murine coronavirus infection of the central nervous system
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. Immune cell infiltration into the brain and spinal cord was determined by flow cytometry. Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and remyelination evaluated by electron microscopy (EM) and calculation of g-ratios. Immune cell infiltration into the CNS and microglia activation were determined by flow cytometry and 10X genomics chromium 3' single cell RNA sequencing (scRNAseq). 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 and monocytes/macrophages into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with amplified demyelination and impaired remyelination. Single-cell RNA-seq of CD45+ cells enriched from spinal cords of infected Cst7-/- and control mice at day 21 post-infection (p.i.) revealed enhanced expression of transcripts encoding macrophage chemoattractant, Ccl2, and T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) transcripts and activation markers 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 macrophage and T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the theory that one mechanism by which cystatin F affects chronic disease in mice persistently infected with JHMV is through regulating expression of pro-inflammatory molecules that impact neuroinflammation and neurologic disease. Overall design: Cst7 knock-out (KO) or wildtype (WT) littermate controls were injected intracranially (i.c.) in the right brain hemisphere with 1200 plaque forming units (PFU) of JHMV and CD45-positive (CD45+) cells were flow sorted from the spinal cords of uninfected and JHMV-infected Cst7 KO and WT mice at day 21 post-infection (p.i.)  FACS sorted CD45+ cells were CellPlex labeled and pooled, then underwent 3' single-cell RNA-sequencing analysis