RESUMO
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.
Assuntos
Infecções por Coronavirus , Cistatinas , Doenças Desmielinizantes , Camundongos Knockout , Vírus da Hepatite Murina , Animais , Camundongos , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/virologia , Doenças Desmielinizantes/imunologia , Vírus da Hepatite Murina/patogenicidade , Cistatinas/genética , Cistatinas/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/metabolismoRESUMO
Purpose: Secreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors. Methods: A lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively. Results: Suppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography. Conclusions: Suppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.