Inducible Nitric Oxide Synthase deficiency leads to early demyelination by altering the balance between pro- and anti-inflammatory responses against Murine-β-Coronavirus

Abstract

The neurological disease Multiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by the activated glial cells, the CNS infiltrating leukocytes, and their reciprocal interaction through inflammatory signals. Inducible nitric oxide synthase (iNOS), an enzyme that catalyzes sustained nitric oxide production in response to an inflammatory stimulus, is a pro-inflammatory marker expressed particularly by the microglia/macrophages (MG/M{varphi}) during neuroinflammation. In MS, iNOS has been reportedly associated with the disease pathology; however, studies dissecting its role in the underlying mechanisms, specifically demyelination, are limited. Therefore, we studied the role of iNOS in a recombinant beta-coronavirus-MHV-RSA59-induced neuroinflammation, which is a prototypic animal model used to investigate the pathological hallmarks of MS, neuroinflammatory demyelination, and axonal degeneration. During the acute phase of infection with RSA59, wildtype C57BL/6 (WT) mice had significantly upregulated iNOS expression in macrophages, natural killer cells, and natural killer T cells suggesting a role for iNOS in RSA59-induced neuroinflammation. Studies comparing RSA59-infected WT and iNOS-deficient mice revealed that iNOS deficiency aggravated the disease with increased CNS infiltration of macrophages and neutrophils and enhanced mortality. As early as 9-10 days after the infection, the CNS of iNOS-deficient mice had substantially higher demyelination marked with morphologically defined MG/M{varphi} in the demyelinating regions. Transcript analysis confirmed the significant upregulation of type2 macrophage (M2) markers-Arginase 1, CD206, and TREM2-in the CNS of iNOS-deficient mice. Corroborating to the phenotype, the iNOS-deficient mice showed a significantly higher expression of TGF{beta}-an anti-inflammatory cytokine- and increased T regulatory (Treg) cell infiltration, indicating an anti-inflammatory milieu established early after the infection. These observations highlight a protective role of iNOS in virus-induced neuroinflammation whereas its absence leads to MG/M{varphi} polarization towards a phenotype that may be involved in the exacerbated demyelination pathology. Author summaryContrary to the reported pathogenic role of inducible nitric oxide synthase (iNOS) in multiple sclerosis and related autoimmune animal models, we show that the mice deficient in iNOS show an exacerbated disease with accelerated demyelination accompanied by heightened production of an anti-inflammatory and phagocytic markers and more numbers of Tregs in a mouse model of a recombinant mouse hepatitis virus RSA59 infection. Therefore, iNOS may play protective and regulatory roles in this beta-coronavirus infection.