Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues

Background: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. Methods: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. Results: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 ⁺ and CD8 ⁺ T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H₂O₂ generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. Conclusion: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.