Expression of human superoxide dismutase (SOD) 1 G93A and chlorovirus ATCV-1 SOD increases the response of macrophages to inflammatory stimulants, including ATCV-1 major capsid protein glycans

One cause of familial Amyotrophic Lateral Sclerosis (ALS) is a mutation in Super Oxide Dismutase 1 (SOD1) whereby amino acid 93 is alanine instead of glycine (SOD1-G93A). Transgenic mice expressing human SOD1-G93A pathogenic variant develop motor neuron disease (MND), similar to ALS. Humans with ALS and SOD1-G93A mice have elevated production of inflammatory cytokines, such as IL-6, which may promote MND. We previously showed that infection with the Chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1), which encodes a SOD1, accelerates onset of MND in these mice and induces macrophages to produce high levels of IL-6. We confirm here that ALS patients compared with healthy controls have significantly elevated levels of plasma IL-6 and Interferon-gamma (IFN-γ), but not IL-17. To determine if expression of ATCV-1 SOD1 or SOD1-G93A in mouse macrophages elevates expression of inflammatory cytokines, we transfected the RAW264.7 mouse macrophage cell line with plasmids encoding ATCV-1 SOD1, wild-type human SOD1, SOD1-G93A, or an empty vector. RAW264.7 cells stably expressing wtSOD1 or G93A-SOD1 were stimulated with poly I:C and Interferon-gamma, alone, or in combination to induce inflammatory factors, such as IL-6 and Nitric Oxide (NO), anti-inflammatory factors, such as IL-10, or activation of Interferon Stimulated Response Elements (ISRE) promoters. After stimulation, production of IL-6 and NO, but not IL-10 or ISRE promoter activity was significantly higher in RAW264.7 cells expressing SOD1-G93A compared with wt SOD1. Moreover, RAW264.7 cells expressing SOD1-G93A compared with wt SOD1 produced higher levels of IL-6 and NO in response to ATCV-1 glycoproteins. Finally, transfection of plasmid encoding ATCV-1 SOD1 into RAW264.7 cells significantly increased expression of inflammatory factors in responses to poly I:C and IFN-γ, primarily in an Interferon regulatory factor 3 (IRF3) dependent fashion. These data clearly show that expression of G93A-SOD1 or ATCV-1 SOD1 in macrophages significantly elevates expression of inflammatory factors following stimulations that mimic virus infection, viral components, or T cell cytokines, thereby suggesting one mechanism by which atypical SOD1 in macrophages can contribute to ALS-MND.