The SOD3 R213G Polymorphism Promotes Resolution of Innate Immune Activation and Blocks Induction of miR29b in Bleomycin Induced Lung Inflammation and Fibrosis

Abstract

A naturally occurring single nucleotide polymorphism (SNP) (R213G) in extracellular superoxide dismutase (SOD3), a key lung and vascular antioxidant enzyme, decreases tissue binding and increases SOD3 in epithelial lining fluid and plasma. Mice harboring the R213G SNP are protected against bleomycin-induced alveolar inflammation and fibrosis. We hypothesized that the redistribution of SOD3 due to the R213G SNP, through site specific changes in the redox environment, modulates key miRNA and mRNA signaling networks responsible for macrophage activation and fibrosis. Plasma, lung and BALF O2•‒ was measured by electron paramagnetic resonance using the CMH spin probe. Bleomycin increased O2•‒ in all three compartments; the increase in O2•‒ was attenuated in the blood of R213G mice. We performed high throughput RNAseq and bioinformatics analyses in lung mRNA and miRNA 7 days post-bleomycin in WT and R213G mice. We identified distinct mRNA and miRNA profiles between strains in response to bleomycin. By pathway analysis, key immune and inflammatory pathways were activated in WT mice but suppressed in R213G mice at 7 days, the timepoint associated with resolution of inflammation in the R213G mice. Predicted upstream regulators included multiple signaling molecules within the TLR4/NF-ks/IKKe/IRF3 pathway. Distinct strain specific miRNA responses were observed, including miR29, a key miRNA implicated in fibrosis, which decreased only in R213G mice. In bone marrow derived macrophages (BMDM) from WT and R213G mice, the response to classic “M1” LPS + IFN polarizing stimuli was similar between strains but attenuated by pretreatment with exogenous extracellular SOD. Using bioinformatics and molecular approaches, we conclude that the redistribution of ecSOD, due to its impact on O2•‒ , can suppress innate immune responses by regulating macrophage plasticity, accounting for the ability of the R213G SNP to protect against bleomycin-induced lung fibrosis. The role of miRNA regulation in this process requires further study.