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A6363 - NOX4 Modulates Lung Macrophage Polarization in Pulmonary Fibrosis Via Regulation of Mitochondrial Biogenesis
Author Block: C. He1, J. Larson-Casey2, L. Gu1, V. J. Thannickal3, A. B. Carter4; 1University of Alabama at Birmingham, Birmingham, AL, United States, 2Univeristy of Alabama at Birmingham, Birmingham, AL, United States, 3Univ of Alabama At Birmingham, Birmingham, AL, United States, 4Birmingham VAMC, University of Alabama at Birmingham, Birmingham, AL, United States.
RATIONALE: The phenotype of macrophages can contribute to fibrotic development. We have previously shown that lung macrophages from subjects with pulmonary fibrosis have elevated pro-fibrotic gene expression and increased mitochondrial dynamics compared with lung macrophages from normal subjects. Here, we found that lung macrophages isolated from subjects with pulmonary fibrosis have a significant increase in nox4 gene expression, suggesting a pivotal role of NOX4 in regulating macrophage phenotype and promoting fibrosis development. We hypothesize that NOX4 is required pro-fibrotic polarization of lung macrophages. METHODS: Pro-fibrotic gene (TGF-β1, Fizz1 and Ym1) expression was analyzed by RT-PCR and ELISA. A dual-luciferase assay was used to measure promoter activity for TGF-β1 and Tfam. A human NOX4 construct or siRNA targeting human NOX4 was used dissect the role of NOX4 in macrophage polarization and mitochondrial biogenesis. TGF-β1 and Ym1 gene expression in lung macrophages was analyzed by RT-PCR in bleomycin-injured mice. Hydroxyproline assay was used to quantitate lung collagen content, and Masson’s trichrome staining was done to evaluate pulmonary fibrosis. RESULTS: We found that NOX4 overexpression increases TGF-β1 promoter activity as well as pro-fibrotic marker expression in vitro suggesting the importance of NOX4 in pro-fibrotic gene regulation. Conversely, macrophages transfected with NOX4 siRNA have decreased TGF-β1 production compared with cells transfected with a scrambled siRNA. These data were recapitulated in vivo with bleomycin-induced injury. Lung macrophages from NOX4-/- mice had a significant decrease in pro-fibrotic gene expression compared to WT mice. Furthermore, NOX4-/- mice were protected from developing pulmonary fibrosis. Mitochondrial dynamics is crucial for macrophage polarization, and lung macrophages from pulmonary fibrosis subjects have increased mitochondrial biogenesis. NOX4 overexpression increased tfam gene expression, whereas inhibition of mitochondrial biogenesis reduced pro-fibrotic polarization of macrophages. CONCLUSIONS: These observations suggest a critical role for NOX4-mediated regulation of pro-fibrotic polarization of macrophages via modulation of mitochondrial biogenesis in pulmonary fibrosis. Research Funding Source: This work was supported, in whole or in part, by National Institutes of Health Grants 2R01ES015981-10 and R56ES027464-01.