H2O2 generated by alveolar macrophages has been linked to the development pulmonary fibrosis, but little is known about its source, mechanism of production and exact role upon alveolar macrophage activation. In this study, we found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H2O2 and have high expression of Cu,Zn-SOD. Cu,Zn-SOD localized to the mitochondrial intermembrane space (IMS) in asbestosis patients and asbestos induced translocation of Cu,Zn-SOD to the IMS. This process was unique to macrophages and dependent on functional mitochondrial respiration. The presence of at least one of the conserved cysteines was required for disulfide bond formation and mitochondrial translocation. These conserved cysteine residues were also necessary for enzyme activation and H2O2 generation. Cu,Zn-SOD-mediated H2O2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant as Cu,Zn-SOD-/- mice generated significantly less H2O2, had less oxidative stress, and were protected from developing pulmonary fibrosis. This protective mechanism is closely related to the alveolar macrophage activation and polarization in Cu,Zn-SOD-/- mice, as they had a dominant pro-inflammatory phenotype. Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. The pro-inflammatory M1 macrophages have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain settings. We demonstrate that overexpression of Cu,Zn-SOD promoted macrophages polarization into an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis. The mechanism which Cu,Zn-SOD-mediated H2O2 utilizes to modulate macrophage M2 polarization is through redox regulation of a critical cysteine in STAT6. The polarization process, at least partially, was regulated by epigenetic modulation. We show that STAT6 was indispensable for Cu,Zn-SOD-mediated M2 polarization. STAT6 upregulated Jmjd3, a histone H3 lysine 27 demethylase, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD plays a paradoxical role. The study highlights the importance of mitochondrial Cu,Zn-SOD and redox signals in macrophage polarization and fibrosis development. These observations demonstrate that the Cu,Zn-SOD-STAT6-Jmjd3 pathway is a novel regulatory mechanism for M2 polarization and that leflunomide is a potential therapeutic agent in the treatment of pulmonary fibrosis.

Evidence from human amyotrophic lateral sclerosis (ALS) patients and ALS-linked Cu/Zn superoxide dismutase (Cu/Zn-SOD) transgenic mice bearing the mutation of glycine to alanine at position 93 (G93A) suggests that the pro-apoptotic protein prostate apoptosis response-4 (Par-4) might be a critical link in the chain of events leading to motor neuron degeneration. We now report that Par-4 is enriched in synaptosomes and post-synaptic density from the ventral horn of the spinal cord. Levels of Par-4 in synaptic compartments increased significantly during rapid and slow declining stages of muscle strength in hSOD1 G93A mutant mice. In the pre-muscle weakness stage, hSOD1 G93A mutation sensitized synaptosomes from the ventral horn of the spinal cord to increased levels of Par-4 expression following excitotoxic and apoptotic insults. In ventral spinal synaptosomes, Par-4-mediated production of pro-apoptotic cytosolic factor(s) was significantly enhanced by the hSOD1 G93A mutation. RNA interference (RNAi) knockdown of Par-4 inhibited mitochondrial dysfunction and caspase-3 activation induced by G93A mutation in synaptosomes from the ventral horn of the spinal cord, and protected spinal motor neurons from apoptosis. These results identify the synapse as a crucial cellular site for the cell death promoting actions of Par-4 in motor neurons, and suggest that targeted inhibition of Par-4 by RNAi may prove to be a neuroprotective strategy for motor neuron degeneration.