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Redistribution of EC-SOD resolves bleomycin-induced inflammation via increased apoptosis of recruited alveolar macrophages.

ABSTRACT: A human single nucleotide polymorphism (SNP) in the matrix-binding domain of extracellular superoxide dismutase (EC-SOD), with arginine to glycine substitution at position 213 (R213G), redistributes EC-SOD from the matrix into extracellular fluids. We reported that, following bleomycin (bleo), knockin mice harboring the human R213G SNP (R213G mice) exhibit enhanced resolution of inflammation and protection against fibrosis, compared with wild-type (WT) littermates. In this study, we tested the hypothesis that the EC-SOD R213G SNP promotes resolution via accelerated apoptosis of recruited alveolar macrophage (AM). RNA sequencing and Ingenuity Pathway Analysis 7 d postbleo in recruited AM implicated increased apoptosis and blunted inflammatory responses in the R213G strain exhibiting accelerated resolution. We validated that the percentage of apoptosis was significantly elevated in R213G recruited AM vs. WT at 3 and 7 d postbleo in vivo. Recruited AM numbers were also significantly decreased in R213G mice vs. WT at 3 and 7 d postbleo. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1), a proapoptotic γ-glutamyl cyclotransferase that depletes glutathione, was increased in the R213G recruited AM. Overexpression of Chac1 in vitro induced apoptosis of macrophages and was blocked by administration of cell-permeable glutathione. In summary, we provide new evidence that redistributed EC-SOD accelerates the resolution of inflammation through redox-regulated mechanisms that increase recruited AM apoptosis.-Allawzi, A., McDermott, I., Delaney, C., Nguyen, K., Banimostafa, L., Trumpie, A., Hernandez-Lagunas, L., Riemondy, K., Gillen, A., Hesselberth, J., El Kasmi, K., Sucharov, C. C., Janssen, W. J., Stenmark, K., Bowler, R., Nozik-Grayck, E. Redistribution of EC-SOD resolves bleomycin-induced inflammation via increased apoptosis of recruited alveolar macrophages.

SUBMITTER: Allawzi A

PROVIDER: S-EPMC6894081 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer's and Parkinson's diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2-3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1-3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Furthermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.

Project description:Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

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Redistribution of EC-SOD resolves bleomycin-induced inflammation via increased apoptosis of recruited alveolar macrophages.

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