Lung microhemorrhage drives macrophage activation and oxidative/inflammatory damage in α-1 antitrypsin deficiency

Background. Animal models of elastase intratracheal instillation have shown that elastase, unopposed by α-1 antitrypsin (AAT), can induce alveolar hemorrhage with ensuing macrophage inflammation and lung damage. Aim. To identify markers of alveolar hemorrhage and damage using bronchoalveolar lavage (BAL) samples and lung tissue explants from AAT deficient (AATD) subjects. Methods. BAL samples (17 patients, 15 controls) were evaluated for free heme and iron concentrations. Alveolar macrophage (AlvMac) activation patterns were assessed by RNA sequencing and validated in vitro using heme-stimulated monocyte-derived macrophages (MDM). Lung explants (7 patients, 4 controls) were evaluated for iron sequestration patterns, by Prussian blue stain and ferritin immunohistochemistry, and for ferritin iron imaging and elemental analysis by electron microscopy (EM). Tissue oxidative damage was assessed by 8-OHdG immunohistochemistry. Results. BAL showed significantly elevated free heme and iron concentrations at lobar level. BAL macrophage RNA sequencing showed innate proinflammatory activation, consistent with free heme activation. AATD tissue alveolar and interstitial macrophages showed elevated iron and ferritin accumulation in large lysosomes packed with iron oxide cores with degraded ferritin protein cages. AATD explants showed massive oxidative DNA damage in lung epithelial cells and macrophages. Conclusions. Identification of BAL and tissue markers of alveolar hemorrhage, together with the molecular and cellular evidence of macrophage innate pro-inflammatory activation and oxidative damage, consistent with free heme stimulation, provides ex vivo evidence for the pathogenetic role of elastase-induced alveolar hemorrhage AATD emphysema as shown in animal studies. Alveolar Macrophages were obtained by bronchoalveolar lavage from 6 patients affected by Alpha-1 Antitrypsin deficiency and 6 helthy controls. Levels of unopposed Neutrophil elastase, Alpha-1 Antitrypsin, free Heme and Iron were measure in bronchoalveolar lavage fluid. Macrophages were isolated from lavage fluid by plastic adherence, RNA was extracted and sequenced for DGE and SGEA. Alveolar macrophage-like monocyte-derive macrophages were exposed to Heme in vitro to validate the expression of of BAL alveolar macrophage innate immune response genes expression identified by DGE and GSEA in Alph-1 Antitrypsin deficiency affected subjectswere stimulat in vitro.