410 Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis

Background: Neutrophil elastase (NE) induces the release of high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP), from macrophages, but the mechanism of NE-induced release of HMGB1 is not known.HMGB1 is present in the nucleus as a chromatin-binding protein, but after lysine acetylation, it is retained in the cytosol and is fated for cellular release.We hypothesize that NE alters the epigenetic program of macrophages by degrading histone deacetylases, resulting in unopposed histone acetyltransferase activity, greater HMGB1 lysine acetylation, and greater cytosolic HMGB1 poised for cell export into the extracellular milieu.Methods: Human blood monocytes were isolated from buffy coats obtained from deidentified healthy subjects from the American Red Cross and cultured with growth media containing granulocyte-macrophage colony-stimulating factor to differentiate into human blood monocytederived macrophages (hBMDMs), which were exposed to control vehicle or NE (200 nM or 500 nM) for 2 hours at 37°C.Nuclear protein or total cell lysate protein was isolated for western blot analysis to determine relative abundance of histone deacetylase (HDAC) Class I (1,2,3,8), Class II (4,5,6,7,8,9,10), Class III (Sirtuins 1-7), and Class IV (HDAC11).Cytosolic lysate protein harvested after control vehicle or NE treatment was used for western blot analysis for HMGB1.Western blots were normalized to β-actin (total cell lysate or cytosolic fraction) or poly (ADP ribose) polymerase 1 (nuclear fraction).Results: NE treatment resulted in significantly greater cytosolic HMGB1 than found in control-treated cytosolic lysates.NE treatment significantly decreased Class II HDACs and Sirtuin 1. NE treatment also decreased Class I HDACs, but the change was not as pronounced as that observed for the Class II HDACs and Sirtuin 1. Conclusions: Active NE taken up by hBMDMs degraded HDACs and Sirtuin 1, shifting HMGB1 localization from nucleus to cytosol, a priming event for HMGB1 release.