(409) Neutrophil elastase (NE) mediated degradation of histone deacetylases in macrophages promotes High Mobility Group Box1 export from nucleus to cytosol.

Background: Neutrophil elastase (NE) induces the release of High Mobility Group Box 1 (HMGB1), a damage associated molecular pattern (DAMP), from macrophages. However, the mechanism of NE-induced release of HMGB1 is not known. HMGB1 is present in the nucleus as a chromatin-binding protein. However, after lysine acetylation, HMGB1 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, increased HMGB1 lysine acetylation, and increased 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 GM-CSF to differentiate into blood monocyte derived macrophages (hBMDM). Human BMDM were exposed to control vehicle or NE (200 or 500 NM) for 2 h at 37°C, and nuclear protein or total cell lysate protein were isolated for western analysis to determine relative abundance of 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 analysis for HMGB1. Western blots were normalized to β-actin (total cell lysate or cytosolic fraction) or poly ADP ribose Polymerase 1 (PARP1) (nuclear fraction).

Results: NE treatment significantly increased cytosolic HMGB1 compared to control-treated cytosolic lysates. NE treatment significantly decreased Class II HDACs and Sirtuin 1. NE treatment also decreased Class I HDACs but the change in abundance was not as pronounced as that observed for the Class II HDACs and Sirtuin 1.

Conclusions: Active NE, taken up by hBMDM, degraded HDACs and Sirtuin 1, resulting in a shift in HMGB1 localization from nucleus to cytosol, a priming event for HMGB1 release.

Acknowledgements: This work was supported by the CFF (VOYNOW19G0), DOD Peer Reviewed Medical Research Program PR180925 (JAV), and NIH R01 HL146811-01A1 (JAV)

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