Mechanisms of pulmonary endothelial permeability and inflammation caused by extracellular histone subunits H3 and H4.

Extracellular DNA-binding proteins such as histones are danger-associated molecular pattern released by the injured tissues in trauma and sepsis settings, which trigger host immune response and vascular dysfunction. Molecular events leading to histone-induced endothelial cell (EC) dysfunction remain poorly understood. This study performed comparative analysis of H1, H2A, H2B, H3, and H4 histone subunits effects on human pulmonary EC permeability and inflammatory response. Analysis of transendothelial electrical resistance and EC monolayer permeability for macromolecues revealed that H3 and H4, but not H1, H2A, or H2B caused dose-dependent EC permeability accompanied by disassembly of adherens junctions. At higher doses, H3 and H4 activated nuclear factor kappa B inflammatory cascade leading to upregulation EC adhesion molecules ICAM1, VCAM1, E-selectin, and release of inflammatory cytokines. Inhibitory receptor analysis showed that toll-like receptor (TLR) 4 but not TLR1/2 or receptor for advanced glycation end inhibition significantly attenuated deleterious effects of H3 and H4 histones. Inhibitor of Rho-kinase was without effect, while inhibition of Src kinase caused partial preservation of cell-cell junctions, H3/H4-induced permeability and inflammation. Deleterious effects of H3/H4 were blocked by heparin. Activation of Epac-Rap1 signaling restored EC barrier properties after histone challenge. Intravenous injection of histones in mice caused elevation of inflammatory markers and increased vascular leak. Post-treatment with pharmacological Epac/Rap1 activator suppressed injurious effects of histones in vitro and in vivo. These results identify H3 and H4 as key histone subunits exhibiting deleterious effects on pulmonary vascular endothelium via TLR4-dependent mechanism. In conclusion, elevation of circulating histones may represent a serious risk of exacerbated acute lung injury (ALI) and multiple organ injury during severe trauma and infection.