Toll‐like Receptor 4 Mediates Histone Subunit H3‐induced Endothelial Dysfunction in Human Lung Endothelium
Authors: Karki Pratap , Zhang Chenou , Li Yue , Promnares Kamoltip , Birukov Konstantin G. , Birukova Anna A. 
Published: May 2022

Background

Extracellular histones released from dying cells during apoptosis, necrosis, or NETosis following trauma, sepsis, ARDS, or other severe organ injuries act as potent endogenous damage‐associated molecular patterns (DAMPs) with profound cytotoxic and inflammatory effects. Studies have reported an increased levels of circulating histones in various lung injuries and accordingly their role in endothelial dysfunction has been suggested but the mechanisms that mediate these pathologies remain to be elucidated. In the present study, we investigated the signaling pathways involved in barrier‐disruptive and inflammatory effects of histone subtype H3 in cultured human lung endothelial cells.

Methods

Human lung endothelial cells were exposed to purified human recombinant histone H3 and endothelial barrier function was determined by monitoring transendothelial electrical resistance and express permeability testing (XPerT) assay. Immunofluorescence staining of endothelial adherens junction protein VE‐cadherin and co‐immunoprecipitation analysis were performed to determine the integrity of endothelial barrier. Cellular fractionation and biotinylation assays were run to determine the membrane/nuclear translocation of junction proteins.

Results

Histone H3 when used at ≥ 30 µg/ml caused a rapid and sustained endothelial barrier disruption in a dose‐dependent manner. Among other histone subtypes tested, only H4 induced endothelial permeability albeit weak as compared to H3, while H1, H2A, and H2B did not have any noticeable barrier disruptive effects. Consistently, immunofluorescence staining with VE‐cadherin showed that only H3 induced a dramatic loss of VE‐cadherin from cell membrane. Cellular fractionation and biotinylation assay data further confirmed H3‐induced disappearance of VE‐cadherin from membrane. Co‐immunoprecipitation analysis demonstrated that H3 stimulation causes a significant reduction in interaction of VE‐cadherin with other junction proteins ZO‐1 and p120‐catenin. Endothelial cells treated with H3 for 24 hours showed a marked reduction in expression of junction proteins VE‐cadherin, p120‐catenin, ZO‐1, JAM‐1, and Claudin‐5. H3 induced the activation of the NF‐kB pathway and increased the protein expression of inflammatory endothelial cell adhesion molecules ICAM‐1, VCAM‐1, and E‐selectin. RT‐PCR analysis showed the upregulation of inflammatory cytokines/chemokines genes TNF‐α, ICAM‐1, VCAM‐1, IL‐6, IL‐8, IL‐1β, and CXCL5. H3 also induced an increase in secretory levels of soluble ICAM‐1, and proinflammatory cytokines IL‐6 and IL‐8 as determined by ELISA. A comprehensive inhibitor analysis showed that only CLI‐095, a TLR4‐specific inhibitor, attenuated H3‐induced permeability and completely blocked H3‐induced upregulation of EC inflammatory mRNA transcripts and proteins. Other inhibitors targeting TLR2, NLRP3, RAGE, MAPK, Src, and Rho signaling pathways did not have any protective effects.

Conclusion

Altogether, our results demonstrate that TLR4 signaling is critically involved in mediating histone H3‐induced endothelial barrier disruption and inflammation.