Hyperoxia results in HMGB1 production and subsequent inflammatory signaling in human pulmonary microvascular endothelial cells

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We have previously shown that neonatal mice exposed to 85% oxygen have substantial elevations in bronchoalveolar lavage levels of HMGB1 protein and that inhibiting TLR4 signaling using TAK‐242, which prevents TLR4 interaction with adaptor proteins like HMGB1, attenuated hyperoxia‐induced lung injury. To begin to understand the molecular and cellular mechanisms underlying these in vivo findings we studied HMGB1 mediated signaling in cultured cells. We hypothesized that hyperoxia would induce HMGB1 production in cells key to lung injury and that HMGB1 would interact with TLR4 to cause pro‐inflammatory cytokine production. We exposed human pulmonary microvascular endothelial cells (hPMVEC), A549 cells (a human lung epithelial cell line), and RAW264.7 cells (a murine macrophage‐like cell line) to hyperoxia, and examined the effect on HMGB1 levels. We found that exposure to 85% oxygen for 48 hours resulted in 16‐fold, 2‐fold, and 14‐fold increases in HMGB1 protein levels in these cells, respectively, relative to the same cells in normoxia. When HMGB1 (100 ng/ml) was added to the media of hPMVEC, there was significant (p<0.005) induction of IL‐8 and MCP‐1 protein levels. Furthermore, HMBG1 added to the media of hPMVEC resulted in significant induction of cleaved caspase‐1 and IL‐1β protein levels compared to vehicle treated hPMVEC. The addition of HMGB1 to the media of hPMVEC also resulted in a significant time‐dependent induction of pNFκB, pIKKα/β, and pIκBα. Hyperoxia exposed hPMVEC had a 3‐fold greater expression of phosphorylated p38 then did normoxic cells and this pp38 induction was significantly attenuated by TAK‐242. In hPMVEC hyperoxia resulted in 2.5‐fold greater IL‐8 protein levels than in normoxia exposed cells (p<0.05) and this effect was completely blocked by TAK‐242. In hPMVEC exposure to hyperoxia resulted in 8‐fold greater levels of cleaved caspase‐1 than in normoxia exposed cells (p<0.05), and in 4‐fold greater levels of cleaved IL‐1β than in normoxia exposed cells (p<0.005). In conclusion, our results suggest that hyperoxic exposure results in HMGB1 mediated TLR4 activation, leading to caspase‐1 activation and induction of pro‐inflammatory cytokines including cleaved IL‐1β. These results suggest that targeting HMGB1‐TLR4 interactions may confer protection from hyperoxia‐induced injury.