A Protective Hsp70–TLR4 Pathway in Lethal Oxidant Lung Injury

Abstract

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that toll-like receptor 4 (TLR4) confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild type, TLR4−/− and Trif−/− (a TLR4 adapter protein) murine lung endothelial cells (MLEC) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from mice lung and MLEC after hyperoxia. We confirmed that Hsp70 and TLR4 co-immunoprecipitate in lung tissue and MLEC. Hsp70-mediated NFκB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif-dependent, MyD88-independent. Hsp70-deficient mice have increased mortality during hyperoxia and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild type mice. Our studies are the first to define an Hsp70-TLR4-Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways

“…Extracellular Hsp70 exert its cytoprotective effect in lung endothelium through toll-like receptor 4 (TLR4) [19]. However, we did not observe increased Hsp70 in the medium of PAECs exposed to hyperoxia (data not shown).…”

Endothelial PINK1 Mediates the Protective Effects of NLRP3 Deficiency during Lethal Oxidant Injury

“…1A). We previously reported 72 h of continuous hyperoxia as a time point of maximal lung injury, which we used as a representative time point (7). NLRP3 −/− , Asc −/− and Caspase 1/11 −/− mice exhibited significantly decreased lung injury (as assessed by bronchoalveolar lavage cell counts, protein and LDH), decreased reactive oxygen species generation (H 2 O 2 release detected by Amplex red assay), decreased lung cell death (assessed by TUNEL) and decreased IL1β secretion compared to WT mice (Fig.…”

“…NLRP3 −/− /PINK1 −/− mice were generated by crossing NLRP3 −/− mice with PINK1 −/− mice for >10 generations. Mice bred and exposure to hyperoxic as described previously (7). All protocols were reviewed and approved by the Animal Care and Use Committee at Yale University.…”

“…Lung or MLEC protein analyses were performed as previously described (7) using LC3B, p62, Caspase3, autophagocytosis-associated protein 3 (ATG3), ATG7, Beclin-1 (Cell Signaling Technology), PINK1 (Millipore, clone N4/15), LAMP2A, mitofusin-1 (MFN1), MFN2, optic atrophy type 1 (OPA1), dynamin-related protein 1 (DRP1) (Abcam), Proteasome 20S α6 subunit (Enzo Life Sci), PARIS (Millipore, clone N196/16), Caspase 1, IL-1β, Parkin, PGC-1α and β-actin (Santa Cruz Biotechnology) antibodies.…”

Preinduction of heat shock protein 70 protects mice against post-infection irritable bowel syndrome via NF-κB and NOS/NO signaling pathways

“…Thus, we speculated that induction of HSP70 plays a protective role in PI-IBS mice via inhibiting NF-κB activation, following with the lower inflammation. This hypothesis has been validated in a mouse model of lethal oxidant lung injury which indicated that HSP70 mediated NF-κB activation to protect against hyperoxiainduced lung injury and the protective property of HSP70 is TLR4 dependent (Zhang et al 2013).…”