Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

Abstract

BackgroundToll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a prototypical pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification.ResultsHere we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein as determined by ELISA. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls.ConclusionsThese results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

Danger-associated molecular patterns in Alzheimer’s disease

“…HSP70, through the stimulation of TLR2 and TLR4 and the activation of MyD88/NF-kB pathways, induces this cytokine production [137]. Similar results were obtained by Luong et al [138]. They found that the ligation of HSP70 with TLR4 leads to increased concentration of TNF-a.…”

Damage-Associated molecular pattern markers HMGB1 and cell-Free fetal telomere fragments in oxidative-Stressed amnion epithelial cell-Derived exosomes

“…DAMPs are molecules with defined intracellular functions, but outside of the cell, DAMPs are proinflammatory mediators. Examples of such DAMPs include high-mobility group box 1 (HMGB1), a nonhistone nuclear protein (Ciucci et al, 2011; Tang et al, 2011); HSP70, a chaperon protein (Andocs et al, 2015; Luong et al, 2012); fragments of cell-free DNA (Girard et al, 2014; Nadeau-Vallée et al, 2016; Phimister and Phillippe, 2014); telomere fragments (Polettini et al, 2015); and uric acid (Mulla et al, 2011). DAMPs are often recognized by pattern recognition receptors (PRRs) that are located in the cell membranes (Santoni et al, 2015).…”

<b><i>Toxoplasma gondii-</i></b>Derived Profilin Triggers Human Toll-Like Receptor 5-Dependent Cytokine Production

“…Peripheral CD14+ blood monocytes were purified from healthy whole blood donors using Ficoll density gradient and highly specific monocyte isolation kit (CD14+ antibody magnetic labeled beads, Miltenyii). Proteinase K digestion of flagellin and profilin were performed as described previously [5,6]. Briefly, proteinase K-agarose was reconstituted in endotoxin-free water to 10 mg/mL, incubated at 4°C for 2 hr, and washed five times with endotoxin-free water.…”