An increasing number of diseases result of a dysregulated inflammatory response, and several inflammatory mediators, including Hsp70, have been implicated. The molecular chaperone Hsp70 has many functions depending on the cell type, and our understanding of the roles it may play, especially the extracellular form, remains incomplete, and is even controversial [27–32]. Two members of the HSP70 family, Hsp73 and Hsp72, share a high degree of sequence homology but differ in their expression pattern: Hsp73 is constitutively expressed, while Hsp72 is stress-inducible . In resting cells, Hsp73 is found mainly in the cytoplasm, the nuclei and nucleoli, while Hsp72 is found in nuclei and nucleoli. However, after a stressful event, such as heat shock, the distribution of both forms is homogenized throughout the cell, suggesting that both isoforms associate with similar molecules to protect essential cell structures . Incubation of human monocytes with Hsp70 –the inducible form- was reported to have a functional consequence by eliciting the rapid expression of pro-inflammatory cytokines ; whereas Hsp73 does not induce any significant release of IL-6 – a pro-inflammatory cytokine by monocytes .
Some authors have reported that extracellular Hsp70 functions as a DAMP and activates monocytes, macrophages and dendritic cells, potentially through cell surface receptors such as CD14, CD40, CD91, Lox1, TLR2 and TLR4 . In different experimental models [14, 22, 37–39], Hsp70 has been found to induce intracellular calcium fluxes and NF-κB activation, with the subsequent production of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8. However, Hsp70 is present in the peripheral circulation of healthy subjects at concentrations that, according to these studies, would elicit cytokine production , suggesting that Hsp70 is not pro-inflammatory in all contexts [40, 41]. In fact, intracellular Hsp70 has a cytoprotective effect, as it inhibits the production of inflammatory mediators during cellular stress . Our results support a cytoprotective role for Hsp70 whereby it diminishes the production of the pro-inflammatory mediator TNF-α, through the recruitment of HSF-1 transcription factor to the cytokine promoter, thus extending its cytoprotective role beyond the intracellular space to the extracellular milieu.
Data shown here bring also evidence that extracellular Hsp70 not only fails to induce TNF-α production in human monocytes, but that it down-regulates their capacity to produce TNF-α and IL-6 in response to LPS. The Hsp70 (LPS-free HSP70) we used in our experiments corresponds to the inducible isoform (Hsp72); as a result of a multi-step chromatography purification process it has very low levels of LPS (<50 endotoxin units/mg) and could not, by itself, induce cytokine production by human monocytes. These results are consistent with reports that attribute the immune-stimulating activity of Hsp70 to contaminating LPS [16, 43], and others that show that in patients with rheumatoid arthritis, extracellular Hsp70 decreased IL-6, IL-8 and MCP-1 production induced by TNF-α . Likewise, recombinant Hsp72 (induced Hsp70) has been reported to lessen the severity of collagen-induced arthritis in mice, by lowering serum TNF-α and IL-6 concentration .
We used a highly purified commercial Hsp70 for our experiments (purified by multi-step chromatography, with <50 endotoxin units/mg); this Hsp70 corresponds to the inducible isoform (Hsp72), and it is not bound to any other molecule. However, we cannot exclude the possibility that Hsp70 bound another protein or peptide in the cell culture supernatants. This binding, as well as its ADP/ATP-binding status, could modify the effects of Hsp70 on human monocytes . These two aspects need to be evaluated in future studies. The recombinant Hsp70 that we used is produced in E. coli, so it is possible that its conformation is different from that of native Hsp70 in cells. However, this Hsp70 from Enzo Life Sciences, formerly Stressgen retains its ATPase activity, and we observed that it activates NF-κB through TLR2 and TLR4 signalling, as has been reported previously which suggest that the recombinant protein retains a conformation compatible with its biological activity [16, 47].
Hsp70 interacts with highly hydrophobic peptides . TLR agonists usually have hydrophobic regions that bind to the leucine-rich-repeats (LRR) domain-groove of TLRs . The binding of a TLR agonist to Hsp70 could interfere with its ability to engage its TLR, and the subsequent signaling cascade leading to cytokine production would be defective. Our results do not support this possibility. Data we report here suggest rather that the effect of Hsp70 on LPS-induced TNF-α production is independent of a physical association with LPS, because the addition of LPS after monocytes were treated with Hsp70 did not lead to TNF-α production, even though Hsp70 was no longer present in the system. Furthermore, our results indicating that Hsp70 activates NF-κB through TLR2 and TLR4 signaling argues against the possibility that Hsp70 inhibit LPS-mediate TNF-α expression by negatively modulating TLR4 expression. In fact, we found that Hsp70 has no effect on the surface expression of TLR2 and TLR4 on monocytes, consistent with a report showing that Hsp70 does not affect TLR2 nor TLR4 expression on mouse macrophages .
TNF-α production by human peripheral blood mononuclear cells was reduced by more than 50% when the cells were cultured with LPS in the presence of Hsp70, but that incubating these cells with 15 to 60 μg/ml of Hsp70 for 24 h increased TNF-α production in response to 1 ng/ml of LPS . This apparent discrepancy with our results could be caused by the different concentrations of Hsp70 and LPS used, and by the use of peripheral blood mononuclear cells instead of enriched monocytes for the experiments.
Endotoxemia stimulates stress responses through HSF-1, as a protective mechanism for the host. HSF-1 is found in the cytoplasm as a monomer devoid of transcriptional activity, but upon exposure to a heat shock or to other stressing insults, it is phosphorylated, trimerized and transported to the nucleus, where it binds to HSEs. Extracellular Hsp70 phosphorylates Akt through the TLR4 signaling pathway, resulting in glycogen synthase kinase (GSK)-3β inactivation . The inactivation of GSK-3β, an inhibitory regulator of HSF-1, leads to increased HSF-1 activity . CHBF interacts also with HSE and competes with HSF1 for binding to DNA . The mouse TNF-α promoter contains an HSE binding site for HSF-1 and binding of HSF-1 this HSE results in repressed transcription . Based on the location of the putative HSE in the human TNF-α gene promoter, HSF-1 and CHBF could hinder the RNA polymerase processivity . In our experimental system, we provide evidence that the decrease in TNF-α production caused by Hsp70 in LPS-activated monocytes results of a change at the gene expression level: the amount of TNF-α mRNA was decreased in the presence of Hsp70. We report that monocyte co-stimulation with LPS and Hsp70 lead to HSF-1 and CHBF binding to the HSE on the TNF-α promoter, and that this binding correlated with a decreased production of TNF-α. As expected, unstressed cells contained mainly the slower migrating complex. These data are consistent with the fact that heat shock, which increases the levels of HSF-1, has a suppressive effect on the inflammatory response, possibly because HSF-1 exerts a competitive inhibition on NF-κB binding . We observed that Hsp70 activates NF-κB through TLR2 and TLR4 signaling (Additional file 1: Figure S1). However, the activation of this transcription factor did not lead to TNF-α production, probably as a result of the strong activation of HSF-1. The reduction of IL-6 production in our model could be explained because HSF-1 inhibits the expression of this cytokine through activating transcription factor 3 (ATF-3) .
Interestingly, we found that Hsp70 treatment prevented LPS-induced IL-6 expression but not that resulting from engaging TL2 (porins) o TLR5 (flagellin). Unlike TLR2 and TLR5, TLR4-induced IL-6 expression involves the activation of the JAK-STAT pathway, thus making IL-6 expression susceptible to the negative regulation of SOCS1 . Noteworthy, T. gondii Hsp70 promotes SOCS1 expression in macrophages . Thus, it is possible that by inducing SOCS1 expression H70 specifically preventsTRL-4 induced JAK-STAT-dependent IL-6 expression, without affecting that resulting from TLR2 or TLR5 signaling.
A previous paper reported that exogenous recombinant Hsp70 interacts with HSF-1 in cell lines. The authors find that Hsp70 binds to HSF-1 and blocks its DNA-binding activity, and they suggest a model in which this association prevents the transcription of HSF-1-regulated genes in resting cells. After a heat shock, miss-folded proteins compete for Hsp70 binding, and this releases HSF-1 . In our study, we did not explore the signalling pathways that lead to HSF-1 activation, but a similar mechanism could operate during inflammation-induced cell stress. Other studies report that HSF-1 deficient mice have increased levels of TNF-α after stimulation with LPS , and that Escherichia coli-induced inflammation is resolved faster when a heat shock precedes inoculation with bacteria . HSF-1 also inhibits the transcription and secretion of G-CSF in response to LPS . Furthermore, HSF-1 has been reported to prevent the overproduction of pro-inflammatory cytokines in conditions such as sepsis , potentially decreasing the inflammation-associated damage. Our results support the idea that exogenous HSP70 provides a mechanism for controlling the excessive expansion of an inflammatory response after monocytes activation by bacterial pathogens through the recruitment of HSF-1 to the human TNF-α promoter, and underscores a potential role for exogenous Hsp70 as a prophylactic agent for different types of inflammatory diseases associated with infections.