Bacterial lipopolysaccharide augments febrile-range hyperthermia-induced heat shock protein 70 expression and extracellular release in human THP1 cells.

Mohan E Tulapurkar,Aparna Ramarathnam,Jeffrey D Hasday,Ishwar S Singh

doi:10.1371/journal.pone.0118010

Mohan E Tulapurkar, Aparna Ramarathnam + Show 2 more

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https://doi.org/10.1371/journal.pone.0118010

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Abstract

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

Highlights

Sepsis is a devastating, often lethal complication of severe infection and injury, characterized by excessive and dysregulated inflammation, multi-organ injury and cardiovascular collapse
To determine whether activation of other Toll-like receptors (TLRs) would synergize with febrile range hyperthermia (FRH) to induce HSPA1A expression, we analyzed HSPA1A mRNA expression level in THP1 cells incubated with a TLR1/2 agonist (Pam3cys; 0.5 μg/ml) or TLR3
Studies from our group and others have shown that HSF1 can be activated by less extreme hyperthermia, including temperatures within the normal febrile range [8, 31], that some inflammatory mediators such as type I interferons and arachidonic acid can lower the thermal threshold for HSF1 activation [32], and that HSF1 and other elements of the heat shock response (HSR) can exert potent immunomodulatory effects [9, 10, 22, 23, 31, 33,34,35,36,37,38,39,40,41,42]

Summary

Introduction

Often lethal complication of severe infection and injury, characterized by excessive and dysregulated inflammation, multi-organ injury and cardiovascular collapse. The mechanisms by which fever reduction improves mortality in septic shock are not yet well understood, fever and febrile-range hyperthermia (FRH) is known to exert many biological effects that may impact on survival during sepsis, including partial activation of the heat shock response (HSR). We have previously shown that HSF1 activation and induction of HSP gene expression occurs at temperatures within the usual febrile range (38.5–40°C) [8]. We and others have shown that HSF1 and certain HSPs can modify innate immune responses, which may shape the inflammatory response during febrile illnesses such as sepsis, trauma, cancer, and drug transfusion reactions (reviewed in [9, 10]), or during hyperthermic states including exertional/environmental or malignant hyperthermia [9,10,11,12]

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