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

Tulapurkar ME, Ramarathnam A, Hasday JD, Singh IS - PLoS ONE (2015)

Bottom Line: 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.The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

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.

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LPS/FRH-induced eHSP70 is predominantly the inducible form and is secreted independent of exosomes:Differentiated THP1 cells were incubated with or without 100 ng/ml LPS at 37°C or 39.5°C for 6 h, cell culture supernatants were collected and half of each were cleared of exosomes (esxosome-depleted) via ultracentrifugation; the remaining sample volume remained unfractionated (total). (A) electron micrograph of exosome fraction (B). HSP70 levels (including both inducible HSP72 and constitutive HSC70 forms) in the total and exosome-depleted cell culture supernatants was assayed by ELISA. (C–E) Total cell extracts and isolated exosomes from cells from panels A and B were immunoblotted using SPA-812 anti-HSP70 antibody (Enzo), which detects only inducible HSP72, H5147 anti-HSP70 antibody (Sigma-Aldrich), which detects both HSP72 and constitutive HSC70, and anti-CD63 antibody (C). Band intensities for CD63, HSPA8 and HSPA1A were quantified and plotted for cell extracts (D) and exosomes (E). Data presented as mean+SE, n = 6. * denotes p<0.05 vs. controls. Images presented are representative of 3 or more independent experiments.
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pone.0118010.g004: LPS/FRH-induced eHSP70 is predominantly the inducible form and is secreted independent of exosomes:Differentiated THP1 cells were incubated with or without 100 ng/ml LPS at 37°C or 39.5°C for 6 h, cell culture supernatants were collected and half of each were cleared of exosomes (esxosome-depleted) via ultracentrifugation; the remaining sample volume remained unfractionated (total). (A) electron micrograph of exosome fraction (B). HSP70 levels (including both inducible HSP72 and constitutive HSC70 forms) in the total and exosome-depleted cell culture supernatants was assayed by ELISA. (C–E) Total cell extracts and isolated exosomes from cells from panels A and B were immunoblotted using SPA-812 anti-HSP70 antibody (Enzo), which detects only inducible HSP72, H5147 anti-HSP70 antibody (Sigma-Aldrich), which detects both HSP72 and constitutive HSC70, and anti-CD63 antibody (C). Band intensities for CD63, HSPA8 and HSPA1A were quantified and plotted for cell extracts (D) and exosomes (E). Data presented as mean+SE, n = 6. * denotes p<0.05 vs. controls. Images presented are representative of 3 or more independent experiments.

Mentions: Previous studies in human peripheral blood mononuclear cells (PBMCs) and rat endothelial cells have shown that eHSP70 is secreted via exosomes [27, 28] and HSP70 is usually found at high levels in exosomal preparations [29, 30]. To determine whether the LPS-induced eHSP70 release in FRH-exposed cells is mediated via the exosomal route, we analyzed whether depleting THP1-conditioned medium of exosomes would reduce their eHSP70 content. Cell culture media from THP1 cells incubated for 6 h with or without LPS at 37° or 39.5°C were sequentially centrifuged, at 3000g for 10 min to remove cells and debris, at 13,000g for 30 min to isolate microvesicles, and at 100,000g for 1h to sediment exosomes and the final supernatants were collected as exosome-depleted media. Sedimented exosomes were verified by their characteristic size and appearance by electron microscopy (Fig. 4a). HSP70 levels in the exosome-sufficient post-3000g and exosome-depleted post-100,000g supernatants were measured by ELISA (Fig. 4b). For each of the four treatments, the exosome-sufficient and exosome-depleted culture media had similar HSP70 content. To analyze the HSP70 content of the exosomes, we immunoblotted the isolated exosomes and the corresponding cell lysates for HSP70 using two different anti-HSP70 antibodies. Using antibody SPA-812 (Enzo) which exclusively identifies inducible HSP72 (coded for by hspa1a and hspa1b) demonstrated a marked increase in HSP72 band intensity in cell extracts from 39.5°C- and LPS/39.5°C-exposed THP1 cells (Fig. 4c, d), but failed to detect HSP72 in the isolated exosomes (Fig. 4c, e). In contrast, antibody H5147 (Sigma), which recognizes both the constitutive (HSC70, HSPA8) and inducible forms of HSP70, showed distinct bands in the isolated exosomes, especially in exosomes isolated from LPS-treated 37° and 39.5°C THP1 cells (Fig. 4c, e). Immunblotting with an antibody against the exosome marker CD63 confirmed the presence of exosomes and demonstrated a modest increase in exosome release during 39.5°C incubation in the absence or presence of LPS.


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

Tulapurkar ME, Ramarathnam A, Hasday JD, Singh IS - PLoS ONE (2015)

LPS/FRH-induced eHSP70 is predominantly the inducible form and is secreted independent of exosomes:Differentiated THP1 cells were incubated with or without 100 ng/ml LPS at 37°C or 39.5°C for 6 h, cell culture supernatants were collected and half of each were cleared of exosomes (esxosome-depleted) via ultracentrifugation; the remaining sample volume remained unfractionated (total). (A) electron micrograph of exosome fraction (B). HSP70 levels (including both inducible HSP72 and constitutive HSC70 forms) in the total and exosome-depleted cell culture supernatants was assayed by ELISA. (C–E) Total cell extracts and isolated exosomes from cells from panels A and B were immunoblotted using SPA-812 anti-HSP70 antibody (Enzo), which detects only inducible HSP72, H5147 anti-HSP70 antibody (Sigma-Aldrich), which detects both HSP72 and constitutive HSC70, and anti-CD63 antibody (C). Band intensities for CD63, HSPA8 and HSPA1A were quantified and plotted for cell extracts (D) and exosomes (E). Data presented as mean+SE, n = 6. * denotes p<0.05 vs. controls. Images presented are representative of 3 or more independent experiments.
© Copyright Policy
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pone.0118010.g004: LPS/FRH-induced eHSP70 is predominantly the inducible form and is secreted independent of exosomes:Differentiated THP1 cells were incubated with or without 100 ng/ml LPS at 37°C or 39.5°C for 6 h, cell culture supernatants were collected and half of each were cleared of exosomes (esxosome-depleted) via ultracentrifugation; the remaining sample volume remained unfractionated (total). (A) electron micrograph of exosome fraction (B). HSP70 levels (including both inducible HSP72 and constitutive HSC70 forms) in the total and exosome-depleted cell culture supernatants was assayed by ELISA. (C–E) Total cell extracts and isolated exosomes from cells from panels A and B were immunoblotted using SPA-812 anti-HSP70 antibody (Enzo), which detects only inducible HSP72, H5147 anti-HSP70 antibody (Sigma-Aldrich), which detects both HSP72 and constitutive HSC70, and anti-CD63 antibody (C). Band intensities for CD63, HSPA8 and HSPA1A were quantified and plotted for cell extracts (D) and exosomes (E). Data presented as mean+SE, n = 6. * denotes p<0.05 vs. controls. Images presented are representative of 3 or more independent experiments.
Mentions: Previous studies in human peripheral blood mononuclear cells (PBMCs) and rat endothelial cells have shown that eHSP70 is secreted via exosomes [27, 28] and HSP70 is usually found at high levels in exosomal preparations [29, 30]. To determine whether the LPS-induced eHSP70 release in FRH-exposed cells is mediated via the exosomal route, we analyzed whether depleting THP1-conditioned medium of exosomes would reduce their eHSP70 content. Cell culture media from THP1 cells incubated for 6 h with or without LPS at 37° or 39.5°C were sequentially centrifuged, at 3000g for 10 min to remove cells and debris, at 13,000g for 30 min to isolate microvesicles, and at 100,000g for 1h to sediment exosomes and the final supernatants were collected as exosome-depleted media. Sedimented exosomes were verified by their characteristic size and appearance by electron microscopy (Fig. 4a). HSP70 levels in the exosome-sufficient post-3000g and exosome-depleted post-100,000g supernatants were measured by ELISA (Fig. 4b). For each of the four treatments, the exosome-sufficient and exosome-depleted culture media had similar HSP70 content. To analyze the HSP70 content of the exosomes, we immunoblotted the isolated exosomes and the corresponding cell lysates for HSP70 using two different anti-HSP70 antibodies. Using antibody SPA-812 (Enzo) which exclusively identifies inducible HSP72 (coded for by hspa1a and hspa1b) demonstrated a marked increase in HSP72 band intensity in cell extracts from 39.5°C- and LPS/39.5°C-exposed THP1 cells (Fig. 4c, d), but failed to detect HSP72 in the isolated exosomes (Fig. 4c, e). In contrast, antibody H5147 (Sigma), which recognizes both the constitutive (HSC70, HSPA8) and inducible forms of HSP70, showed distinct bands in the isolated exosomes, especially in exosomes isolated from LPS-treated 37° and 39.5°C THP1 cells (Fig. 4c, e). Immunblotting with an antibody against the exosome marker CD63 confirmed the presence of exosomes and demonstrated a modest increase in exosome release during 39.5°C incubation in the absence or presence of LPS.

Bottom Line: 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.The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus