Katherine E. Dunsmore

Extracellular Heat Shock Protein-70 Induces Endotoxin Tolerance in THP-1 Cells

Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-κB activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 μg/ml HSP-70) for 18 h, followed by LPS stimulation (1 μg/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-κB-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP-70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100°C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor κB-α and inhibited activation of inhibitor κB kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-κB following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.

Curcumin, a medicinal herbal compound capable of inducing the heat shock response.

Objective There is interest in developing pharmacologic inducers of the heat shock response as a means to confer cytoprotection in the clinical setting. We propose that a potential strategy for screening novel pharmacologic inducers of the heat shock response is to examine known inhibitors of the transcription factor nuclear factor-&kgr;B. Curcumin, derived from the tropical herb Curcuma longa, is a recently described inhibitor of nuclear factor-&kgr;B and is widely used in Eastern medicinal practices. We tested the hypothesis that curcumin can induce expression of heat shock protein 70. Design Experimental. Setting University laboratory. Subjects HeLa cells. Interventions HeLa cells were exposed to varying concentrations of curcumin and analyzed for expression of heat shock protein 70 by Western blot. Measurements and Main Results Activation of the transcription factor, heat shock factor-1, was analyzed by electromobility shift assays. Curcumin-mediated inhibition of nuclear factor-&kgr;B activation was measured by transiently transfecting cells with a nuclear factor-&kgr;B luciferase reporter plasmid. The role of heat shock factor-1 in curcumin-mediated expression of heat shock protein 70 was tested in embryonic fibroblasts derived from heat shock factor-1 knockout mice. Induction of the heat shock response was quantified by transiently transfecting cells with a heat shock protein 70 promoter-luciferase reporter plasmid. Cell viability was measured by using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Curcumin induced expression of heat shock protein 70, the major inducible heat shock protein in cells undergoing the heat shock response, in a dose-dependent and time-dependent manner. Curcumin induced specific nuclear translocation and activation of heat shock factor-1. Curcumin-mediated expression of heat shock protein 70 was reduced substantially in fibroblasts having genetic ablation of heat shock factor-1. The extent of induction of the heat shock response correlated, in part, with cellular toxicity. Conclusions Curcumin, a widely used medicinal compound, induces the heat shock response in vitro as measured by expression of heat shock protein 70. The mechanism of heat shock protein 70 induction depends on activation of heat shock factor-1. Examining known inhibitors of nuclear factor-&kgr;B for their ability to induce heat shock protein 70 may be a valid screening method to discover novel pharmacologic inducers of the heat shock response.

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain.

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.

Selectively increasing inducible heat shock protein 70 via TAT‐protein transduction protects neurons from nitrosative stress and excitotoxicity

Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT‐Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT‐Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre‐incubated with 300 nm TAT‐Hsp 70 overnight, then exposed to either peroxynitrite (ONOO–) or glutamate. TAT‐Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild‐type Hsp70, and TAT‐β‐galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT‐Hsp70 is protective in in vivo models of brain injury.

A novel role for matrix metalloproteinase-8 in sepsis.

Objectives: Matrix metalloproteinase-8 messenger RNA expression was previously found to be increased in whole blood of children with septic shock. The impact of this finding on the severity and inflammatory response to sepsis is unknown. Here, we investigate the relationship between matrix metalloproteinase-8 and disease severity in children with septic shock. We further corroborate the role of matrix metalloproteinase-8 in sepsis in a murine model. Design: Retrospective observational clinical study and randomized controlled laboratory experiments. Setting: Pediatric intensive care units and an animal research facility at an academic childrens hospital. Patients and Subjects: Patients age ⩽10 yrs admitted to the intensive care unit with a diagnosis of septic shock. For laboratory studies, we utilized male mice deficient for matrix metalloproteinase-8 and male wild-type C57BL/6J mice. Interventions: Blood from children with septic shock was analyzed for matrix metalloproteinase-8 messenger RNA expression and matrix metalloproteinase-8 activity, and correlated with disease severity based on mortality and degree of organ failure. A murine model of sepsis was used to explore the effect of genetic and pharmacologic inhibition of matrix metalloproteinase-8 on the inflammatory response to sepsis. Finally, activation of nuclear factor-&kgr;B was assessed both in vitro and in vivo. Measurements and Main Results: Increased matrix metalloproteinase-8 mRNA expression and activity in septic shock correlates with decreased survival and increased organ failure in pediatric patients. Genetic and pharmacologic inhibition of matrix metalloproteinase-8 leads to improved survival and a blunted inflammatory profile in a murine model of sepsis. We also identify matrix metalloproteinase-8 as a direct in vitro activator of the proinflammatory transcription factor, nuclear factor-&kgr;B. Conclusions: Matrix metalloproteinase-8 is a novel modulator of inflammation during sepsis and a potential therapeutic target.

Temporal and Mechanistic Effects of Heat Shock on LPS-Mediated Degradation of IκBα in Macrophages

Previous studies demonstrated important interactions between the heat shock response and the IκBα/NF-κB pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IκBα in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IκBα degradation, and the proximal mechanisms by which heat shock inhibits degradation of IκBα in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IκBα degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-κB. Beyond these recovery periods, heat shock did not inhibit IκBα degradation. IκB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IκBα and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IκBα is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IκBα involves dual modulation of IKK and intracellular phosphatase activity.

The Role Of Endogenously Produced Extracellular Hsp72 In Mononuclear Cell Reprogramming

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-&kgr;B activation. Heat shock conditions consisted of incubation at 43°C for 1 h. Control cells were incubated at 37°C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 &mgr;g/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-&kgr;B luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-&kgr;B-DNA binding and I&kgr;B&agr; degradation. Heat shock protein 72-mediated inhibition of NF-&kgr;B activation was further corroborated by a significant decrease in TNF-&agr; production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-&kgr;B was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.

Biological activity of truncated C-terminus human heat shock protein 72.

Heat shock protein 72 (Hsp72), a canonical intracellular molecular chaperone, may also function as an extracellular danger signal for the innate immune system. To further delineate the biological role of Hsp72 in the innate immune system, we generated two truncated versions of the full length human Hsp72 (N-terminus Hsp72, amino acids 1-430; and C-terminus Hsp72 amino acids 420-641) and directly compared their ability to activate cells from the macrophage/monocyte lineage. In RAW 264.7 macrophages transfected with a NF-κB-dependent luciferase reporter plasmid, C-terminus Hsp72 was a more potent inducer of NF-κB activity than N-terminus Hsp72, and this effect did not seem to be secondary to endotoxin contamination. C-terminus Hsp72-mediated activation of the NF-κB pathway was corroborated by increased activation of IκB kinase, degradation of IκBα, and increased NF-κB-DNA binding. C-terminus Hsp72 was a more potent inducer of tumor necrosis factor-α (TNFα) expression in RAW 264.7 macrophages and in primary murine peritoneal macrophages from wild-type mice. C-terminus Hsp72 did not induce TNFα expression in primary murine peritoneal macrophages from Toll-like receptor (TLR4) mutant mice, indicating a role for TLR4. In human THP-1 mononuclear cells, C-terminus Hsp72 induced tolerance to subsequent LPS stimulation, whereas N-terminus Hsp72 did not induce tolerance. Finally, control experiments using equimolar amounts of N-terminus or C-terminus Hsp72 demonstrated a higher biological potency for C-terminus Hsp72. These data demonstrate that the ability of human Hsp72 to serve as an activator for cells of the macrophage/monocyte lineage primarily lies in the C-terminus region spanning amino acids 420-641.