Exposure to febrile-range hyperthermia potentiates Wnt signalling and epithelial-mesenchymal transition gene expression in lung epithelium.

BACKGROUND: As environmental and body temperatures vary, lung epithelial cells experience temperatures significantly different from normal core temperature. Our previous studies in human lung epithelium showed that: (i) heat shock accelerates wound healing and activates profibrotic gene expression through heat shock factor-1 (HSF1); (ii) HSF1 is activated at febrile temperatures (38-41 °C) and (iii) hypothermia (32 °C) activates and hyperthermia (39.5 °C) reduces expression of a subset of miRNAs that target protein kinase-Cα (PKCα) and enhance proliferation. METHODS: We analysed the effect of hypo- and hyperthermia exposure on Wnt signalling by exposing human small airway epithelial cells (SAECs) and HEK293T cells to 32, 37 or 39.5 °C for 24 h, then analysing Wnt-3a-induced epithelial-mesenchymal transition (EMT) gene expression by qRT-PCR and TOPFlash reporter plasmid activity. Effects of miRNA mimics and inhibitors and the HSF1 inhibitor, KNK437, were evaluated. RESULTS: Exposure to 39.5 °C for 24 h increased subsequent Wnt-3a-induced EMT gene expression in SAECs and Wnt-3a-induced TOPFlash activity in HEK293T cells. Increased Wnt responsiveness was associated with HSF1 activation and blocked by KNK437. Overexpressing temperature-responsive miRNA mimics reduced Wnt responsiveness in 39.5 °C-exposed HEK293T cells, but inhibitors of the same miRNAs failed to restore Wnt responsiveness in 32 °C-exposed HEK293T cells. CONCLUSIONS: Wnt responsiveness, including expression of genes associated with EMT, increases after exposure to febrile-range temperature through an HSF1-dependent mechanism that is independent of previously identified temperature-dependent miRNAs. This process may be relevant to febrile fibrosing lung diseases, including the fibroproliferative phase of acute respiratory distress syndrome (ARDS) and exacerbations of idiopathic pulmonary fibrosis (IPF).

Novel Noncatalytic Substrate-Selective p38α-Specific MAPK Inhibitors with Endothelial-Stabilizing and Anti-Inflammatory Activity.

The p38 MAPK family is composed of four kinases of which p38α/MAPK14 is the major proinflammatory member. These kinases contribute to many inflammatory diseases, but the currently available p38 catalytic inhibitors (e.g., SB203580) are poorly effective and cause toxicity. We reasoned that the failure of catalytic p38 inhibitors may derive from their activity against noninflammatory p38 isoforms (e.g., p38ß/MAPK11) and loss of all p38α-dependent responses, including anti-inflammatory, counterregulatory responses via mitogen- and stress-activated kinase (MSK) 1/2 and Smad3. We used computer-aided drug design to target small molecules to a pocket near the p38α glutamate-aspartate (ED) substrate-docking site rather than the catalytic site, the sequence of which had only modest homology among p38 isoforms. We identified a lead compound, UM101, that was at least as effective as SB203580 in stabilizing endothelial barrier function, reducing inflammation, and mitigating LPS-induced mouse lung injury. Differential scanning fluorimetry and saturation transfer difference-nuclear magnetic resonance demonstrated specific binding of UM101 to the computer-aided drug design-targeted pockets in p38α but not p38ß. RNA sequencing analysis of TNF-α-stimulated gene expression revealed that UM101 inhibited only 28 of 61 SB203580-inhibited genes and 7 of 15 SB203580-inhibited transcription factors, but spared the anti-inflammatory MSK1/2 pathway. We provide proof of principle that small molecules that target the ED substrate-docking site may exert anti-inflammatory effects similar to the catalytic p38 inhibitors, but their isoform specificity and substrate selectivity may confer inherent advantages over catalytic inhibitors for treating inflammatory diseases.

Activation of heat shock response augments fibroblast growth factor-1 expression in wounded lung epithelium.

We previously showed that coincident exposure to heat shock (HS; 42°C for 2 h) and TNF-α synergistically induces apoptosis in mouse lung epithelium. We extended this work by analyzing HS effects on human lung epithelial responses to clinically relevant injury. Cotreatment with TNF-α and HS induced little caspase-3 and poly(ADP-ribose) polymerase cleavage in human small airway epithelial cells, A549 cells, and BEAS2B cells. Scratch wound closure rates almost doubled when A549 and BEAS2B cells and air-liquid interface cultures of human bronchial epithelial cells were heat shocked immediately after wounding. Microarray, qRT-PCR, and immunoblotting showed fibroblast growth factor 1 (FGF1) to be synergistically induced by HS and wounding. Enhanced FGF1 expression in HS/wounded A549 was blocked by inhibitors of p38 MAPK (SB203580) or HS factor (HSF)-1 (KNK-437) and in HSF1 knockout BEAS2B cells. PCR demonstrated FGF1 to be expressed from the two most distal promoters in wounded/HS cells. Wound closure in HS A549 and BEAS2B cells was reduced by FGF receptor-1/3 inhibition (SU-5402) or FGF1 depletion. Exogenous FGF1 accelerated A549 wound closure in the absence but not presence of HS. In the presence of exogenous FGF1, HS slowed wound closure, suggesting that it increases FGF1 expression but impairs FGF1-stimulated wound closure. Frozen sections from normal and idiopathic pulmonary fibrosis (IPF) lung were analyzed for FGF1 and HSP70 by immunofluorescence confocal microscopy and qRT-PCR. FGF1 and HSP70 mRNA levels were 7.5- and 5.9-fold higher in IPF than normal lung, and the proteins colocalized to fibroblastic foci in IPF lung. We conclude that HS signaling may have an important impact on gene expression contributing to lung injury, healing, and fibrosis.

TrkB agonist, 7,8-dihydroxyflavone, reduces the clinical and pathological severity of a murine model of multiple sclerosis.

7,8-Dihydroxyflavone (DHF), is a recently described TrkB agonist that readily crosses the blood brain barrier. We treated C57Bl/6 mice with MOG--induced EAE daily with DHF starting on the day of disease induction. Clinical severity of impairment was reduced throughout the course of disease. Pathological examination of brains and spinal cords on day 28 showed that DHF treatment increased the phosphorylation of TrkB and activated downstream signaling pathways including AKT and STAT3 and reduced inflammation, demyelination and axonal loss compared to EAE controls. DHF treatment duplicated the central nervous system effects of brain derived neurotrophic factor in the EAE.

Shifts in temperature within the physiologic range modify strand-specific expression of select human microRNAs.

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes.

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

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.

Celastrol, an oral heat shock activator, ameliorates multiple animal disease models of cell death.

Protein homeostatic regulators have been shown to ameliorate single, loss-of-function protein diseases but not to treat broader animal disease models that may involve cell death. Diseases often trigger protein homeostatic instability that disrupts the delicate balance of normal cellular viability. Furthermore, protein homeostatic regulators have been delivered invasively and not with simple oral administration. Here, we report the potent homeostatic abilities of celastrol to promote cell survival, decrease inflammation, and maintain cellular homeostasis in three different disease models of apoptosis and inflammation involving hepatocytes and cardiomyocytes. We show that celastrol significantly recovers the left ventricular function and myocardial remodeling following models of acute myocardial infarction and doxorubicin-induced cardiomyopathy by diminishing infarct size, apoptosis, and inflammation. Celastrol prevents acute liver dysfunction and promotes hepatocyte survival after toxic doses of thioacetamide. Finally, we show that heat shock response (HSR) is necessary and sufficient for the recovery abilities of celastrol. Our observations may have dramatic clinical implications to ameliorate entire disease processes even after cellular injury initiation by using an orally delivered HSR activator.

Polymorphisms in human heat shock factor-1 and analysis of potential biological consequences.

The stress-activated transcription factor, heat shock factor-1 (HSF1), regulates many genes including cytoprotective heat shock proteins (HSPs). We hypothesized that polymorphisms in HSF1 may alter the level or function of HSF1 protein accounting for interindividual viability in disease susceptibility or prognosis. We searched for exomic variants in HSF1 by querying human genome databases and directly sequencing DNA from 80 anonymous genomic DNA samples. Overall, HSF1 sequence was highly conserved, with no common variations. We found 31 validated deviations from a reference sequence in the dbSNP database and an additional 5 novel variants by sequencing, with allele frequencies that were 0.06 or less. Of these 36, 2 were in 5'-untranslated region (5'UTR), 10 in 3'UTR, and 24 in the coding region. The potential effects of 5'UTR on secondary structure, protein structure/function, and 3'UTR targets of microRNAs were analyzed using RNAFold, PolyPhen-2, SIFT, and MicroSNiper. One of the 5'UTR variants was predicted to strengthen secondary structure. Eight of 3'UTR variants were predicted to modify microRNA target sequences. Eight of the coding region variants were predicted to modify HSF1 structure/function. Reducing HSF1 levels in A549 cells using short hairpin RNA (shRNA) increased sensitivity to heat-induced killing demonstrating the impact that genetic variants that reduce HSF1 levels might have. Using the pmirGLO expression system, we found that the wild-type HSF1 3'UTR suppressed translation of a firefly luciferase reporter plasmid by 65 %. Introducing two of four 3'UTR single nucleotide polymorphisms (SNPs) increased HSF1 3'UTR translational suppression by 27-44 % compared with the wild-type HSF1 3'UTR sequence while a third SNP reduced suppression by 25 %. HSF1 variants may alter HSF1 protein levels or function with potential effects on cell functions, including sensitivity to stress.

Fever, immunity, and molecular adaptations.

The heat shock response (HSR) is an ancient and highly conserved process that is essential for coping with environmental stresses, including extremes of temperature. Fever is a more recently evolved response, during which organisms temporarily subject themselves to thermal stress in the face of infections. We review the phylogenetically conserved mechanisms that regulate fever and discuss the effects that febrile-range temperatures have on multiple biological processes involved in host defense and cell death and survival, including the HSR and its implications for patients with severe sepsis, trauma, and other acute systemic inflammatory states. Heat shock factor-1, a heat-induced transcriptional enhancer is not only the central regulator of the HSR but also regulates expression of pivotal cytokines and early response genes. Febrile-range temperatures exert additional immunomodulatory effects by activating mitogen-activated protein kinase cascades and accelerating apoptosis in some cell types. This results in accelerated pathogen clearance, but increased collateral tissue injury, thus the net effect of exposure to febrile range temperature depends in part on the site and nature of the pathologic process and the specific treatment provided.

Fever, hyperthermia and the heat shock response.

The heat shock response is a highly conserved primitive response that is essential for survival against a wide range of stresses, including extremes of temperature. Fever is a more recently evolved response, during which organisms raise their core body temperature and temporarily subject themselves to thermal stress in the face of infections. The present review documents studies showing the potential overlap between the febrile response and the heat shock response and how both activate the same common transcriptional programme (although with different magnitudes) including the stress-activated transcription factor, heat shock factor-1, to modify host defences in the context of infection, inflammation and injury. The review focuses primarily on how hyperthermia within the febrile range that often accompanies infections and inflammation acts as a biological response modifier and modifies innate immune responses. The characteristic 2-3 °C increase in core body temperature during fever activates and utilises elements of the heat shock response pathway to modify cytokine and chemokine gene expression, cellular signalling and immune cell mobilisation to sites of inflammation, infection and injury. Interestingly, typical proinflammatory agonists such as Toll-like receptor agonists modify the heat shock-induced transcriptional programme and expression of HSP genes following co-exposure to febrile range hyperthermia or heat shock, suggesting a complex reciprocal regulation between the inflammatory pathway and the heat shock response pathway.

Toll-like receptor agonists and febrile range hyperthermia synergize to induce heat shock protein 70 expression and extracellular release.

Heat shock protein (Hsp) 70 expression can be stimulated by febrile range temperature (FRT). Hsp70 has been shown to be elevated in serum of patients with sepsis, and when released from cells, extracellular Hsp70 exerts endotoxin-like effects through Toll-like receptor 4 (TLR4) receptors. Circulating TLR agonists and fever both persist for the first several days of sepsis, and each can activate Hsp70 expression; however, the effect of combined exposure to FRT and TLR agonists on Hsp70 expression is unknown. We found that concurrent exposure to FRT (39.5 °C) and agonists for TLR4 (LPS), TLR2 (Pam3Cys), or TLR3 (poly(IC)) synergized to increase Hsp70 expression and extracellular release in RAW264.7 macrophages. The increase in Hsp70 expression was associated with activation of p38 and ERK MAP kinases, phosphorylation of histone H3, and increased recruitment of HSF1 to the Hsp70 promoter. Pretreatment with the p38 MAPK inhibitor SB283580 but not the ERK pathway inhibitor UO126 significantly reduced Hsp70 gene modification and Hsp70 expression in RAW cells co-exposed to LPS and FRT. In mice challenged with intratracheal LPS and then exposed to febrile range hyperthermia (core temperature, ∼39.5 °C), Hsp70 levels in lung tissue and in cell-free lung lavage were increased compared with mice exposed to either hyperthermia or LPS alone. We propose a model of how enhanced Hsp70 expression and extracellular release in patients concurrently exposed to fever and TLR agonists may contribute to the pathogenesis of sepsis.

Tolerance for chronic heat exposure is greater in female than male mice.

PURPOSE: Chronic heat exposure in mice has cellular and physiological effects that improve thermal tolerance [1], but also modifies innate immune responses with potential adverse consequences [2]. While male and female mice are known to respond differently to acute exposure to severe hyperthermia, sex-based differences in responses to chronic moderate heat exposure have not been reported. The major objective of this study was to compare the tolerance of male and female mice for chronic heat exposure. MATERIALS AND METHODS: We used a mouse model of 5-day moderate heat exposure (ambient temperature ∼37°C) to compare the physiological and cellular heat shock response in male and female mice. Core temperature, heart rate, and activity were monitored telemetrically and heat shock protein levels were measured in brain and lung by western blotting. RESULTS: Adult CD-1 female mice maintained a 1.2°C lower core temperature (38.31 ± 0.64 versus 39.51 ± 0.72°C; p = 0.002), experienced less weight loss (1.54 ± 0.45 versus 4.54 ± 1.97 g; p = 0.0007), and had improved survival (16/16 survived versus 13/21, p < 0.006) than male mice of the same age. After 5 days of moderate heat exposure Hsp72 levels in brain and lung increased 2.1-fold (p = 0.007) and 5-fold (p = 0.048) in male mice compared with 1.3- (p = 0.054) and 1.5-fold (p = 0.134) in female mice. CONCLUSIONS: This study reveals previously unknown and potentially important differences between male and female mice in physiological and cellular responses to chronic heat exposure, which had consequences for survival. Future studies may identify biomarkers of differential heat tolerance and treatments to improve heat tolerance in humans.

Hyperthermia promotes and prevents respiratory epithelial apoptosis through distinct mechanisms.

Hyperthermia has been shown to confer cytoprotection and to augment apoptosis in different experimental models. We analyzed the mechanisms of both effects in the same mouse lung epithelial (MLE) cell line (MLE15). Exposing MLE15 cells to heat shock (HS; 42°C, 2 h) or febrile-range hyperthermia (39.5°C) concurrent with activation of the death receptors, TNF receptor 1 or Fas, greatly accelerated apoptosis, which was detectable within 30 minutes and was associated with accelerated activation of caspase-2, -8, and -10, and the proapoptotic protein, Bcl2-interacting domain (Bid). Caspase-3 activation and cell death were partially blocked by inhibitors targeting all three initiator caspases. Cells expressing the IκB superrepessor were more susceptible than wild-type cells to TNF-α-induced apoptosis at 37°C, but HS and febrile-range hyperthermia still increased apoptosis in these cells. Delaying HS for 3 hours after TNF-α treatment abrogated its proapoptotic effect in wild-type cells, but not in IκB superrepressor-expression cells, suggesting that TNF-α stimulates delayed resistance to the proapoptotic effects of HS through an NF-κB-dependent mechanism. Pre-exposure to 2-hour HS beginning 6 to16 hours before TNF-α treatment or Fas activation reduced apoptosis in MLE15 cells. The antiapoptotic effects of HS pretreatment were reduced in TNF-α-treated embryonic fibroblasts from heat shock factor-1 (HSF1)-deficient mice, but the proapoptotic effects of concurrent HS were preserved. Thus, depending on the temperature and timing relative to death receptor activation, hyperthermia can exert pro- and antiapoptotic effects through distinct mechanisms.

Febrile-range hyperthermia augments reversible TNF-α-induced hyperpermeability in human microvascular lung endothelial cells.

Fever commonly occurs in acute lung injury (ALI) and ALI occurs in 25% of victims of heat stroke. We have shown in mouse models of ALI that exposure to febrile-range hyperthermia (FRH), 39.5°C, increases non-cardiogenic pulmonary oedema. In this study we studied the direct effects of FRH on endothelial barrier integrity using human microvascular endothelial cells (HMVEC-Ls). We analysed the effect of exposure to culture temperatures between 38.5° and 41°C with and without tumour necrosis factor-α (TNF-α) up to 250 U/mL for 6-24 h. We found that exposure to 2.5-250 U/mL TNF-α increased HMVEC-L permeability by 4.1-15.8-fold at 37°C. Exposure to 39.5°C alone caused variable, modest, lot-specific increases in HMVEC-L permeability, however raising culture temperature to 39.5°C in the presence of TNF-α increased permeability an additional 1.6-4.5-fold compared with cells incubated with the same TNF-α concentration at 37°C. Permeability occurred without measurable cytotoxicity and was reversible upon removal of TNF-α and reduction in temperature to 37°C. Exposure to 39.5°C or TNF-α each stimulated rapid activation of p38 and ERK but the effects were not additive. Treatment with inhibitors of ERK (U0126) or p38 (SB203580) each reduced TNF-α-induced permeability in 39.5°C monolayers to levels in 37°C cells, but did not alter TNF-α-induced permeability in the 37°C cells. These results demonstrate that FRH directly increases paracellular pathway opening through a process that requires ERK and p38 MAPKs. A better understanding of this mechanism may provide new understanding about how fever may contribute to the pathogenesis of ALI and provide new therapeutic targets to improve clinical outcomes.

Febrile-range hyperthermia modifies endothelial and neutrophilic functions to promote extravasation.

Acute respiratory distress syndrome (ARDS) is a neutrophil (polymorphonuclear leukocyte; PMN)-driven lung injury that is associated with fever and heat-stroke, and involves approximately 40% mortality. In murine models of acute lung injury (ALI), febrile-range hyperthermia (FRH) enhanced PMN accumulation, vascular permeability, and epithelial injury, in part by augmenting pulmonary cysteine-x-cysteine (CXC) chemokine expression. To determine whether FRH increases chemokine responsiveness within the lung, we used in vivo and in vitro models that bypass the endogenous generation of chemokines. We measured PMN transalveolar migration (TAM) in mice after intratracheal instillations of the human CXC chemokine IL-8 in vivo, and of IL-8-directed PMN transendothelial migration (TEM) through human lung microvascular endothelial cell (HMVEC-L) monolayers in vitro. Pre-exposure to FRH increased in vivo IL-8-directed PMN TAM by 23.5-fold and in vitro TEM by 7-fold. Adoptive PMN transfer demonstrated that enhanced PMN TAM required both PMN donors and recipients to be exposed to FRH, suggesting interdependent effects on PMNs and endothelium. FRH exposure caused the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in lung homogenates and circulating PMNs, with an associated increase in HSP27 phosphorylation and stress-fiber formation. The inhibition of these signaling pathways with U0126 and SB203580 blocked the effects of FRH on PMN extravasation in vivo and in vitro. Collectively, these results (1) demonstrate that FRH augments chemokine-directed PMN extravasation through direct effects on endothelium and PMNs, (2) identify ERK and p38 signaling pathways in the effect, and (3) underscore the complex effects of physiologic temperature change on innate immune function and its potential consequences for lung injury.

Prolonged exposure to hyperthermic stress augments neutrophil recruitment to lung during the post-exposure recovery period.

The effects of heat, especially long-term heat exposure, are complex and incompletely understood and few studies have analysed the immunological consequences of such exposures. In the present study we analysed how long-term hyperthermia modified the pulmonary immune responses, especially recruitment of neutrophils to sites of inflammation, infection and injury. Using our mouse model of long-term whole body hyperthermia (continuous 5-day passive febrile range hyperthermia (5d-FRH)) we found that bacterial lipopolysaccharide (LPS) challenge greatly increased neutrophil accumulation in bronchoalveolar lavage and lung parenchyma in 5d-FRH exposed mice in comparison to LPS-treated controls. Moreover, the effect was sustained, and persisted during the post-exposure recovery period, and LPS challenge on days 5-7 post-recovery also exhibited similarly augmented neutrophil response. Lung lavage from 5d-FRH mice, either immediately or up to 7 days post-exposure, showed significantly increased levels of ELR + CXC chemokines, KC or LIX in response to LPS challenge, indicating that enhanced chemokines could contribute to the increased recruitment of neutrophils to the lung. However, an in vivo neutrophil migration assay following 5d-FRH and during the post-exposure recovery period also showed persistently enhanced neutrophil influx in response to a fixed chemotactic gradient generated by recombinant human IL-8, suggesting that additional mechanisms besides increased ELR + CXC chemokines contributed to the augmented neutrophil response caused by 5d-FRH exposure. These previously unappreciated profound and lasting effects of long-term hyperthermia may have important consequences and may help explain the increased risk of respiratory illnesses in active duty personnel and returning veterans.

Distinct, gene-specific effect of heat shock on heat shock factor-1 recruitment and gene expression of CXC chemokine genes.

The heat shock (HS) response, a phylogenetically conserved ubiquitous response to stress, is generally characterized by the induced expression of heat shock protein (HSP) genes. Our earlier studies showed that the stress-activated transcription factor, heat shock factor-1 (HSF1), activated at febrile range or HS temperatures also modified expression of non-HSP genes including cytokine and chemokine genes. We also showed by in silico analysis that 28 among 29 human and mouse CXC chemokine genes had multiple putative heat shock response elements (HSEs) present in their gene promoters. To further determine whether these potential HSEs were functional and bound HSF1, we analyzed the recruitment of HSF1 to promoters of 5 human CXC chemokine genes (CXCL-1, 2, 3, 5 and 8) by chromatin immunoprecipitation (ChIP) assay and analyzed the effect of HS exposure on tumor necrosis factor-α (TNFα)-induced expression of these genes in human lung epithelial-like A549 cells. HSF1 ChIP analysis showed that HSF1 was recruited to all but one of these CXC chemokine genes (CXCL-3) and HS caused a significant increase in recruitment of HSF1 to one or multiple HSEs present in the promoters of CXCL-1, 2, 5 and 8 genes. However, the effect of HS exposure on expression of these genes showed a variable gene-specific effect. For example, CXCL8 expression was markedly enhanced (p<0.05) whereas CXCL5 expression was significantly repressed (p<0.05) in cells exposed to HS coincident with TNFα stimulation. In contrast, expression of CXCL1 and CXCL2, despite HSF1 recruitment to their promoters, was not affected by HS exposure. Our results indicate that some, if not all, putative HSEs present in the CXC chemokine gene promoters are functional and recruit HSF1 in vivo but the effects on gene expression are variable and gene specific. We speculate, the physical proximity and interactions of other transcription factors and co-regulators with HSF1 could be critical to determining the effects of HS on the expression of these genes.

Response of mice to continuous 5-day passive hyperthermia resembles human heat acclimation.

Chronic repeated exposure to hyperthermia in humans results in heat acclimation (HA), an adaptive process that is attained in humans by repeated exposure to hyperthermia and is characterized by improved heat elimination and increased exercise capacity, and acquired thermal tolerance (ATT), a cellular response characterized by increased baseline heat shock protein (HSP) expression and blunting of the acute increase in HSP expression stimulated by re-exposure to thermal stress. Epidemiologic studies in military personnel operating in hot environments and elite athletes suggest that repeated exposure to hyperthermia may also exert long-term health effects. Animal models demonstrate that coincident exposure to mild hyperthermia or prior exposure to severe hyperthermia can profoundly affect the course of experimental infection and injury, but these models do not represent HA. In this study, we demonstrate that CD-1 mice continuously exposed to mild hyperthermia (ambient temperature ~37°C causing ~2°C increase in core temperature) for 5 days and then exposed to a thermal stress (42°C ambient temperature for 40 min) exhibited some of the salient features of human HA, including (1) slower warming during thermal stress and more rapid cooling during recovery and (2) increased activity during thermal stress, as well as some of the features of ATT, including (1) increased baseline expression of HSP72 and HSP90 in lung, heart, spleen, liver, and brain; and (2) blunted incremental increase in HSP72 expression following acute thermal stress. This study suggests that continuous 5-day exposure of CD-1 mice to mild hyperthermia induces a state that resembles the physiologic and cellular responses of human HA. This model may be useful for analyzing the molecular mechanisms of HA and its consequences on host responsiveness to subsequent stresses.

Prostaglandin E2 potentiates heat shock-induced heat shock protein 72 expression in A549 cells.

The heat shock (HS) response is an important cytoprotective response comprising the expression of heat shock proteins (HSPs) and orchestrated by the heat/stress-induced transcription factor, heat shock factor-1 (HSF-1). Previous studies suggest that the activation threshold and magnitude of the HS response may be modified by treatment with arachidonic acid (AA). We analyzed the effect of exogenous AA and its metabolites, PGE(2), LTD(4), and 15-HETE on HSF-1-dependent gene expression in A549 human respiratory epithelial-like cells. When added at 1microM, PGE(2) much more than LTD(4), but not 15-HETE increased activity of a synthetic HSF-1-dependent reporter after HS exposure (42 degrees C for 2h), but had no effect in the absence of HS. Exposing A549 cells to HS stimulated the release of PGE(2) and treatment with the cyclooxygenase inhibitor, ibuprofen, reduced HS-induced HSF-1-dependent transcription. PGE(2) increased HS-induced HSP72 mRNA and protein expression but EMSA and Western blot analysis failed to show an effect on HSF-1 DNA binding activity or post-translational modification. In summary, we showed that HS stimulates the generation of PGE(2), which augments the generation of HSPs. The clinical consequences of this pathway have yet to be determined.

Febrile range temperature represses TNF-alpha gene expression in LPS-stimulated macrophages by selectively blocking recruitment of Sp1 to the TNF-alpha promoter.

We have previously shown that exposure to febrile-range temperature (FRT, 39.5 degrees C) reduces LPS-induced TNF-alpha transcription in mouse macrophages through at least two mechanisms: (1) by directly recruiting heat shock factor-1 (HSF-1) to a heat shock response element present in the TNF-alpha 5'-UTR and (2) by markedly reducing LPS-induced recruitment of NFkappaB-p65 to the kappaB enhancer (at -510) in the TNF-alpha gene. In the present study, we used EMSA and chromatin immunoprecipitation assays to further analyze the complex effects of FRT on the recruitment of transcription factors and co-activators on the TNF-alpha gene in LPS-stimulated RAW 264.7 mouse macrophages. Our results showed that in FRT-exposed RAW cells, HSF-1 was recruited only to the 5'-UTR site, and no additional interaction was evident in the TNF-alpha gene up to 1,300 nt upstream of the transcription start site. Similarly, FRT exposure selectively reduced LPS-induced NFkappaB-p65 recruitment to the kappaB enhancer site at -510 without affecting the other three kappaB enhancer sites present in the TNF-alpha 5'-flanking sequence. Finally, we found that FRT exposure abrogated LPS-stimulated recruitment of Sp1 to the proximal TNF-alpha promoter without any change in associated histone H3 acetylation around the TNF-alpha promoter and despite a marked increase in the total intra-nuclear Sp1 DNA binding activity. In conclusion, our studies further emphasize the complex and redundant control of TNF-alpha transcription and identify additional potential mechanisms through which FRT exposure may reduce TNF-alpha expression by selectively modifying gene-specific recruitment of transcription factors to the proximal TNF-alpha promoter.