TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching.

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.

A temperature-dependent conformational shift in p38α MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38ß is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-α (TNFα)-induced p38 activation. In in vitro kinase assays containing activated p38α and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1α (STAT1α) and a 7.7-fold difference for p38ß phosphorylation of MK2. The temperature dependence of p38α:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38α performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an α helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38ß did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38α-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38α may contribute to the temperature dependence of acute lung injury.

An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13.

BACKGROUND: Pulmonary fibrosis is one of the leading indications for lung transplantation. The disease, which is of unknown aetiology, can be progressive, resulting in distortion of the extracellular matrix (ECM), inflammation, fibrosis and eventual death. METHODS: 13 patients born to consanguineous parents from two unrelated families presenting with interstitial lung disease were clinically investigated. Nine patients developed respiratory failure and subsequently died. Molecular genetic investigations were performed on patients' whole blood or archived tissues, and cell biological investigations were performed on patient-derived fibroblasts. RESULTS: The combination of a unique pattern of early-onset lung fibrosis (at 12-15 years old) with distinctive radiological findings, including 1) traction bronchiectasis, 2) intralobular septal thickening, 3) shrinkage of the secondary pulmonary lobules mainly around the bronchovascular bundles and 4) early type 2 respiratory failure (elevated blood carbon dioxide levels), represents a novel clinical subtype of familial pulmonary fibrosis. Molecular genetic investigation of families revealed a hypomorphic variant in S100A3 and a novel truncating mutation in S100A13, both segregating with the disease in an autosomal recessive manner. Family members that were either heterozygous carriers or wild-type normal for both variants were unaffected. Analysis of patient-derived fibroblasts demonstrated significantly reduced S100A3 and S100A13 expression. Further analysis demonstrated aberrant intracellular calcium homeostasis, mitochondrial dysregulation and differential expression of ECM components. CONCLUSION: Our data demonstrate that digenic inheritance of mutations in S100A3 and S100A13 underlie the pathophysiology of pulmonary fibrosis associated with a significant reduction of both proteins, which suggests a calcium-dependent therapeutic approach for management of the disease.

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.

Transcriptomic evidence of immune activation in macroscopically normal-appearing and scarred lung tissues in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease manifested by overtly scarred peripheral and basilar regions and more normal-appearing central lung areas. Lung tissues from macroscopically normal-appearing (IPFn) and scarred (IPFs) areas of explanted IPF lungs were analyzed by RNASeq and compared with healthy control (HC) lung tissues. There were profound transcriptomic changes in IPFn compared with HC tissues, which included elevated expression of numerous immune-, inflammation-, and extracellular matrix-related mRNAs, and these changes were similar to those observed with IPFs compared to HC. Comparing IPFn directly to IPFs, elevated expression of epithelial mucociliary mRNAs was observed in the IPFs tissues. Thus, despite the known geographic tissue heterogeneity in IPF, the entire lung is actively involved in the disease process, and demonstrates pronounced elevated expression of numerous immune-related genes. Differences between normal-appearing and scarred tissues may thus be driven by deranged epithelial homeostasis or possibly non-transcriptomic factors.

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).

Pilot Feasibility Study of Therapeutic Hypothermia for Moderate to Severe Acute Respiratory Distress Syndrome.

OBJECTIVES: Prior studies suggest hypothermia may be beneficial in acute respiratory distress syndrome, but cooling causes shivering and increases metabolism. The objective of this study was to assess the feasibility of performing a randomized clinical trial of hypothermia in patients with acute respiratory distress syndrome receiving treatment with neuromuscular blockade because they cannot shiver. DESIGN: Retrospective study and pilot, prospective, open-label, feasibility study. SETTING: Medical ICU. PATIENTS: Retrospective review of 58 patients with acute respiratory distress syndrome based on Berlin criteria and PaO2/FIO2 less than 150 who received neuromuscular blockade. Prospective hypothermia treatment in eight acute respiratory distress syndrome patients with PaO2/FIO2 less than 150 receiving neuromuscular blockade. INTERVENTION: Cooling to 34-36°C for 48 hours. MEASUREMENTS AND MAIN RESULTS: Core temperature, hemodynamics, serum glucose and electrolytes, and P/F were sequentially measured, and medians (interquartile ranges) presented, 28-day ventilator-free days, and hospital mortality were calculated in historical controls and eight cooled patients. Average patient core temperature was 36.7°C (36-37.3°C), and fever occurred during neuromuscular blockade in 30 of 58 retrospective patients. In the prospectively cooled patients, core temperature reached target range less than or equal to 4 hours of initiating cooling, remained less than 36°C for 92% of the 48 hours cooling period without adverse events, and was lower than the controls (34.35°C [34-34.8°C]; p < 0.0001). Compared with historical controls, the cooled patients tended to have lower hospital mortality (75% vs 53.4%; p = 0.26), more ventilator-free days (9 [0-21.5] vs 0 [0-12]; p = 0.16), and higher day 3 P/F (255 [160-270] vs 171 [120-214]; p = 0.024). CONCLUSIONS: Neuromuscular blockade alone does not cause hypothermia but allowed acute respiratory distress syndrome patients to be effectively cooled. Results support conducting a randomized clinical trial of hypothermia in acute respiratory distress syndrome and the feasibility of studying acute respiratory distress syndrome patients receiving neuromuscular blockade.

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.

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.

Myofiber damage precedes macrophage infiltration after in vivo injury in dysferlin-deficient A/J mouse skeletal muscle.

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage.

IFN-γ directly controls IL-33 protein level through a STAT1- and LMP2-dependent mechanism.

IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion.

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.

Interleukin-33 potentiates bleomycin-induced lung injury.

The mechanisms of interstitial lung disease (ILD) remain incompletely understood, although recent observations have suggested an important contribution by IL-33. Substantial elevations in IL-33 expression were found in the lungs of patients with idiopathic pulmonary fibrosis and scleroderma lung disease, as well as in the bleomycin injury mouse model. Most of the observed IL-33 expression was intracellular and intranuclear, suggesting involvement of the full-length (fl) protein, but not of the proteolytically processed mature IL-33 cytokine. The effects of flIL-33 on mouse lungs were assessed independently and in combination with bleomycin injury, using recombinant adenovirus-mediated gene delivery. Bleomycin-induced changes were not affected by gene deficiency of the IL-33 receptor T1/ST2. Combined flIL-33 expression and bleomycin injury exerted a synergistic effect on pulmonary lymphocyte and collagen accumulation, which could be explained by synergistic regulation of the cytokines transforming growth factor-ß, IL-6, monocyte chemotactic protein-1, macrophage inflammatory protein\x{2013}1α, and tumor necrosis factor-α. By contrast, no increase in the levels of the Th2 cytokines IL-4, IL-5, or IL-13 was evident. Moreover, flIL-33 was found to increase the expression of several heat shock proteins (HSPs) significantly, and in particular HSP70, which is known to be associated with ILD. Thus, flIL-33 is a synergistic proinflammatory and profibrotic regulator that acts by stimulating the expression of several non-Th2 cytokines, and activates the expression of HSP70.

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.

Management of two circulations in a COVID-19 patient with secondary superinfection.

Optimal oxygenation in the intensive care unit requires adequate pulmonary gas exchange, oxygen-carrying capacity in the form of hemoglobin, sufficient delivery of oxygenated hemoglobin to the tissue, and an appropriate tissue oxygen demand. In this Case Study in Physiology, we describe a patient with COVID-19 whose pulmonary gas exchange and oxygen delivery were severely compromised by COVID-19 pneumonia requiring extracorporeal membrane oxygenation (ECMO) support. His clinical course was complicated by a secondary superinfection with staphylococcus aureus and sepsis. This case study is provided with two goals in mind (1) We outline how basic physiology was used to address life-threatening consequences of a novel infection-COVID-19. (2) We describe a strategy of whole-body cooling to lower the cardiac output and oxygen consumption, use of the shunt equation to optimize flow to the ECMO circuit, and transfusion to improve oxygen-carrying capacity when ECMO alone failed to provide sufficient oxygenation.

Design and rationale of the CHILL phase II trial of hypothermia and neuromuscular blockade for acute respiratory distress syndrome.

The Cooling to Help Injured Lungs (CHILL) trial is an open label, two group, parallel design multicenter, randomized phase IIB clinical trial assessing the efficacy and safety of targeted temperature management with combined external cooling and neuromuscular blockade to block shivering in patients with early moderate-severe acute respiratory distress syndrome (ARDS). This report provides the background and rationale for the clinical trial and outlines the methods using the Consolidated Standards of Reporting Trials guidelines. Key design challenges include: [1] protocolizing important co-interventions; [2] incorporation of patients with COVID-19 as the cause of ARDS; [3] inability to blind the investigators; and [4] ability to obtain timely informed consent from patients or legally authorized representatives early in the disease process. Results of the Reevaluation of Systemic Early Neuromuscular Blockade (ROSE) trial informed the decision to mandate sedation and neuromuscular blockade only in the group assigned to therapeutic hypothermia and proceed without this mandate in the control group assigned to a usual temperature management protocol. Previous trials conducted in National Heart, Lung, and Blood Institute ARDS Clinical Trials (ARDSNet) and Prevention and Early Treatment of Acute Lung Injury (PETAL) Networks informed ventilator management, ventilation liberation and fluid management protocols. Since ARDS due to COVID-19 is a common cause of ARDS during pandemic surges and shares many features with ARDS from other causes, patients with ARDS due to COVID-19 are included. Finally, a stepwise approach to obtaining informed consent prior to documenting critical hypoxemia was adopted to facilitate enrollment and reduce the number of candidates excluded because eligibility time window expiration.

Wild-type S100A3 and S100A13 restore calcium homeostasis and mitigate mitochondrial dysregulation in pulmonary fibrosis patient-derived cells.

Patients with digenic S100A3 and S100A13 mutations exhibited an atypical and progressive interstitial pulmonary fibrosis, with impaired intracellular calcium homeostasis and mitochondrial dysfunction. Here we provide direct evidence of a causative effect of the mutation on receptor mediated calcium signaling and calcium store responses in control cells transfected with mutant S100A3 and mutant S100A13. We demonstrate that the mutations lead to increased mitochondrial mass and hyperpolarization, both of which were reversed by transfecting patient-derived cells with the wild type S100A3 and S100A13, or extracellular treatment with the recombinant proteins. In addition, we demonstrate increased secretion of inflammatory mediators in patient-derived cells and in control cells transfected with the mutant-encoding constructs. These findings indicate that treatment of patients' cells with recombinant S100A3 and S100A13 proteins is sufficient to normalize most of cellular responses, and may therefore suggest the use of these recombinant proteins in the treatment of this devastating disease.

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.

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.