Impaired Tumor-Necrosis-Factor-α-driven Dendritic Cell Activation Limits Lipopolysaccharide-Induced Protection from Allergic Inflammation in Infants.

Infants have a higher risk of developing allergic asthma than adults. However, the underlying mechanism remains unknown. We show here that sensitization of mice with house-dust mites (HDMs) in the presence of low-dose lipopolysaccharide (LPS) prevented T helper 2 (Th2) cell allergic responses in adult, but not infant, mice. Mechanistically, adult CD11b+ migratory dendritic cells (mDCs) upregulated the transcription factor T-bet in response to tumor necrosis factor-α (TNF-α), which was rapidly induced after HDM + LPS sensitization. Consequently, adult CD11b+ mDCs produced interleukin-12 (IL-12), which prevented Th2 cell development by promoting T-bet upregulation in responding T cells. Conversely, infants failed to induce TNF-α after HDM + LPS sensitization. Therefore, CD11b+ mDCs failed to upregulate T-bet and did not secrete IL-12 and Th2 cell responses normally developed in infant mice. Thus, the availability of TNF-α dictates the ability of CD11b+ mDCs to suppress allergic Th2-cell responses upon dose-dependent endotoxin sensitization and is a key mediator governing susceptibility to allergic airway inflammation in infant mice.

Elevated Plasmin(ogen) as a Common Risk Factor for COVID-19 Susceptibility.

Patients with hypertension, diabetes, coronary heart disease, cerebrovascular illness, chronic obstructive pulmonary disease, and kidney dysfunction have worse clinical outcomes when infected with SARS-CoV-2, for unknown reasons. The purpose of this review is to summarize the evidence for the existence of elevated plasmin(ogen) in COVID-19 patients with these comorbid conditions. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-2, extracellularly, which increases its infectivity and virulence. Hyperfibrinolysis associated with plasmin leads to elevated D-dimer in severe patients. The plasmin(ogen) system may prove a promising therapeutic target for combating COVID-19.

Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

The SARS-CoV-2 spike S1 protein induces global proteomic changes in ATII-like rat L2 cells that are attenuated by hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants, COVID-19 has led to 651,918,402 confirmed cases and 6,656,601 deaths worldwide (as of December 27, 2022; https://covid19.who.int/). Despite advances in our understanding of COVID-19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in the proteome of alveolar type II (ATII)-like rat L2 cells that lack ACE2 receptors. Systems biology analysis revealed that the S1-induced proteomics changes were associated with three significant network hubs: E2F1, CREB1/RelA, and ROCK2/RhoA. We also found that pretreatment of L2 cells with high molecular weight hyaluronan (HMW-HA) greatly attenuated the S1 effects on the proteome. Western blotting analysis and cell cycle measurements confirmed the S1 upregulation of E2F1 and ROCK2/RhoA in L2 cells and the protective effects of HMW-HA. Taken as a whole, our studies revealed profound and novel biological changes that contribute to our current understanding of both S1 and hyaluronan biology. These data show that the S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV-2-induced cell injury.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Halogen-Induced Chemical Injury to the Mammalian Cardiopulmonary Systems.

The halogens chlorine (Cl2) and bromine (Br2) are highly reactive oxidizing elements with widespread industrial applications and a history of development and use as chemical weapons. When inhaled, depending on the dose and duration of exposure, they cause acute and chronic injury to both the lungs and systemic organs that may result in the development of chronic changes (such as fibrosis) and death from cardiopulmonary failure. A number of conditions, such as viral infections, coexposure to other toxic gases, and pregnancy increase susceptibility to halogens significantly. Herein we review their danger to public health, their mechanisms of action, and the development of pharmacological agents that when administered post-exposure decrease morbidity and mortality.

The chemokine CX3CL1/fractalkine regulates immunopathogenesis during fungal-associated allergic airway inflammation.

Individuals that present with difficult-to-control asthma and sensitivity to one or more fungal species are categorized as a subset of severe asthma patients belonging to a group herein referred to as severe asthma with fungal sensitization (SAFS). We have previously reported the identification of numerous cytokines and chemokines that were elevated in human asthmatics that were sensitized to fungi vs. nonfungal sensitized asthmatics. Here, we show that the unique chemokine CX3CL1 (fractalkine) is elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with CX3CL1 in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that the absence of CX3CR1 signaling unexpectedly resulted in a profound impairment in lung function. Histological assessment of lung tissue revealed an unrestricted inflammatory response that was subsequently characterized by enhanced levels of neutrophils, eosinophils, and inflammatory monocytes. Neutrophilic inflammation correlated with elevated IL-17A, proinflammatory cytokines (TNF-α, IL-1α, and IL-1ß), neutrophil survival factors (granulocyte colony-stimulating factor), and neutrophil-targeting chemokines (CCL3 and CCL4). Eosinophilia correlated with elevated type 2 responses (IL-5 and IL-13) whereas inflammatory monocyte levels correlated with elevated type 1 responses (IFN-γ and CXCL9) and survival factors (macrophage colony-stimulating factor). Despite enhanced inflammatory responses, the immunoregulatory cytokine IL-10 and the natural inhibitor of IL-1 signaling, IL-1RA, were significantly elevated rather than impaired. Regulatory T-cell levels were unchanged, as were levels of the anti-inflammatory cytokines IL-35 and IL-38. Taken together, the CX3CL1/CX3CR1 axis preserves lung function during fungal-associated allergic airway inflammation through a nonclassical immunoregulatory mechanism.

AICAR decreases acute lung injury by phosphorylating AMPK and upregulating heme oxygenase-1.

AIM: We investigated the mechanisms by which N1-(ß-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase (AMPK), decreases lung injury and mortality when administered to mice post exposure to bromine gas (Br2). METHODS: We exposed male C57BL/6 mice and heme oxygenase-1 (HO-1)-deficient (HO-1-/-) and corresponding wild-type (WT) littermate mice to Br2 (600 ppm for 45 or 30 min, respectively) in environmental chambers and returned them to room air. AICAR was administered 6 h post exposure (10 mg·kg-1, intraperitoneal). We assessed survival, indices of lung injury, high mobility group box 1 (HMGB1) in the plasma, HO-1 levels in lung tissues and phosphorylation of AMPK and its upstream liver kinase B1 (LKB1). Rat alveolar type II epithelial (L2) cells and human club-like epithelial (H441) cells were also exposed to Br2 (100 ppm for 10 min). After 24 h we measured apoptosis and necrosis, AMPK and LKB1 phosphorylation, and HO-1 expression. RESULTS: There was a marked downregulation of phosphorylated AMPK and LKB1 in lung tissues and in L2 and H441 cells post exposure. AICAR increased survival in C57BL/6 but not in HO-1-/- mice. In WT mice, AICAR decreased lung injury and restored phosphorylated AMPK and phosphorylated LKB1 to control levels and increased HO-1 levels in both lung tissues and cells exposed to Br2. Treatment of L2 and H441 cells with small interfering RNAs against nuclear factor erythroid 2-related factor 2 or HO-1 abrogated the protective effects of AICAR. CONCLUSIONS: Our data indicate that the primary mechanism for the protective action of AICAR in toxic gas injury is the upregulation of lung HO-1 levels.

Halogen gas exposure: toxic effects on the parturient.

The elemental halogens include chlorine, bromine, and phosgene. Halogen gas can be directly weaponized and employed in warfare or terrorism. Industrial stockpiles or halogen transport can provide targets for terrorist attack as well as an origin for accidental release creating a risk for potential mass-casualty incidents. Pregnant and post-partum women represent a substantial and vulnerable subset of the population who may be at particular risk during an attack or accidental exposure. We review the effects of halogen exposure on the parturient with a focus on bromine toxicity. Bromine is the most extensively studied agent in the context of pregnancy and to-date murine models form the basis for the majority of current knowledge. Pregnancy potentiates the acute lung injury after halogen exposure. In addition, halogen exposure precipitates a preeclamptic-like syndrome in mice. This phenotype is characterized by systemic and pulmonary hypertension, endothelial dysfunction, decreased cardiac output, placental injury and fetal growth restriction. This constellation contributes to increased maternal and fetal mortality observed after bromine exposure. Angiogenic imbalance is noted with overexpression of the soluble fms-like tyrosine kinase-1 (sFlt-1) form of the vascular endothelial growth factor receptor 1 reminiscent of human preeclampsia. Additional research is needed to further explore the effect of halogen gas exposure in pregnancy and to develop therapeutic interventions to mitigate risk to this unique population.

SARS-CoV-2 may regulate cellular responses through depletion of specific host miRNAs.

Cold viruses have generally been considered fairly innocuous until the appearance of the severe acute respiratory coronavirus 2 (SARS-CoV-2) in 2019, which caused the coronavirus disease 2019 (COVID-19) global pandemic. Two previous viruses foreshadowed that a coronavirus could potentially have devastating consequences in 2002 [severe acute respiratory coronavirus (SARS-CoV)] and in 2012 [Middle East respiratory syndrome coronavirus (MERS-CoV)]. The question that arises is why these viruses are so different from the relatively harmless cold viruses. On the basis of an analysis of the current literature and using bioinformatic approaches, we examined the potential human miRNA interactions with the SARS-CoV-2's genome and compared the miRNA target sites in seven coronavirus genomes that include SARS-CoV-2, MERS-CoV, SARS-CoV, and four nonpathogenic coronaviruses. Here, we discuss the possibility that pathogenic human coronaviruses, including SARS-CoV-2, could modulate host miRNA levels by acting as miRNA sponges to facilitate viral replication and/or to avoid immune responses.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Role of fibroblast growth factor 23 and klotho cross talk in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia that mainly affects the elderly. Several reports have demonstrated that aging is involved in the underlying pathogenic mechanisms of IPF. α-Klotho (KL) has been well characterized as an "age-suppressing" hormone and can provide protection against cellular senescence and oxidative stress. In this study, KL levels were assessed in human plasma and primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF-FB) and in lung tissue from mice exposed to bleomycin, which showed significant downregulation when compared with controls. Conversely, transgenic mice overexpressing KL were protected against bleomycin-induced lung fibrosis. Treatment of human lung fibroblasts with recombinant KL alone was not sufficient to inhibit transforming growth factor-ß (TGF-ß)-induced collagen deposition and inflammatory marker expression. Interestingly, fibroblast growth factor 23 (FGF23), a proinflammatory circulating protein for which KL is a coreceptor, was upregulated in IPF and bleomycin lungs. To our surprise, FGF23 and KL coadministration led to a significant reduction in fibrosis and inflammation in IPF-FB; FGF23 administration alone or in combination with KL stimulated KL upregulation. We conclude that in IPF downregulation of KL may contribute to fibrosis and inflammation and FGF23 may act as a compensatory antifibrotic and anti-inflammatory mediator via inhibition of TGF-ß signaling. Upon restoration of KL levels, the combination of FGF23 and KL leads to resolution of inflammation and fibrosis. Altogether, these data provide novel insight into the FGF23/KL axis and its antifibrotic/anti-inflammatory properties, which opens new avenues for potential therapies in aging-related diseases like IPF.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein.

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

Acidic Mammalian Chitinase Negatively Affects Immune Responses during Acute and Chronic Aspergillus fumigatus Exposure.

Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) (Chia) deficiency on host defense during acute exposure to the fungal pathogen Aspergillus fumigatus as well as its contribution to A. fumigatus-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in A. fumigatus conidia, which emerged at the highest level during hyphal transition. In response to acute A. fumigatus challenge, Chia-/- mice unexpectedly demonstrated lower A. fumigatus lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1ß and prostaglandin E2 (PGE2) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic A. fumigatus exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.