Myocardial SERCA2 Protects Against Cardiac Damage and Dysfunction Caused by Inhaled Bromine.

Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen-induced myocardial damage, we used bromine-exposed cardiac-specific SERCA2 knockout (KO) mice [tamoxifen-administered SERCA2 (flox/flox) Tg (αMHC-MerCreMer) mice] and compared them to the oil-administered controls. We performed echocardiography and hemodynamic analysis to investigate cardiac function 24 hours after bromine (600 ppm for 30 minutes) exposure and measured cardiac injury markers in plasma and proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac-specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine-exposed KO mice revealed significantly enhanced mean arterial pressure and sphericity index and decreased LV end diastolic diameter and LV end systolic pressure when compared with the bromine-exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures. SIGNIFICANCE STATEMENT: Due to their increased industrial production and transportation, halogens such as chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac-specific SERCA2 KO mice, these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.

A Murine Model of Vesicant-Induced Acute Lung Injury.

Burn injuries including those caused by chemicals can result in systemic effects and acute lung injury (ALI). Cutaneous exposure to Lewisite, a warfare and chemical burn agent, also causes ALI. To overcome the limitations in conducting direct research on Lewisite-induced ALI in a laboratory setting, an animal model was developed using phenylarsine oxide (PAO) as a surrogate for Lewisite. Due to lack of a reliable animal model mimicking the effects of such exposures, development of effective therapies to treat such injuries is challenging. We demonstrated that a single cutaneous exposure to PAO resulted in disruption of the alveolar-capillary barrier as evidenced by elevated protein levels in the bronchoalveolar lavage fluid (BALF). BALF supernatant of PAO-exposed animals had increased levels of high mobility group box 1, a damage associated molecular pattern molecule. Arterial blood-gas measurements showed decreased pH, increased PaCO2, and decreased partial pressure of arterial O2, indicative of respiratory acidosis, hypercapnia, and hypoxemia. Increased protein levels of interleukin (IL)-6, CXCL-1, CXCL-2, CXCL-5, granulocyte-macrophage colony-stimulating factor, CXCL-10, leukemia inhibitory factor, leptin, IL-18, CCL-2, CCL-3, and CCL-7 were observed in the lung of PAO-exposed mice. Further, vascular endothelial growth factor levels were reduced in the lung. Pulmonary function evaluated using a flexiVent showed a downward shift in the pressure-volume loop, decreases in static compliance and inspiratory capacity, increases in respiratory elastance and tissue elastance. These changes are consistent with an ALI phenotype. These results demonstrate that cutaneous PAO exposure leads to ALI and that the model can be used as an effective surrogate to investigate vesicant-induced ALI. SIGNIFICANCE STATEMENT: This study presents a robust model for studying ALI resulting from cutaneous exposure to PAO, a surrogate for the toxic vesicating agent Lewisite. The findings in this study mimic the effects of cutaneous Lewisite exposure, providing a reliable model for investigating mechanisms underlying toxicity. The model can also be used to develop medical countermeasures to mitigate ALI associated with cutaneous Lewisite exposure.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

Epigenetic underpinnings of inflammation: Connecting the dots between pulmonary diseases, lung cancer and COVID-19.

Inflammation is an essential component of several respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma and acute respiratory distress syndrome (ARDS). It is central to lung cancer, the leading cancer in terms of associated mortality that has affected millions of individuals worldwide. Inflammation and pulmonary manifestations are also the major causes of COVID-19 related deaths. Acute hyperinflammation plays an important role in the COVID-19 disease progression and severity, and development of protective immunity against the virus is greatly sought. Further, the severity of COVID-19 is greatly enhanced in lung cancer patients, probably due to the genes such as ACE2, TMPRSS2, PAI-1 and furin that are commonly involved in cancer progression as well as SAR-CoV-2 infection. The importance of inflammation in pulmonary manifestations, cancer and COVID-19 calls for a closer look at the underlying processes, particularly the associated increase in IL-6 and other cytokines, the dysregulation of immune cells and the coagulation pathway. Towards this end, several reports have identified epigenetic regulation of inflammation at different levels. Expression of several key inflammation-related cytokines, chemokines and other genes is affected by methylation and acetylation while non-coding RNAs, including microRNAs as well as long non-coding RNAs, also affect the overall inflammatory responses. Select miRNAs can regulate inflammation in COVID-19 infection, lung cancer as well as other inflammatory lung diseases, and can serve as epigenetic links that can be therapeutically targeted. Furthermore, epigenetic changes also mediate the environmental factors-induced inflammation. Therefore, a better understanding of epigenetic regulation of inflammation can potentially help develop novel strategies to prevent, diagnose and treat chronic pulmonary diseases, lung cancer and COVID-19.

Sex differences in cardiopulmonary effects of acute bromine exposure.

Accidental occupational bromine (Br>2>) exposures are common, leading to significant morbidity and mortality; however, the specific effects of Br>2> inhalation in female victims are unclear. Our studies demonstrated that acute high-concentration Br>2> inhalation is fatal, and cardiac injury and dysfunction play an important role in Br>2> toxicity in males. In this study, we exposed female Sprague Dawley rats, age-matched to those males from previously studied, to 600 ppm Br>2> for 45 min and assessed their survival, cardiopulmonary injury and cardiac function after exposure. Br>2> exposure caused serious mortality in female rats (59%) 48 h after exposure. Rats had severe clinical distress, reduced heart rates and oxygen saturation after Br>2> inhalation as was previously reported with male animals. There was significant lung injury and edema when measured 24 h after exposure. Cardiac injury biomarkers were also significantly elevated 24 h after Br>2> inhalation. Echocardiography and hemodynamic studies were also performed and revealed that the mean arterial pressure was not significantly elevated in females. Other functional cardiac parameters were also altered. Aside from the lack of elevation of blood pressure, all other changes observed in female animals were also present in male animals as reported in our previous study. These studies are important to understand the toxicity mechanisms to generate therapies and better-equip first responders to deal with these specific scenarios after bromine spill disasters.>.

Echocardiographic, Biochemical, and Electrocardiographic Correlates Associated With Progressive Pulmonary Arterial Hypertension.

Background: Pulmonary arterial hypertension (PAH) is a progressive proliferative vasculopathy associated with mechanical and electrical changes, culminating in increased vascular resistance, right ventricular (RV) failure, and death. With a main focus on invasive tools, there has been an underutilization of echocardiography, electrocardiography, and biomarkers to non-invasively assess the changes in myocardial and pulmonary vascular structure and function during the course of PAH. Methods: A SU5416-hypoxia rat model was used for inducing PAH. Biventricular functions were measured using transthoracic two-dimensional (2D) echocardiography/Doppler (echo/Doppler) at disease onset (0 week), during progression (3 weeks), and establishment (5 weeks). Similarly, electrocardiography was performed at 0, 3, and 5 weeks. Invasive hemodynamic measurements and markers of cardiac injury in plasma were assessed at 0, 3, and 5 weeks. Results: Increased RV systolic pressure (RVSP) and rate of isovolumic pressure rise and decline were observed at 0, 3, and 5 weeks in PAH animals. EKG showed a steady increase in QT-interval with progression of PAH, whereas P-wave height and RS width were increased only during the initial stages of PAH progression. Echocardiographic markers of PAH progression and severity were also identified. Three echocardiographic patterns were observed: a steady pattern (0-5 weeks) in which echo parameter changed progressively with severity [inferior vena cava (IVC) expiratory diameter and pulmonary artery acceleration time (PAAT)], an early pattern (0-3 weeks) where there is an early change in parameters [RV fractional area change (RV-FAC), transmitral flow, left ventricle (LV) output, estimated mean PA pressure, RV performance index, and LV systolic eccentricity index], and a late pattern (3-5 weeks) in which there is only a late rise at advanced stages of PAH (LV diastolic eccentricity index). RVSP correlated with PAAT, PAAT/PA ejection times, IVC diameters, RV-FAC, tricuspid systolic excursion, LV systolic eccentricity and output, and transmitral flow. Plasma myosin light chain (Myl-3) and cardiac troponin I (cTnI) increased progressively across the three time points. Cardiac troponin T (cTnT) and fatty acid-binding protein-3 (FABP-3) were significantly elevated only at the 5-week time point. Conclusion: Distinct electrocardiographic and echocardiographic patterns along with plasma biomarkers were identified as useful non-invasive tools for monitoring PAH progression.

Behavioral and Neuronal Effects of Inhaled Bromine Gas: Oxidative Brain Stem Damage.

The risk of accidental bromine (Br2) exposure to the public has increased due to its enhanced industrial use. Inhaled Br2 damages the lungs and the heart; however, adverse effects on the brain are unknown. In this study, we examined the neurological effects of inhaled Br2 in Sprague Dawley rats. Rats were exposed to Br2 (600 ppm for 45 min) and transferred to room air and cage behavior, and levels of glial fibrillary acidic protein (GFAP) in plasma were examined at various time intervals. Bromine exposure resulted in abnormal cage behavior such as head hitting, biting and aggression, hypervigilance, and hyperactivity. An increase in plasma GFAP and brain 4-hydroxynonenal (4-HNE) content also was observed in the exposed animals. Acute and delayed sympathetic nervous system activation was also evaluated by assessing the expression of catecholamine biosynthesizing enzymes, tryptophan hydroxylase (TrpH1 and TrpH2), and tyrosine hydroxylase (TyrH), along with an assessment of catecholamines and their metabolites. TyrH was found to be increased in a time-dependent manner. TrpH1 and TrpH2 were significantly decreased upon Br2 exposure in the brainstem. The neurotransmitter content evaluation indicated an increase in 5-HT and dopamine at early timepoints after exposure; however, other metabolites were not significantly altered. Taken together, our results predict brain damage and autonomic dysfunction upon Br2 exposure.

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats.

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

MicroRNA-mediated inflammation and coagulation effects in rats exposed to an inhaled analog of sulfur mustard.

Exposure of rats to 2-chloroethyl ethyl sulfide (CEES), an analog of sulfur mustard, can cause acute lung injury (ALI), resulting in increased inflammation and coagulation and altered levels of plasma microRNAs (miRNAs). Rats were exposed to aerosolized CEES and euthanized 12 h later for collection of tissue and plasma. Profiling of miRNAs in plasma, using a TaqMan-based RT-PCR array, revealed 14 differentially expressed miRNAs. Target gene prediction and pathway analysis revealed miRNA-mediated regulation of organismal injury, inflammation, and respiratory diseases. miR-140-5p, a marker of ALI, was downregulated in the plasma, lung, liver, and kidney of CEES-exposed rats, with a concomitant increase in the expression of the inflammation markers IL-6 and IL-1α and the coagulation marker tissue factor (F3). Exposure of rat airway epithelial cells (RL-65) to CEES (0.5 mM) caused cell death and a decrease in miR-140-5p both in cells and media supernatant. This was accompanied by an increase in cellular mRNA levels of IL-6, IL-1α, and F3, as well as FGF9 and EGR2, putative targets of miR-140. Knockdown of miR-140 by specific oligos in RL-65 cells mimicked the in vivo CEES-mediated effects, leading to significantly increased mRNA levels of IL-6, IL-1α, F3, FGF9, and EGR2. Our study identifies miR-140-5p as a mediator of CEES-induced ALI, which could potentially be targeted for therapy.

Cutaneous lewisite exposure causes acute lung injury.

Lewisite is a strong vesicating and chemical warfare agent. Because of the rapid transdermal absorption, cutaneous exposure to lewisite can also elicit severe systemic injury. Lewisite (2.5, 5.0, and 7.5 mg/kg) was applied to the skin of Ptch1+/- /SKH-1 mice and acute lung injury (ALI) was assessed after 24 hours. Arterial blood gas measurements showed hypercapnia and hypoxemia in the lewisite-exposed group. Histological evaluation of lung tissue revealed increased levels of proinflammatory neutrophils and a dose-dependent increase in structural changes indicative of injury. Increased inflammation was also confirmed by altered expression of cytokines, including increased IL-33, and a dose-dependent elevation of CXCL1, CXCL5, and GCSF was observed in the lung tissue. In the bronchoalveolar lavage fluid of lewisite-exposed animals, there was a significant increase in HMGB1, a damage-associated molecular pattern molecule, as well as elevated CXCL1 and CXCL5, which coincided with an influx of neutrophils to the lungs. Complete blood cell analysis revealed eosinophilia and altered neutrophil-lymphocyte ratios as a consequence of lewisite exposure. Mean platelet volume and RBC distribution width, which are predictors of lung injury, were also increased in the lewisite group. These data demonstrate that cutaneous lewisite exposure causes ALI and may contribute to mortality in exposed populations.

Extracellular nucleic acid scavenging rescues rats from sulfur mustard analog-induced lung injury and mortality.

Sulfur mustard (SM) is a highly toxic war chemical that causes significant morbidity and mortality and lacks any effective therapy. Rats exposed to aerosolized CEES (2-chloroethyl ethyl sulfide; 10% in ethanol), an analog of SM, developed acute respiratory distress syndrome (ARDS), which is characterized by increased inflammation, hypoxemia and impaired gas exchange. We observed elevated levels of extracellular nucleic acids (eNA) in the bronchoalveolar lavage fluid (BALF) of CEES-exposed animals. eNA can induce inflammation, coagulation and barrier dysfunction. Treatment with hexadimethrine bromide (HDMBr; 10 mg/kg), an eNA neutralizing agent, 2 h post-exposure, reduced lung injury, inhibited disruption of alveolar-capillary barrier, improved blood oxygenation (PaO2/FiO2 ratio), thus reversing ARDS symptoms. HDMBr treatment also reduced lung inflammation in the CEES-exposed animals by decreasing IL-6, IL-1A, CXCL-1 and CCL-2 mRNA levels in lung tissues and HMGB1 protein in BALF. Furthermore, HDMBr treatment also reduced levels of lung tissue factor and plasminogen activator inhibitor-1 indicating reduction in clot formation and increased fibrinolysis. Fibrin was reduced in BALF of the HDMBr-treated animals. This was further confirmed by histology that revealed diminished airway fibrin, epithelial sloughing and hyaline membrane in the lungs of HDMBr-treated animals. HDMBr completely rescued the CEES-associated mortality 12 h post-exposure when the survival rate in CEES-only group was just 50%. Experimental eNA treatment of cells caused increased inflammation that was reversed by HDMBr. These results demonstrate a role of eNA in the pathogenesis of CEES/SM-induced injury and that its neutralization can serve as a potential therapeutic approach in treating SM toxicity.

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.

Acute pulmonary effects of aerosolized nicotine.

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 µm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.

Mechanism of TH2/TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma.

BACKGROUND: The mechanism of TH2/TH17-predominant and TH2/TH17-low asthma is unknown. OBJECTIVE: We sought to study the immune mechanism of TH2/TH17-predominant and TH2/TH17-low asthma. METHODS: In a previously reported cohort of 60 asthmatic patients, 16 patients were immunophenotyped with TH2/TH17-predominant asthma and 22 patients with TH2/TH17-low asthma. We examined bronchoalveolar lavage (BAL) fluid leukocytes, cytokines, mediators, and epithelial cell function for these asthma subgroups. RESULTS: Patients with TH2/TH17-predominant asthma had increased IL-1ß, IL-6, IL-23, C3a, and serum amyloid A levels in BAL fluid, and these correlated with IL-1ß and C3a levels. TH2/TH17 cells expressed higher levels of the IL-1 receptor and phospho-p38 mitogen-activated protein kinase. Anakinra, an IL-1 receptor antagonist protein, inhibited BAL TH2/TH17 cell counts. TH2/TH17-low asthma had 2 distinct subgroups: neutrophilic asthma (45%) and pauci-inflammatory asthma (55%). This contrasted with patients with TH2/TH17-predominant and TH2-predominant asthma, which included neutrophilic asthma in 6% and 0% of patients, respectively. BAL fluid neutrophils strongly correlated with BAL fluid myeloperoxidase, IL-8, IL-1α, IL-6, granulocyte colony-stimulating factor, and GM-CSF levels. Sixty percent of the patients with neutrophilic asthma had a pathogenic microorganism in BAL culture, which suggested a subclinical infection. CONCLUSION: We uncovered a critical role for the IL-1ß pathway in patients with TH2/TH17-predminant asthma. A subgroup of patients with TH2/TH17-low asthma had neutrophilic asthma and increased BAL fluid IL-1α, IL-6, IL-8, granulocyte colony-stimulating factor, and GM-CSF levels. IL-1α was directly involved in IL-8 production and likely contributed to neutrophilic asthma. Sixty percent of neutrophilic patients had a subclinical infection.

Chlorine inhalation-induced myocardial depression and failure.

Victims of chlorine (Cl2) inhalation that die demonstrate significant cardiac pathology. However, a gap exists in the understanding of Cl2-induced cardiac dysfunction. This study was performed to characterize cardiac dysfunction occurring after Cl2 exposure in rats at concentrations mimicking accidental human exposures (in the range of 500 or 600 ppm for 30 min). Inhalation of 500 ppm Cl2 for 30 min resulted in increased lactate in the coronary sinus of the rats suggesting an increase in anaerobic metabolism by the heart. There was also an attenuation of myocardial contractile force in an ex vivo (Langendorff technique) retrograde perfused heart preparation. After 20 h of return to room air, Cl2 exposure at 500 ppm was associated with a reduction in systolic and diastolic blood pressure as well echocardiographic/Doppler evidence of significant left ventricular systolic and diastolic dysfunction. Cl2 exposure at 600 ppm (30 min) was associated with biventricular failure (observed at 2 h after exposure) and death. Cardiac mechanical dysfunction persisted despite increasing the inspired oxygen fraction concentration in Cl2-exposed rats (500 ppm) to ameliorate hypoxia that occurs after Cl2 inhalation. Similarly ex vivo cardiac mechanical dysfunction was reproduced by sole exposure to chloramine (a potential circulating Cl2 reactant product). These results suggest an independent and distinctive role of Cl2 (and its reactants) in inducing cardiac toxicity and potentially contributing to mortality.

Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33.

BACKGROUND: Asthma in a mouse model spontaneously resolves after cessation of allergen exposure. We developed a mouse model in which asthma features persisted for 6 months after cessation of allergen exposure. OBJECTIVE: We sought to elucidate factors contributing to the persistence of asthma. METHODS: We used a combination of immunologic, genetic, microarray, and pharmacologic approaches to dissect the mechanism of asthma persistence. RESULTS: Elimination of T cells though antibody-mediated depletion or lethal irradiation and transplantation of recombination-activating gene (Rag1)(-/-) bone marrow in mice with chronic asthma resulted in resolution of airway inflammation but not airway hyperreactivity or remodeling. Elimination of T cells and type 2 innate lymphoid cells (ILC2s) through lethal irradiation and transplantation of Rag2(-/-)γc(-/-) bone marrow or blockade of IL-33 resulted in resolution of airway inflammation and hyperreactivity. Persistence of asthma required multiple interconnected feedback and feed-forward circuits between ILC2s and epithelial cells. Epithelial IL-33 induced ILC2s, a rich source of IL-13. The latter directly induced epithelial IL-33, establishing a positive feedback circuit. IL-33 autoinduced, generating another feedback circuit. IL-13 upregulated IL-33 receptors and facilitated IL-33 autoinduction, thus establishing a feed-forward circuit. Elimination of any component of these circuits resulted in resolution of chronic asthma. In agreement with the foregoing, IL-33 and ILC2 levels were increased in the airways of asthmatic patients. IL-33 levels correlated with disease severity. CONCLUSIONS: We present a critical network of feedback and feed-forward interactions between epithelial cells and ILC2s involved in maintaining chronic asthma. Although T cells contributed to the severity of chronic asthma, they were redundant in maintaining airway hyperreactivity and remodeling.

An mTOR kinase inhibitor slows disease progression in a rat model of polycystic kidney disease.

BACKGROUND: The mTOR pathway, which consists of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), is activated in polycystic kidney disease (PKD) kidneys. Sirolimus and everolimus indirectly bind and inhibit mTORC1. A novel group of drugs, the mTOR kinase inhibitors, directly bind to mTOR kinase, thus inhibiting both mTORC1 and 2. The aim of the study was to determine the therapeutic effect of an mTOR kinase inhibitor, PP242, in the Han:SPRD rat (Cy/+) model of PKD. METHODS: Male rats were treated with PP242 5 mg/kg/day IP or vehicle for 5 weeks. RESULTS: PP242 significantly reduced the kidney enlargement, the cyst density and the blood urea nitrogen in Cy/+ rats. On immunoblot of kidneys, PP242 resulted in a decrease in pS6, a marker of mTORC1 signaling and pAkt(Ser473), a marker of mTORC2 signaling. mTORC plays an important role in regulating cytokine production. There was an increase in IL-1, IL-6, CXCL1 and TNF-α in Cy/+ rat kidneys that was unaffected by PP242. Apoptosis or proliferation is known to play a causal role in cyst growth. PP242 had no effect on caspase-3 activity, TUNEL positive or active caspase-3-positive tubular cells in Cy/+ kidneys. PP242 reduced the number of proliferating cells per cyst and per non-cystic tubule in Cy/+ rats. CONCLUSIONS: In a rat model of autosomal dominant polycystic kidney disease, PP242 treatment (i) decreases proliferation in cystic and non-cystic tubules; (ii) inhibits renal enlargement and cystogenesis and (iii) significantly reduces the loss of kidney function.

Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma.

BACKGROUND: TH2 cells can further differentiate into dual-positive TH2/TH17 cells. The presence of dual-positive TH2/TH17 cells in the airways and their effect on asthma severity are unknown. OBJECTIVE: We sought to study dual-positive TH2/TH17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients, examine their response to glucocorticoids, and define their relevance for disease severity. METHODS: Bronchoscopy and lavage were performed in 52 asthmatic patients and 25 disease control subjects. TH2 and TH2/TH17 cells were analyzed by using multicolor flow cytometry and confocal immunofluorescence microscopy. Cytokines were assayed by means of ELISA. RESULTS: Dual-positive TH2/TH17 cells were present at a higher frequency in BAL fluid from asthmatic patients compared with numbers seen in disease control subjects. High-level IL-4 production was typically accompanied by high-level IL-17 production and coexpression of GATA3 and retinoic acid receptor-related orphan receptor γt. Increased presence of TH2/TH17 cells was associated with increased IL-17 production in lavage fluid. TH2/TH17 cell counts and IL-17 production correlated with PC20 for methacholine, eosinophil counts, and FEV1. TH2/TH17 cells, unlike TH2 cells, were resistant to dexamethasone-induced cell death. They expressed higher levels of mitogen-activated protein-extracellular signal-regulated kinase kinase 1, a molecule that induces glucocorticoid resistance. On the basis of the dominance of BAL fluid TH2 or TH2/TH17 cells, we identified 3 subgroups of asthma: TH2(predominant), TH2/TH17(predominant), and TH2/TH17(low). The TH2/TH17(predominant) subgroup manifested the most severe form of asthma, whereas the TH2/TH17(low) subgroup had the mildest asthma. CONCLUSION: Asthma is associated with a higher frequency of dual-positive TH2/TH17 cells in BAL fluid. The TH2/TH17(predominant) subgroup of asthmatic patients manifested glucocorticoid resistance in vitro. They also had the greatest airway obstruction and hyperreactivity compared with the TH2(predominant) and TH2/TH17(low) subgroups.

An mTOR anti-sense oligonucleotide decreases polycystic kidney disease in mice with a targeted mutation in Pkd2.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disease in the USA. In human ADPKD studies, sirolimus, a mammalian target of rapamycin complex 1 (mTORC1) inhibitor, had little therapeutic effect. While sirolimus robustly inhibits mTORC1, it has a minimal effect on mTOR complex 2 (mTORC2). Polycystic kidneys of Pkd2WS25/- mice, an orthologous model of human ADPKD caused by a mutation in the Pkd2 gene, had an early increase in pS6 (marker of mTORC1) and pAktSer(473) (marker of mTORC2). To investigate the effect of combined mTORC1 and 2 inhibition, Pkd2WS25/- mice were treated with an mTOR anti-sense oligonucleotide (ASO) that blocks mTOR expression thus inhibiting both mTORC1 and 2. The mTOR ASO resulted in a significant decrease in mTOR protein, pS6 and pAktSer(473). Pkd2WS25/- mice treated with the ASO had a normalization of kidney weights and kidney function and a marked decrease in cyst volume. The mTOR ASO resulted in a significant decrease in proliferation and apoptosis of tubular epithelial cells. To demonstrate the role of mTORC2 on cyst growth, Rictor, the functional component of mTORC2, was silenced in Madin-Darby canine kidney cell cysts grown in 3D cultures. Silencing Rictor significantly decreased cyst volume and expression of pAktSer(473). The decreased cyst size in the Rictor silenced cells was reversed by introduction of a constitutively active Akt1. In vitro, combined mTORC1 and 2 inhibition reduced cyst growth more than inhibition of mTORC1 or 2 alone. In conclusion, combined mTORC1 and 2 inhibition has therapeutic potential in ADPKD.

Effects of lovastatin treatment on the metabolic distributions in the Han:SPRD rat model of polycystic kidney disease.

BACKGROUND: We previously demonstrated that lovastatin decreases cyst volume and improves kidney function in the Han:SPRD (Cy/+) rat model of ADPKD. Since endothelial dysfunction and inflammatory activity are evident in patients with ADPKD, we investigated whether lovastatin reduces the inflammation and vascular dysfunction and improves kidney cell energy metabolism of Cy/+ rats. METHODS: Cy/+ and normal littermate control animals (+/+) were treated with either lovastatin (4 mg/kg/day) or vehicle (ethanol) from 3-8 weeks of age. 1H-NMR analysis was performed on water-soluble and lipid kidney fractions following perchloric acid extraction. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess endothelial dysfunction, oxidative stress and inflammation markers in plasma and kidney tissue extracts. RESULTS: Cy/+ rats showed perturbations in fatty acid metabolism and increased synthesis of pro-inflammatory lipoxygenases-produced bioactive lipids was observed. Lovastatin decreased inflammatory markers, specifically 13-HODE, 12-HETE and leukotriene B4. In Cy/+ rats, lovastatin reduced the elevated homocysteine and allantoin plasma levels and increased arginine, that is known to positively affect NO production. CONCLUSION: As previously described, lovastatin was able to decrease kidney weight and cyst volume density in Cy/+ rats. The decrease in cyst volume was accompanied by a reduction in arachidonic acid-mediated inflammation markers, the normalization of metabolism of NO precursors and the improvement of kidney energy cell metabolism.