Airway epithelial injury is the hallmark of various respiratory diseases, but its mechanisms remain poorly understood. While 13-S-hydroxyoctadecadienoic acid (13-S-HODE) is produced in high concentration during mitochondrial degradation in reticulocytes little is known about its role in asthma pathogenesis. Here, we show that extracellular 13-S-HODE induces mitochondrial dysfunction and airway epithelial apoptosis. This is associated with features of severe airway obstruction, lung remodeling, increase in epithelial stress related proinflammatory cytokines and drastic airway neutrophilia in mouse. Further, 13-S-HODE induced features are attenuated by inhibiting Transient Receptor Potential Cation Channel, Vanilloid-type 1 (TRPV1) both in mouse model and human bronchial epithelial cells. These findings are relevant to human asthma, as 13-S-HODE levels are increased in human asthmatic airways. Blocking of 13-S-HODE activity or disruption of TRPV1 activity attenuated airway injury and asthma mimicking features in murine allergic airway inflammation. These findings indicate that 13-S-HODE induces mitochondrial dysfunction and airway epithelial injury.

Discovery of exosomes as modulator of cellular communication has added a new dimension to our understanding of biological processes. Exosomes influence the biological systems by mediating trans-communication across tissues and cells, which has important implication for health and disease. In absence of well-characterized modulators of exosome biogenesis, an alternative option is to target pathways generating important exosomal components. Cholesterol represents one such essential component required for exosomal biogenesis. We initiated this study to test the hypothesis that owing to its cholesterol lowering effect, simvastatin, a HMG CoA inhibitor, might be able to alter exosome formation and secretion. Simvastatin was tested for its effect on exosome secretion under various in-vitro and in-vivo settings and was found to reduce the secretion of exosome from various cell-types. It was also found to alter the levels of various proteins important for exosome production. Murine model of Acute Airway Inflammation was used for further validation of our findings. We believe that the knowledge acquired in this study holds potential for extension to other exosome dominated pathologies and model systems.

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Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the SARS-CoV-2 spike protein is an envelope glycoprotein that binds angiotensin converting enzyme 2 as an entry receptor. The capacity of enveloped viruses to infect host cells depends on a precise thiol/disulfide balance in their surface glycoprotein complexes. To determine if cystines in the SARS-CoV-2 spike protein maintain a native binding interface that can be disrupted by drugs that cleave cystines, we tested if thiol-based drugs have efficacy in receptor binding and cell infection assays. We found that thiol-based drugs, cysteamine and WR-1065 (the active metabolite of amifostine) in particular, decrease binding of SARS-CoV-2 spike protein to its receptor, decrease the entry efficiency of SARS-CoV-2 spike pseudotyped virus, and inhibit SARS-CoV-2 live virus infection. Our findings uncover a vulnerability of SARS-CoV-2 to thiol-based drugs and provide rationale to test thiol-based drugs, especially cysteamine and amifostine, as novel treatments for COVID-19.One Sentence SummaryThiol-based drugs decrease binding of SARS-CoV-2 spike protein to its receptor and inhibit SARS-CoV-2 cell entry.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for binding of SARS-2-S to ACE2, suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally (IP) to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with IP delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

The asthma candidate gene inositol polyphosphate 4-phosphatase type I A (INPP4A) is a lipid phosphatase that negatively regulates the PI3K/Akt pathway. Destabilizing genetic variants of INPP4A increase the risk of asthma, and lung-specific INPP4A knockdown induces asthma-like features. INPP4A is known to localize intracellularly, and its extracellular presence has not been reported yet. Here we show for the first time that INPP4A is secreted by airway epithelial cells and that extracellular INPP4A critically inhibits airway inflammation and remodeling. INPP4A was present in blood and BAL fluid, and this extracellular INPP4A was reduced in patients with asthma and mice with allergic airway inflammation. In both naive mice and mice with allergic airway inflammation, antibody-mediated neutralization of extracellular INPP4A potentiated PI3K/Akt signaling and induced airway hyperresponsiveness, with prominent airway remodeling, subepithelial fibroblast proliferation, and collagen deposition. The link between extracellular INPP4A and fibroblasts was investigated in vitro. Cultured airway epithelial cells secreted enzymatically active INPP4A in extracellular vesicles and in a free form. Extracellular vesicle-mediated transfer of labeled INPP4A, from epithelial cells to fibroblasts, was observed. Inhibition of such transfer by anti-INPP4A antibody increased fibroblast proliferation. We propose that secretory INPP4A is a novel "paracrine" layer of the intricate regulation of lung homeostasis, by which airway epithelium dampens PI3K/Akt signaling in inflammatory cells or local fibroblasts, thereby limiting inflammation and remodeling.

Severe asthma is chronic airway disease, exhibit poor response to conventional asthma therapies.Growing evidence suggests that elevated hypoxia increases the severity of asthmatic inflammation among patient and model systems. In this study, we elucidate the therapeutic effect and mechanistic basis of Adhatoda Vasica (AV) aqueous extract on acute allergic as well as severe asthma subtypes, at physiological, histopathological and molecular levels using mouse models.We observed, oral administration of AV extract not only attenuates the increased airway resistance and inflammation in acute allergic asthmatic mice but also alleviates the molecular signatures of steroid (dexamethasone) resistance like IL-17A, KC and HIF-1 (hypoxia inducible factor-1alpha) in severe asthmatic mice. The reversal of pathophysiological features after AV treatment is associated with inhibition of elevated HIF-1 levels by restoring the expression of its negative regulator-PHD2 (prolyl hydroxylase domain-2). This was further confirmed in acute and severe asthma model developed by augmented hypoxic response. Further, AV treatment reverses cellular hypoxia-induced mitochondrial dysfunction in human bronchial epithelial cells -evident from bioenergetic profiles and morphological analysis of mitochondria. Involvement of hypoxia and mitochondrial dysfunction in asthma severity is being increasingly realised. Extract of AV although widely used in Ayurveda practice for the treatment of diverse respiratory ailments, including asthma, its molecular basis of action and effect on severe asthma subtype is still unclear. This study, demonstrates therapeutic mechanism of Adhatoda Vasica through hypoxia-induced mitochondrial dysfunction and highlights its potential in the treatment of severe steroid-resistant asthma. Significance StatementSevere asthma is a global health concern found to be unresponsive to current treatment modalities involving corticosteroids. Recent findings suggest that elevated hypoxia has a critical role in severity of asthma. Here, we report therapeutic treatment with aqueous extract of Adhatoda Vasica (AV), an ayurvedic medicine, is effective in attenuation of severe steroid-insensitive asthmatic features in mice. The observed anti-asthmatic effects of AV was through inhibition of hypoxic response, both in vivo and in vitro. AV also reverses mitochondrial dysfunction, a key consequence associated with hypoxia, and asthma. This study not only highlights the translational potential of AV for the treatment of severe asthma but also provides opportunities for its usage in other disease conditions where hypoxia is pertinent. 250,000 annual deaths, asthma significantly contributes to the global health burden (3). Initially, it was considered to be primarily an allergic, eosinophilic Th2 biased disease with involvement of its cytokines (IL-4, IL-5 and IL-13) in the inflammatory cascade leading to allergic exacerbations (4).However, there are non-Th2 asthma patho-phenotypes, where cells like Th17, Th1 and their respective cytokines play a ...

Severe asthma is a chronic airway disease that exhibits poor response to conventional asthma therapies. Growing evidence suggests that elevated hypoxia increases the severity of asthmatic inflammation among patients and in model systems. In this study, we elucidate the therapeutic effects and mechanistic basis of Adhatoda Vasica (AV) aqueous extract on mouse models of acute allergic as well as severe asthma subtypes at physiological, histopathological, and molecular levels. Oral administration of AV extract attenuates the increased airway resistance and inflammation in acute allergic asthmatic mice and alleviates the molecular signatures of steroid (dexamethasone) resistance like IL-17A, KC, and HIF-1α (hypoxia inducible factor-1alpha) in severe asthmatic mice. AV inhibits HIF-1α levels through restoration of expression of its negative regulator-PHD2 (prolyl hydroxylase domain-2). Alleviation of hypoxic response mediated by AV is further confirmed in the acute and severe asthma model. AV reverses cellular hypoxia-induced mitochondrial dysfunction in human bronchial epithelial cells - evident from bioenergetic profiles and morphological analysis of mitochondria. In silico docking of AV constituents reveal higher negative binding affinity for C and O- glycosides for HIF-1a, IL-6, Janus kinase 1/3, TNF-α and TGF- β -key players of hypoxia-inflammation. This study for the first time provides a molecular basis of action and effect of AV whole extract that is widely used in Ayurveda practice for diverse respiratory ailments. Further, through its effect on hypoxia-induced mitochondrial dysfunction, the study highlights its potential to treat severe steroid-resistant asthma.