Corrigendum: Standardised Sonneratia apetala Buch.-Ham. fruit extract inhibits human neutrophil elastase and attenuates elastase-induced lung injury in mice.

[This corrects the article DOI: 10.3389/fphar.2022.1011216.].

Obesity impairs cardiolipin-dependent mitophagy and therapeutic intercellular mitochondrial transfer ability of mesenchymal stem cells.

Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on IMT and thereby on the therapeutic efficacy of MSCs has largely remained unexplored. Here we found impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mouse (MSC-Ob). MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue mitochondrial dysfunction and cell death in stress-induced airway epithelial cells. Pharmacological modulation of MSCs enhanced cardiolipin-dependent mitophagy and restored their IMT ability to airway epithelial cells. Therapeutically, these modulated MSCs attenuated features of allergic airway inflammation (AAI) in two independent mouse models by restoring healthy IMT. However, unmodulated MSC-Ob failed to do so. Notably, in human (h)MSCs, induced metabolic stress associated impaired cardiolipin-dependent mitophagy was restored upon pharmacological modulation. In summary, we have provided the first comprehensive molecular understanding of impaired mitophagy in obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention. A MSCs obtained from (HFD)-induced obese mice (MSC-Ob) show underlying mitochondrial dysfunction with a concomitant decrease in cardiolipin content. These changes prevent LC3-cardiolipin interaction, thereby reducing dysfunctional mitochondria sequestration into LC3-autophagosomes and thus impaired mitophagy. The impaired mitophagy is associated with reduced intercellular mitochondrial transport (IMT) via tunneling nanotubes (TNTs) between MSC-Ob and epithelial cells in co-culture or in vivo. B Pyrroloquinoline quinone (PQQ) modulation in MSC-Ob restores mitochondrial health, cardiolipin content, and thereby sequestration of depolarized mitochondria into the autophagosomes to alleviate impaired mitophagy. Concomitantly, MSC-Ob shows restoration of mitochondrial health upon PQQ treatment (MSC-ObPQQ). During co-culture with epithelial cells or transplantation in vivo into the mice lungs, MSC-ObPQQ restores IMT and prevents epithelial cell death. C Upon transplantation in two independent allergic airway inflammatory mouse models, MSC-Ob failed to rescue the airway inflammation, hyperactivity, metabolic changes in epithelial cells. D PQQ modulated MSCs restored these metabolic defects and restored lung physiology and airway remodeling parameters.

Cellular Distribution of Secreted Phospholipase A2 in Lungs of IPF Patients and Its Inhibition in Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a very poor prognosis as it has a 2.5 to 5 years mean survival after proper diagnosis. Even nintedanib and pirfenidone cannot halt the progression, though they slow the progression of IPF. Hence, there is a need to understand the novel pathophysiology. Phospholipase A2 (PLA2) could be the ideal candidate to study in IPF, as they have a role in both inflammation and fibrosis. In the present study, we have shown the expression profile of various secretory Phospholipase A2 (PLA2) isoforms by analyzing publicly available transcriptome data of single cells from the lungs of healthy individuals and IPF patients. Among 11 members of sPLA2, PLA2G2A is found to be increased in the fibroblasts and mesothelial cells while PLA2G5 is found to be increased in the fibroblasts of IPF patients. We identified a subset of fibroblasts expressing high PLA2G2A with moderate expression of PLA2G5 and which are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast. Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have upregulation of both inflammatory and fibrosis-related pathways like the TGF-ß signaling pathway, IL-17 signaling, the arachidonic acid metabolism pathway and ECM-receptor interaction. In addition to this, we found elevated levels of sPLA2-IIA in plasma samples of IPF patients in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be increased in certain epithelial cells of IPF patients. Thus, these findings indicate that these five isoforms have a disease-dominant role along with innate immune roles as these isoforms are found predominantly in structural cells of IPF patients. Further, we have targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis induced by bleomycin along with a reduction in TGF-ß and deposition of extracellular matrix in lung. Thus, these findings indicate that these sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in lung fibrosis.

Airway Epithelium: A Neglected but Crucial Cell Type in Asthma Pathobiology.

The features of allergic asthma are believed to be mediated mostly through the Th2 immune response. In this Th2-dominant concept, the airway epithelium is presented as the helpless victim of Th2 cytokines. However, this Th2-dominant concept is inadequate to fill some of the vital knowledge gaps in asthma pathogenesis, like the poor correlation between airway inflammation and airway remodeling and severe asthma endotypes, including Th2-low asthma, therapy resistance, etc. Since the discovery of type 2 innate lymphoid cells in 2010, asthma researchers started believing in that the airway epithelium played a crucial role, as alarmins, which are the inducers of ILC2, are almost exclusively secreted by the airway epithelium. This underscores the eminence of airway epithelium in asthma pathogenesis. However, the airway epithelium has a bipartite functionality in sustaining healthy lung homeostasis and asthmatic lungs. On the one hand, the airway epithelium maintains lung homeostasis against environmental irritants/pollutants with the aid of its various armamentaria, including its chemosensory apparatus and detoxification system. Alternatively, it induces an ILC2-mediated type 2 immune response through alarmins to amplify the inflammatory response. However, the available evidence indicates that restoring epithelial health may attenuate asthmatic features. Thus, we conjecture that an epithelium-driven concept in asthma pathogenesis could fill most of the gaps in current asthma knowledge, and the incorporation of epithelial-protective agents to enhance the robustness of the epithelial barrier and the combative capacity of the airway epithelium against exogenous irritants/allergens may mitigate asthma incidence and severity, resulting in better asthma control.

Allergic diseases in India - Prevalence, risk factors and current challenges.

Epidemiological studies have shown a rise in the prevalence of allergic diseases in India during the last two decades. However, recent evidence from the Global Asthma Network study has observed a decrease in allergic rhinitis, asthma and atopic dermatitis in children. Still, with a population over 1.3 billion, there is a huge burden of allergic rhinitis, asthma and atopic dermatitis, and this is compounded by an unmet demand for trained allergy specialists and poor health service framework. There is wide variation in the prevalence of allergic diseases between different geographical locations in India, and the reasons are unclear at present. This may at least in part be attributable to considerable heterogeneity in aero-biology, weather, air pollution levels, cultural and religious factors, diet, socioeconomic strata and literacy. At present, factors enhancing risks and those protecting from development of atopy and allergic diseases have not been well delineated, although there is some evidence for the influence of genetic factors alongside cultural and environmental variables such as diet, exposure to tobacco smoke and air pollution and residence in urban areas. This narrative review provides an overview of data from India regarding epidemiology, risk factors and genetics and highlights gaps in evidence as well as areas for future research.

Standardised Sonneratia apetala Buch.-Ham. fruit extract inhibits human neutrophil elastase and attenuates elastase-induced lung injury in mice.

Chronic obstructive pulmonary disease (COPD) along with asthma is a major and increasing global health problem. Smoking contributes to about 80%-90% of total COPD cases in the world. COPD leads to the narrowing of small airways and destruction of lung tissue leading to emphysema primarily caused by neutrophil elastase. Neutrophil elastase plays an important role in disease progression in COPD patients and has emerged as an important target for drug discovery. Sonneratia apetala Buch.-Ham. is a mangrove plant belonging to family Sonneratiaceae. It is widely found in the Sundarban regions of India. While the fruits of this plant have antibacterial, antifungal, antioxidant and astringent activities, fruit and leaf extracts have been shown to reduce the symptoms of asthma and cough. The aim of this study is to find whether hydro alcoholic fruit extracts of S. apetala inhibit neutrophil elastase and thus prevent the progression of neutrophil elastase-driven lung emphysema. The hydroalcoholic extract, ethanol: water (90:10), of the S. apetala Buch.-Ham. fresh fruits (SAM) were used for neutrophil elastase enzyme kinetic assay and IC50 of the extract was determined. The novel HPLC method has been developed and the extract was standardized with gallic acid and ellagic acid as standards. The extract was further subjected to LC-MS2 profiling to identify key phytochemicals. The standardized SAM extract contains 53 µg/mg of gallic acid and 95 µg/mg of ellagic acid, based on the HPLC calibration curve. SAM also reversed the elastase-induced morphological change of human epithelial cells and prevented the release of ICAM-1 in vitro and an MTT assay was conducted to assess the viability. Further, 10 mg/kg SAM had reduced alveolar collapse induced by neutrophil elastase in the mice model. Thus, in this study, we reported for the first time that S. apetala fruit extract has the potential to inhibit human neutrophil elastase in vitro and in vivo.

Novel benzoxazinone derivative as potent human neutrophil elastase inhibitor: Potential implications in lung injury.

Neutrophil elastase, a powerful physiological defence tool, may serve as drug target for diverse diseases due to its bystander effect on host cells like chronic obstructive pulmonary disease (COPD). Here, we synthesised seven novel benzoxazinone derivatives and identified that these synthetic compounds are human neutrophil elastase inhibitor that was demonstrated by enzyme substrate kinetic assay. One such compound, PD05, emerged as the most potent inhibitor with lower IC50 as compared to control drug sivelestat. While this inhibition is competitive based on substrate dilution assay, PD05 showed a high binding affinity for human neutrophil elastase (Kd = 1.63 nM) with faster association and dissociation rate compared to notable elastase inhibitors like ONO 6818 and AZD9668, and its interaction with human neutrophil elastase was fully reversible.Preclinical pharmacokinetic studies were performed in vitro where protein binding was found to be 72% with a high recovery rate, aqueous solubility of 194.7 µM, low permeability along with a favourable hERG. Experiments with cell line revealed that the molecule successfully prevented elastase induced rounding and retracted cell morphology and cell cytotoxicity. In mouse model PD05 is able to reduce the alveolar collapse induced by neutrophil elastase. In summary, we demonstrate the in situ, in vitro and in vivo anti-elastase potential of the newly synthesised benzoxazinone derivative PD05 and thus this could be promising candidate for further investigation as a drug for the treatment of COPD.

Systemic deficiency of vitronectin is associated with aortic inflammation and plaque progression in ApoE-Knockout mice.

Optimal cell spreading and interplay of vascular smooth muscle cells (VSMC), inflammatory cells, and cell adhesion molecules (CAM) are critical for progressive atherosclerosis and cardiovascular complications. The role of vitronectin (VTN), a major cell attachment glycoprotein, in the pathogenesis of atherosclerosis remains elusive. In this study, we attempt to examine the pathological role of VTN in arterial plaque progression and inflammation. We found that, relative expression analysis of VTN from the liver of Apolipoprotein E (ApoE) Knockout mice revealed that atherosclerotic progression induced by feeding mice with high cholesterol diet (HCD) causes a significant downregulation of VTN mRNA as well as protein after 60 days. Promoter assay confirmed that cholesterol modulates the expression of VTN by influencing its promoter. Mimicking VTN reduction with siRNA in HCD fed ApoE Knockout mice, accelerated athero-inflammation with an increase in NF-kB, ICAM-1, and VCAM-1 at the site of the plaque along with upregulation of inflammatory proteins like MCP-1 and IL-1ß in the plasma. Also, matrix metalloprotease (MMP)-9 and MMP-12 expression were increased and collagen content was decreased in the plaque, in VTN deficient condition. This might pose a challenge to plaque integrity. Human subjects with acute coronary syndrome or having risk factors of atherosclerosis have lower levels of VTN compared to healthy controls suggesting a clinical significance of plasma VTN in the pathophysiology of coronary artery disease. We establish that, VTN plays a pivotal role in cholesterol-driven atherosclerosis and aortic inflammation and might be a useful indicator for atherosclerotic plaque burden and stability.

Mitochondrial calcium in command of juggling myriads of cellular functions.

The puzzling traits related to the evolutionary aspect of mitochondria, still positions the mitochondrion at the center of the research. The theory of endosymbiosis popularized by Lynn Margulis in 1967 gained prominence wherein the mitochondrion is believed to have emerged as a prokaryote and later integrated into the eukaryotic system. This semi-autonomous organelle has bagged two responsible but perilous cellular functions: a) energy metabolism, and b) calcium buffering, though both are interdependent. While most of the mitochondrial functions are saliently regulated by calcium ions, the calcium buffering role of mitochondria decides the cellular fate. Though calcium overload in few mitochondria makes them dysfunctional at the early stage of cellular stress, this doesn't lead to sudden cell death due to critical checkpoints like mitophagy, mitochondrial fusion, etc. Thus, mitochondrion juggles with multiple crucial cellular functions with its calcium buffering skill.

Ku70 modulation alleviates murine allergic asthma features and restores mitochondrial function in lungs.

The airway epithelium is continuously exposed to a variety of pollutants and allergens, thanks to both natural and manmade environmental pollution. With numerous protective mechanisms, the airway epithelium protects the lungs. DNA repair mechanism is one such protective response and its failure could lead to the accumulation of DNA mutations. Our lab had earlier demonstrated the dysfunctional mitochondria in airway epithelium of the asthmatic mice lungs. Here, we show that Ku70 modulation by the administration of Ku70 plasmid attenuates asthma features and reduces mitochondrial dysfunction in the lungs of allergen exposed mice. Ku70 is a key DNA repair protein with diverse roles including VDJ recombination, telomere maintenance, and maintenance of cell homeostasis. Recently, we found a reduction in Ku70 expression in asthmatic airway epithelium, and this was associated with mitochondrial dysfunction in asthmatic condition. In this study, we have shown that Ku70 over-expression in asthmatic mice attenuated airway hyperresponsiveness, airway inflammation, sub-epithelial fibrosis along with reduction in TGF-ß with no effect in IL-13 levels and goblet cell metaplasia. Ku70 over-expression in asthmatic mice reduced 8-isoprostane, a marker of oxidative stress, and restored the mitochondrial function in asthmatic mice. We further found these roles of Ku70 to be independent of DNA damage as Ku70 overexpressed mice did not show any reduction in DNA tail, an index of DNA damage. Thus, our findings indicate that Ku70 can attenuate crucial features of asthma along with the restoration of mitochondrial function. This implies that Ku70 could be a therapeutic target for asthma without affecting DNA repair function.

Noncanonical role for Ku70/80 in the prevention of allergic airway inflammation via maintenance of airway epithelial cell organelle homeostasis.

Airway epithelial homeostasis is under constant threat due to continuous exposure to the external environment, and abnormally robust sensitivity to external stimuli is critical to the development of airway diseases, including asthma. Ku is a key nonhomologous end-joining DNA repair protein with diverse cellular functions such as VDJ recombination and telomere length maintenance. Here, we show a novel function of Ku in alleviating features of allergic airway inflammation via the regulation of mitochondrial and endoplasmic reticulum (ER) stress. We first determined that airway epithelial cells derived from both asthmatic lungs and murine asthma models demonstrate increased expression of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. Ku protein expression was dramatically reduced in the bronchial epithelium of patients with asthma as well as in human bronchial epithelial cells exposed to oxidative stress. Knockdown of Ku70 or Ku80 in naïve mice elicited mitochondrial collapse or ER stress, leading to bronchial epithelial cell apoptosis and spontaneous development of asthma-like features, including airway hyperresponsiveness, airway inflammation, and subepithelial fibrosis. These findings demonstrate an essential noncanonical role for Ku proteins in asthma pathogenesis, likely via maintenance of organelle homeostasis. This novel function of Ku proteins may also be important in other disease processes associated with organelle stress.

A looming role of mitochondrial calcium in dictating the lung epithelial integrity and pathophysiology of lung diseases.

With the increasing appreciation of mitochondria in modulating cellular homeostasis, various disease biology researchers have started exploring the detailed role of mitochondria in multiple diseases beyond neuronal and muscular diseases. In this context, emerging shreds of evidence in lung biology indicated the meticulous role of lung epithelia in provoking a plethora of lung diseases in contrast to earlier beliefs. As lung epithelia are ceaselessly exposed to the environment, they need to have multiple protective mechanisms to maintain the integrity of lung structure and function. As ciliated airway epithelium and type 2 alveolar epithelia require intense energy for executing their key functions like ciliary beating and surfactant production, it is no surprise that defects in mitochondrial function in these cells could perturb lung homeostasis and engage in the pathophysiology of lung diseases. On one hand, intracellular calcium plays the central role in executing key functions of lung epithelia, and on the other hand maintenance of intracellular calcium needs the buffering role of mitochondria. Thus, the regulation of mitochondrial calcium in lung epithelia seems to be critical in lung homeostasis and could be decisive in the pathogenesis of various lung diseases.

Clinical, Epidemiological and Experimental Approaches to Assess Adverse Health Outcomes of Indoor Biomass Smoke Exposure: Conclusions from An Indo-Swedish Workshop in Mysuru, January 2020.

This report summarizes the outcome of a workshop held in Mysuru, India in January 2020 addressing the adverse health effects of exposure to biomass smoke (BMS). The aim of the workshop was to identify uncertainties and gaps in knowledge and possible methods to address them in the Mysuru study on Determinants of Health in Rural Adults (MUDHRA) cohort. Specific aims were to discuss the possibility to improve and introduce new screening methods for exposure and effect, logistic limitations and other potential obstacles, and plausible strategies to overcome these in future studies. Field visits were included in the workshop prior to discussing these issues. The workshop concluded that multi-disciplinary approaches to perform: (a) indoor and personalized exposure assessment; (b) clinical and epidemiological field studies among children, adolescents, and adults; (c) controlled exposure experiments using physiologically relevant in vitro and in vivo models to understand molecular patho-mechanisms are warranted to dissect BMS-induced adverse health effects. It was perceived that assessment of dietary exposure (like phytochemical index) may serve as an important indicator for understanding potential protective mechanisms. Well trained field teams and close collaboration with the participating hospital were identified as the key requirements to successfully carry out the study objectives.

Secretory Inositol Polyphosphate 4-Phosphatase Protects against Airway Inflammation and Remodeling.

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.

House dust mite allergen causes certain features of steroid resistant asthma in high fat fed obese mice.

Obesity is a high risk factor for diseases such as cardiovascular, metabolic syndrome and asthma. Obese-asthma is another emerging phenotype in asthma which is typically refractive to steroid treatment due to its non-classical features such as non-eosinophilic cellular inflammation. The overall increased morbidity, mortality and economical burden in asthma is mainly due to steroid resistant asthma. In the present study, we used high fat diet induced obese mice which when sensitized with house dust mite (HDM) showed steroid resistant features. While the steroid, dexamethasone (DEX), treatment to high fat fed naïve mice could not reduce the airway hyperresponsiveness (AHR) induced by high fat, DEX treatment to high fat fed allergic mice could not reduce the HDM allergen induced airway remodeling features though it reduced airway inflammation. Further, these HDM induced high fat fed mice with or without DEX treatment had shown the increased activity and expression of arginase as well as the inducible nitric oxide synthase (iNOS) expression. However, DEX treatment had reduced the expressions of high iNOS and arginase I in control chow diet fed mice. Thus, we speculate that the steroid resistance seen in human obese asthmatics could be stemming from altered NO metabolism and its induced airway remodeling and with further investigations, it would encourage new treatments specific to obese-asthma phenotype.

Linoleic acid metabolite leads to steroid resistant asthma features partially through NF-κB.

Studies have highlighted the role of nutritional and metabolic modulators in asthma pathobiology. Steroid resistance is an important clinical problem in asthma but lacks good experimental models. Linoleic acid, a polyunsaturated fatty acid, has been linked to asthma and glucocorticoid sensitivity. Its 12/15-lipoxygenase metabolite, 13-S-hydroxyoctadecadienoic acid (HODE) induces mitochondrial dysfunction, with severe airway obstruction and neutrophilic airway inflammation. Here we show that HODE administration leads to steroid unresponsiveness in an otherwise steroid responsive model of allergic airway inflammation (AAI). HODE treatment to allergic mice further increased airway hyperresponsiveness and goblet metaplasia. Treatment with dexamethasone was associated with increased neutrophilic inflammation in HODE treated allergic mice; unlike control allergic mice that showed resolution of inflammation. HODE induced loss of steroid sensitivity was associated with increased p-NFkB in mice and reduced GR-α transcript levels in cultured human bronchial epithelia. In summary, HODE modifies typical AAI to recapitulate many of the phenotypic features seen in severe steroid unresponsive asthma. We speculate that since HODE is a natural metabolite, it may be relevant to the increased asthma severity and steroid insensitivity in patients who are obese or consume high fat diets. Further characterization of HODE induced steroid insensitivity may clarify the mechanisms.