HIF1A reduces acute lung injury by optimizing carbohydrate metabolism in the alveolar epithelium

Background: While acute lung injury (ALI) contributes significantly to critical illness, it resolves spontaneously in many instances. The majority of patients experiencing ALI require mechanical ventilation. Therefore, we hypothesized that mechanical ventilation and concomitant stretch-exposure of pulmonary epithelia could activate endogenous pathways important in lung protection. Methods and Findings: To examine transcriptional responses during ALI, we exposed pulmonary epithelia to cyclic mechanical stretch conditions—an in vitro model resembling mechanical ventilation. A genome-wide screen revealed a transcriptional response similar to hypoxia signaling. Surprisingly, we found that stabilization of hypoxia-inducible factor 1A (HIF1A) during stretch conditions in vitro or during ventilator-induced ALI in vivo occurs under normoxic conditions. Extension of these findings identified a functional role for stretch-induced inhibition of succinate dehydrogenase (SDH) in mediating normoxic HIF1A stabilization, concomitant increases in glycolytic capacity, and improved tricarboxylic acid (TCA) cycle function. Pharmacologic studies with HIF activator or inhibitor treatment implicated HIF1A-stabilization in attenuating pulmonary edema and lung inflammation during ALI in vivo. Systematic deletion of HIF1A in the lungs, endothelia, myeloid cells, or pulmonary epithelia linked these findings to alveolar-epithelial HIF1A. In vivo analysis of 13C-glucose metabolites utilizing liquid-chromatography tandem mass-spectrometry demonstrated that increases in glycolytic capacity, improvement of mitochondrial respiration, and concomitant attenuation of lung inflammation during ALI were specific for alveolar-epithelial expressed HIF1A. Conclusions: These studies reveal a surprising role for HIF1A in lung protection during ALI, where normoxic HIF1A stabilization and HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation

Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1beta Release by Pulmonary Epithelial Cells

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1beta. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1beta release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits alpha7, alpha9, and/or alpha10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae

Screening of siRNA Nanoparticles for Delivery to Airway Epithelial Cells Using High Content Analysis

Background: Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop novel methods of treating a range of poorly treated respiratory conditions. However progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery Methodology: Following physico-chemical analysis, the ability of PEI-PEG/siRNA nanoparticles to facilitate siRNA delivery was determined using high content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI-PEG/siRNA particles was analysed by HCA using the Cellomics® multiparamter cytotoxicity assay. Conclusions: PEI-PEG/siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared to PEI/siRNA

Alpha-tocopherol exerts protective function against the mucotoxicity of particulate matter in amphibian and human goblet cells

Exposure to particulate matter (PM) in ambient air is known to increase the risk of cardiovascular disorders and mortality. The cytotoxicity of PM is mainly due to the abnormal increase of reactive oxygen species (ROS), which damage cellular components such as DNA, RNA, and proteins. The correlation between PM exposure and human disorders, including mortality, is based on long-term exposure. In this study we have investigated acute responses of mucus-secreting goblet cells upon exposure to PM derived from a heavy diesel engine. To this end, we employed the mucociliary epithelium of amphibian embryos and human Calu-3 cells to examine PM mucotoxicity. Our data suggest that acute exposure to PM significantly impairs mucus secretion and results in the accumulation of mucus vesicles in the cytoplasm of goblet cells. RNA-seq analysis revealed that acute responses to PM exposure significantly altered gene expression patterns; however, known regulators of mucus production and the secretory pathway were not significantly altered. Interestingly, pretreatment with alpha-tocopherol nearly recovered the hyposecretion of mucus from both amphibian and human goblet cells. We believe this study demonstrates the mucotoxicity of PM and the protective function of alpha-tocopherol on mucotoxicity caused by acute PM exposure from heavy diesel engines

Elevated Paracellular Glucose Flux across Cystic Fibrosis Airway Epithelial Monolayers Is an Important Factor for Pseudomonas aeruginosa Growth.

People with cystic fibrosis (CF) who develop related diabetes (CFRD) have accelerated pulmonary decline, increased infection with antibiotic-resistant Pseudomonas aeruginosa and increased pulmonary exacerbations. We have previously shown that glucose concentrations are elevated in airway surface liquid (ASL) of people with CF, particularly in those with CFRD. We therefore explored the hypotheses that glucose homeostasis is altered in CF airway epithelia and that elevation of glucose flux into ASL drives increased bacterial growth, with an effect over and above other cystic fibrosis transmembrane conductance regulator (CFTR)-related ASL abnormalities. The aim of this study was to compare the mechanisms governing airway glucose homeostasis in CF and non-CF primary human bronchial epithelial (HBE) monolayers, under normal conditions and in the presence of Ps. aeruginosa filtrate. HBE-bacterial co-cultures were performed in the presence of 5 mM or 15 mM basolateral glucose to investigate how changes in blood glucose, such as those seen in CFRD, affects luminal Ps. aeruginosa growth. Calu-3 cell monolayers were used to evaluate the potential importance of glucose on Ps. aeruginosa growth, in comparison to other hallmarks of the CF ASL, namely mucus hyperviscosity and impaired CFTR-dependent fluid secretions. We show that elevation of basolateral glucose promotes the apical growth of Ps. aeruginosa on CF airway epithelial monolayers more than non-CF monolayers. Ps. aeruginosa secretions elicited more glucose flux across CF airway epithelial monolayers compared to non-CF monolayers which we propose increases glucose availability in ASL for bacterial growth. In addition, elevating basolateral glucose increased Ps. aeruginosa growth over and above any CFTR-dependent effects and the presence or absence of mucus in Calu-3 airway epithelia-bacteria co-cultures. Together these studies highlight the importance of glucose as an additional factor in promoting Ps. aeruginosa growth and respiratory infection in CF disease

A Systematic Review of therapeutic agents for the treatment of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Background The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was first described in 2012 and attracted a great international attention due to multiple healthcare associated outbreaks. The disease carries a high case fatality rate of 34.5%, and there is no internationally or nationally recommended therapy. Method We searched MEDLINE, Science Direct, Embase and Scopus databases for relevant papers published till March 2019 describing in vitro, in vivo or human therapy of MERS. Results Initial search identified 62 articles: 52 articles were from Medline, 6 from Embase, and 4 from Science Direct. Based on the inclusions and exclusions criteria, 30 articles were included in the final review and comprised: 22 in vitro studies, 8 studies utilizing animal models, 13 studies in humans, and one study included both in vitro and animal model. There are a few promising therapeutic agents on the horizon. The combination of lopinavir/ritonavir and interferon-beta- 1b showed excellent results in common marmosets and currently is in a randomized control trial. Ribavirin and interferon were the most widely used combination and experience comes from a number of observational studies. Although, the data are heterogenous, this combination might be of potential benefit and deserve further investigation. There were no randomized clinical trials to recommend specific therapy for the treatment of MERS-CoV infection. Only one such study is planned for randomization and is pending completion. The study is based on a combination of lopinavir/ritonavir and interferon-beta- 1b. A fully human polyclonal IgG antibody (SAB-301) was safe and well tolerated in healthy individuals and this agent may deserve further testing for efficacy. Conclusion Despite multiple studies in humans there is no consensus on the optimal therapy for MERS-CoV. Randomized clinical trials are needed and potential therapies should be evaluated only in such clinical trials. In order to further enhance the therapeutic aroma for MERS-CoV infection, repurposing old drugs against MERS-CoV is an interesting strategy and deserves further consideration and use in clinical settings

A small synthetic molecule functions as a chloride–bicarbonate dual-transporter and induces chloride secretion in cells

A C2 symmetric small molecule composed of L-phenylalanine and isophthalamide was found to function as a Cl−/HCO3− dual transporter and self-assemble into chloride channels. In Ussing-chamber based short-circuit current measurements, this molecule elicited chloride-dependent short-circuit current (Isc) increase in both Calu-3 cell and CFBE41o-cell (with F508del mutant CFTR) monolayers.postprin

MAP17 predicts sensitivity to platinum-based therapy, EGFR inhibitors and the proteasome inhibitor bortezomib in lung adenocarcinoma

Background The high incidence and mortality of lung tumours is a major health problem. Therefore, the identification both of biomarkers predicting efficacy for therapies in use and of novel efficacious therapeutic agents is crucial to increase patient survival. MAP17 (PDZK1IP1) is a small membrane-bound protein whose upregulation is reported as a common feature in tumours from diverse histological origins. Furthermore, MAP17 is correlated with tumour progression. Go to: Methods We assessed the expression of MAP17 in preclinical models, including cell lines and patient-derived xenografts (PDXs), assessing its correlation with sensitivity to different standard-of-care drugs in lung adenocarcinoma, as well as novel drugs. At the clinical level, we subsequently correlated MAP17 expression in human tumours with patient response to these therapies. Go to: Results We show that MAP17 expression is induced during lung tumourigenesis, particularly in lung adenocarcinomas, and provide in vitro and in vivo evidence that MAP17 levels predict sensitivity to therapies currently under clinical use in adenocarcinoma tumours, including cisplatin, carboplatin and EGFR inhibitors. In addition, we show that MAP17 expression predicts proteasome inhibitor efficacy in this context and that bortezomib, an FDA-approved drug, may be a novel therapeutic approach for MAP17-overexpressing lung adenocarcinomas. Go to: Conclusions Our results indicate a potential prognostic role for MAP17 in lung tumours, with particular relevance in lung adenocarcinomas, and highlight the predictive pot0065ntial of this membrane-associated protein for platinum-based therapy and EGFR inhibitor efficacy. Furthermore, we propose bortezomib treatment as a novel and efficacious therapy for lung adenocarcinomas exhibiting high MAP17 expression

BanLec, a banana lectin, is a potent inhibitor of Middle East respiratory syndrome coronavirus in in vitro assays

Poster Abstract Session - Viral Infections: Treatment and Prevention: no. 1159BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause human infections with multiple clusters two years after the onset of the epidemic. Though mild cases have been recognized, the infection is severe in those with co-morbidities and >30% of patients die from the infection. Our recent structure-based development of a fusion inhibitor is one of the few treatment options for MERS and it led us to hypothesize that other existing antivirals that block cellular entry may also be active against MERS-CoV. BanLec is a jacalin-related banana lectin that has potent anti-HIV activity through binding to glycosylated viral envelope proteins and blocking cellular entry. We assessed the anti-MER-CoV activity of BanLec in cell culture assays. METHODS: The anti-MERS-CoV activity of BanLec was assessed by cytopathic effect (CPE) inhibition, viral yield reduction, and plaque reduction (PRA) assays in Vero, Calu-3, and/or HK2 cells. The cytotoxicity of BanLec was also assessed. RESULTS: The CC50 of BanLec was >10 nM in Vero and Calu-3 cells. CPE was completely absent in Vero and HK2 cells infected with MERS-CoV on 3 dpi with 30.00 nM of BanLec. In Calu-3 cells, CPE was completely absent at 90.00 nM of the drug. The EC50 of BanLec ranged from 3.99-4.82 nM (Table 1). The mean viral loads reduced by 7.13, 3.40, and 3.63 log10 copies/ml in Vero, Calu-3, and HK2 cells respectively (Fig. 1A to C). The highest percentage of plaque reduction at a concentration of >10 nM of BanLec were 100% and 59.5% in Vero cells and HK2 cells respectively (Fig. 2A & B). CONCLUSION: BanLec exhibits potent in vitro anti-MERS-CoV activity. The detailed mechanism and in vivo correlation of its antiviral activity should be further tested in animal models. The potential advantages of using BanLec for MERS include its high stability and the prospect of using it as a topical treatment or prophylaxis for exposed patients. (Table see attachment)BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause human infections with multiple clusters two years after the onset of the epidemic. Though mild cases have been recognized, the infection is severe in those with co-morbidities and >30% of patients die from the infection. Our recent structure-based development of a fusion inhibitor is one of the few treatment options for MERS and it led us to hypothesize that other existing antivirals that block cellular entry may also be active against MERS-CoV. BanLec is a jacalin-related banana lectin that has potent anti-HIV activity through binding to glycosylated viral envelope proteins and blocking cellular entry. We assessed the anti-MER-CoV activity of BanLec in cell culture assays. METHODS: The anti-MERS-CoV activity of BanLec was assessed by cytopathic effect (CPE) inhibition, viral yield reduction, and plaque reduction (PRA) assays in Vero, Calu-3, and/or HK2 cells. The cytotoxicity of BanLec was also assessed. RESULTS: The CC50 of BanLec was >10 nM in Vero and Calu-3 cells. CPE was completely absent in Vero and HK2 cells infected with MERS-CoV on 3 dpi with 30.00 nM of BanLec. In Calu-3 cells, CPE was completely absent at 90.00 nM of the drug. The EC50 of BanLec ranged from 3.99-4.82 nM (Table 1). The mean viral loads reduced by 7.13, 3.40, and 3.63 log10 copies/ml in Vero, Calu-3, and HK2 cells respectively (Fig. 1A to C). The highest percentage of plaque reduction at a concentration of >10 nM of BanLec were 100% and 59.5% in Vero cells and HK2 cells respectively (Fig. 2A & B). CONCLUSION: BanLec exhibits potent in vitro anti-MERS-CoV activity. The detailed mechanism and in vivo correlation of its antiviral activity should be further tested in animal models. The potential advantages of using BanLec for MERS include its high stability and the prospect of using it as a topical treatment or prophylaxis for exposed patients