Characterization of alveolar epithelial lineage heterogeneity during the late pseudoglandular stage of mouse lung development

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (Fgfr2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury. We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool. Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular development, and that lineage commitment is regulated in part by Fgfr2b signalling. We have characterized a set of direct Fgfr2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes concentrate on a subpopulation of AT2 cells later in development, and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.

COVID-19: Salient Aspects of Coronavirus Infection, Vaccines and Vaccination Testing and their Implications

In the present study, three basic aspects related to COVID-19 are presented. (a) The occurrence of coronavirus infection is analyzed statistically as number of coronaviruses infected alveolar cells compared to normal alveolar cells in human lungs. The mole concept is used to estimate the number of normal alveolar cells per human lung. The number of coronavirus infections in infected alveolar cells is estimated from the published Lower Respiratory Tract (LRT) load data. The Poisson probability distribution is aptly applied to imply the incubation period of the coronavirus infection to be within day-3 to day-7, with the cumulative probability of 75%. The incubation period within day-0 to day-10 has a cumulative probability of 98%. It implies a 10-day quarantine to isolate an uninfected individual as a precautionary measure. (b) Three vaccines to combat COVID-19, which adopt distinct paradigms while preparing them, are analyzed. These are Moderna's mRNA-1273, Oxford-AstraZeneca's ChAdOx1 nCoV-19 and Bharat BioTech's COVAXIN. The mole concept is used to estimate the antigen mass density per dose of each of these vaccines as 10, 0.1 and 1 (g per cubic-cm), respectively. The vaccines are deemed to be compatible to neutralize the infection. (c) A statistical analysis is performed of the Moderna's mRNA-1273 vaccine efficacy of 94.1% and Oxford's ChAdOx1 nCoV-19 vaccine efficacy of 62.1% in terms of groups of volunteers testing negative to vaccine by chance. In the Moderna vaccination testing scenario, since the probability of negative response of vaccine is small, the Poisson probability distribution for 95% cumulative probability is used to describe the vaccination testing in 300 samples of 47 volunteers each. Thus, 87% of samples have average group of 3 volunteers testing negative to vaccine. About 6% of samples have all volunteers testing positive to vaccine. In the Oxford vaccination testing scenario, since the probability of negative response of vaccine is finite, the Gaussian probability distribution for 95% probability is used to describe the vaccination testing in 75 samples of 120 volunteers each. Thus, 68% of samples have average group of 45 volunteers testing negative to vaccine. No sample has all volunteers testing positive to vaccine. A vaccine, irrespective of its efficacy being high or low, is necessary for mass immunization.

Extracellular Vesicle Associated miRNAs Regulate Signaling Pathways Involved in COVID-19 Pneumonia and the Progression to Severe Acute Respiratory Corona Virus-2 Syndrome

BackgroundExtracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19.MethodsWe prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS.ResultsDGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted<0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, padjusted=0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p.ConclusionEV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets.

The Normal and Fibrotic Mouse Lung in Situ Classified by High Dimensional Single Cell Analysis on Routinely Processed Tissue

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis

Background Staphylococcus aureus (S. aureus) mastitis is one of the most difficult diseases to treat in lactating dairy cows worldwide. S. aureus with different lineages leads to different host immune responses. Long non-coding RNAs (lncRNAs) are reported to be widely involved in the progress of inflammation. However, no research has identified stable lncRNAs among different S. aureus strain infections. In addition, folic acid (FA) can effectively reduce inflammation, and whether the inflammatory response caused by S. aureus can be reduced by FA remains to be explored. Methods lncRNA transcripts were identified from Holstein mammary gland tissues infected with different concentrations of S. aureus (in vivo) and mammary alveolar cells (Mac-T cells, in vitro) challenged with different S. aureus strains. Differentially expressed (DE) lncRNAs were evaluated, and stable DE lncRNAs were identified in vivo and in vitro. On the basis of the gene sequence conservation and function conservation across species, key lncRNAs with the function of potentially immune regulation were retained for further analysis. The function of FA on inflammation induced by S. aureus challenge was also investigated. Then, the association analysis between these keys lncRNA transcripts and hematological parameters (HPs) was carried out. Lastly, the knockdown and overexpression of the important lncRNA were performed to validate the gene function on the regulation of cell immune response. Results Linear regression analysis showed a significant correlation between the expression levels of lncRNA shared by mammary tissue and Mac-T cells (P < 0.001, R2 = 0.3517). lncRNAs PRANCR and TNK2–AS1 could be regarded as stable markers associated with bovine S. aureus mastitis. Several HPs could be influenced by SNPs around lncRNAs PRANCR and TNK2–AS1. The results of gene function validation showed PRANCR regulates the mRNA expression of SELPLG and ITGB2 within the S. aureus infection pathway and the Mac-T cells apoptosis. In addition, FA regulated the expression change of DE lncRNA involved in toxin metabolism and inflammation to fight against S. aureus infection. Conclusions The remarkable association between SNPs around these two lncRNAs and partial HP indicates the potentially important role of PRANCR and TNK2–AS1 in immune regulation. Stable DE lncRNAs PRANCR and TNK2–AS1 can be regarded as potential targets for the prevention of bovine S. aureus mastitis. FA supplementation can reduce the negative effect of S. aureus challenge by regulating the expression of lncRNAs.

Follow Your Nose: A Key Clue to Understanding and Treating COVID-19

This paper suggests that ATP release induced by the SARS-CoV-2 virus plays a key role in the genesis of the major symptoms and complications of COVID-19. Infection of specific cells which contain the Angiotensin-Converting Enzyme 2 (ACE2) receptor results in a loss of protection of the Mineralocorticoid Receptor (MR). Local activation by cortisol stimulates the release of ATP initially into the basolateral compartment and then by lysosomal exocytosis from the cell surface. This then acts on adjacent cells. In the nose ATP acts as a nociceptive stimulus which results in anosmia. It is suggested that a similar paracrine mechanism is responsible for the loss of taste. In the lung ATP release from type 2 alveolar cells produces the non-productive cough by acting on purinergic receptors on adjacent neuroepithelial cells and activating, via the vagus, the cough reflex. Infection of endothelial cells results in the exocytosis of WeibelPalade bodies. These contain the Von Willebrand Factor responsible for micro-clotting and angiopoietin-2 which increases vascular permeability and plays a key role in the Acute Respiratory Distress Syndrome. To test this hypothesis this paper reports proof of concept studies in which MR blockade using spironolactone and low dose dexamethasone (SpiDex) was given to PCR-confirmed COVID-19 patients. In 80 patients with moderate to severe respiratory failure 40 were given SpiDex and 40 conventional treatment with high dose dexamethasone (HiDex). There was 1 death in the HiDex group and none in the SpiDex. As judged by clinical, biochemical and radiological parameters there were clear statistically significant benefits of SpiDex in comparison to HiDex. A further 20 outpatients with COVID-19 were given SpiDex. There was no control group and the aim was to demonstrate safety. No adverse effects were noted and no patient became hyperkalaemic. 90% were asymptomatic at 10 days. The very positive results suggest that blockade of the MR can produce major benefit in COVID19 patients. Further larger controlled studies of inpatients and outpatients are required not only for SARS-CoV-2 infection per se but also to determine if this treatment affects the incidence of Long COVID.

Effects of Hypocalcemic Vitamin D Analogs in the Expression of DNA Damage Induced in Minilungs from Hescs: Implications for Lung Fibrosis

BackgroundIn our previous work, we evaluated the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of bleomycin-induced lung fibrosis. Contrary to the expected, vitamin D supplementation increased DNA damage expression and cellular senescence in alveolar epithelial type II cells and aggravated the overall lung pathology induced in mice by bleomycin. These effects were probably due to an alteration of the cellular DNA double-strand breaks repair capability. In the present work we have evaluated the effects of two hypocalcemic vitamin D analogs (calcipotriol and paricalcitol) in the expression of DNA damage in the context of minilungs derived from human embryonic stem cells and in the cell line A549.ResultsAs in the case of the cell line A549, bleomycin can induce DNA damage in the generated minilungs enriched in alveolar cells. The results indicate that, in contrast to vitamin D, the treatment of the minilungs with the hypocalcemic analogs reduce significantly the bulk of DNA damage expression in both bidimensional arrays of epithelial cells (2D minilungs) and lung bud organoids (3D minilungs). The initial evaluation of a battery of commercially available vitamin D analogs shows a significant reduction in A549 cells of gH2AFX expression levels, a marker of DNA damage, cell senescence and aging.ConclusionsThe treatments based in hypocalcemic vitamin D analogs might be used to reduce the bulk of DNA damage and eventually the subsequent cell senescence expression that underlie lung conditions as those that can evolve with fibrosis.

Surfactant-based therapy against COVID-19: A review

The coronavirus disease 2019 (COVID-19) has led to serious health and economic damage to all over the world, and it still remains unstoppable. The SARS-CoV-2, by using its S-glycoprotein, binds with an angiotensin-converting enzyme 2 receptor, mostly present in alveolar epithelial type II cells. Eventually pulmonary surfactant depletion occurs. The pulmonary surfactant is necessary for maintaining the natural immunity as well as the surface tension reduction within the lung alveoli during the expiration. Its insufficiency results in the reduction of blood oxygenation, poor pulmonary regeneration, lung fibrosis, and finally the respiratory system collapses. Exogenous surfactants have previously shown great promise in the treatment of infant respiratory distress syndrome, and they may also aid in the healing of damaged alveolar cells and the prevention of respiratory failure. Surfactant based therapy has been advised for the prevention of COVID-19, and the trials have begun around the world. Furthermore, greater research on the timing, dose, and the distribution of surfactant to the COVID-19 patients is required before this technique can be implemented in clinical practice.

Cough. The place of herbal medicine in treatment

Cough is one of the auxiliary mechanisms for cleaning the airways from mucus, foreign particles, microorganisms. The physiological cough reflex allows the mechanism of airway cleansing, provided that mucociliary clearance works sufficiently. However, sometimes the cough loses its protective function, becomes persistent, and impairs the quality of life of the patient. In this regard, in the treatment of cough, attention is paid to both secretomotor and secretolytic therapy. Medicinal plants are among the drugs with such properties. Numerous group of drugs containing herbal components has a reflex action, which allows coping most effectively with cough in the initial stages of diseases accompanied by respiratory symptoms. The most common among them and widely used are plantain leaf, coltsfoot leaf, thermopsis herb, ipecacuanha root, marshmallow root, licorice root, anise fruit, thyme (thyme) herb extract, ivy leaf extract. A well-known drug, the active ingredient of which is ivy leaf extract. Its mechanism of action consists in increasing the production of surfactant and increasing the number of β2-adrenoreceptors on the surface of alveolar cells of the bronchial tree, to which ivy active substance α-hederin is attached, which has a bronchospasmodic and expectorant action. Numerous clinical studies have proven a high efficacy and safety of the product based on ivy leaf extract, which allows us to recommend it as the drug of choice for symptomatic cough therapy in both children and adults during acute respiratory infections.

ACE2 and Innate Immunity in the Regulation of SARS-CoV-2-Induced Acute Lung Injury: A Review

Despite the protracted battle against coronavirus acute respiratory infection (COVID-19) and the rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), no specific and effective drugs have to date been reported. Angiotensin-converting enzyme 2 (ACE2) is a zinc metalloproteinase and a critical modulator of the renin-angiotensin system (RAS). In addition, ACE2 has anti-inflammatory and antifibrosis functions. ACE has become widely known in the past decade as it has been identified as the primary receptor for SARS-CoV and SARS-CoV-2, being closely associated with their infection. SARS-CoV-2 primarily targets the lung, which induces a cytokine storm by infecting alveolar cells, resulting in tissue damage and eventually severe acute respiratory syndrome. In the lung, innate immunity acts as a critical line of defense against pathogens, including SARS-CoV-2. This review aims to summarize the regulation of ACE2, and lung host cells resist SARS-CoV-2 invasion by activating innate immunity response. Finally, we discuss ACE2 as a therapeutic target, providing reference and enlightenment for the clinical treatment of COVID-19.