Alveolar Epithelial Type Research Articles

BORCS6 is involved in the enlargement of lung lamellar bodies in Lrrk2 knockout mice.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson disease. It has been shown that Lrrk2 knockout (KO) rodents have enlarged lamellar bodies (LBs) in their alveolar epithelial type II cells, although the underlying mechanisms remain unclear. Here we performed proteomic analyses on LBs isolated from Lrrk2 KO mice and found that the LB proteome is substantially different in Lrrk2 KO mice compared with wild-type mice. In Lrrk2 KO LBs, several Rab proteins were increased, and subunit proteins of BLOC-1-related complex (BORC) were decreased. The amount of surfactant protein C was significantly decreased in the bronchoalveolar lavage fluid obtained from Lrrk2 KO mice, suggesting that LB exocytosis is impaired in Lrrk2 KO mice. We also found that the enlargement of LBs is recapitulated in A549 cells upon KO of LRRK2 or by treating cells with LRRK2 inhibitors. Using this model, we show that KO of BORCS6, a BORC subunit gene, but not other BORC genes, causes LB enlargement. Our findings implicate the LRRK2-BORCS6 pathway in the maintenance of LB morphology.

GRK6 Depletion Induces HIF Activity in Lung Adenocarcinoma.

G protein-coupled receptor kinase 6 (GRK6) is expressed in various tissues and is involved in the development of several diseases including lung cancer. We previously reported that GRK6 is down-regulated in lung adenocarcinoma patients, which induces cell invasion and metastasis. However, further understanding of the role of GRK6 in lung adenocarcinoma is required. Here we explored the functional consequence of GRK6 inhibition in lung epithelial cells. Analysis of TCGA data was coupled with RNA sequencing (RNA-seq) in alveolar epithelial type II (ATII) cells following depletion of GRK6 with RNA interference (RNAi). Findings were validated in ATII cells followed by tissue microarray analysis. Pathway analysis suggested that one of the Hallmark pathways enriched upon GRK6 inhibition is ‘Hallmark_Hypoxia’ (FDR = 0.014). We demonstrated that GRK6 depletion induces HIF1α (hypoxia-inducible factor 1 alpha) levels and activity in ATII cells. The findings were further confirmed in lung adenocarcinoma samples, in which GRK6 expression levels negatively and positively correlate with HIF1α expression (P = 0.015) and VHL expression (P < 0.0001), respectively. Mechanistically, we showed the impact of GRK6 on HIF activity could be achieved via regulation of VHL levels. Taken together, targeting the HIF pathway may provide new strategies for therapy in GRK6-depleted lung adenocarcinoma patients.

Exosomal lncRNA TCONS_00064356 derived from injured alveolar epithelial type II cells affects the biological characteristics of mesenchymal stem cells

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disease, and a leading cause of morbidity and mortality worldwide. There is still a lack of effective treatment to improve pulmonary structural abnormality and reverse the progression of COPD. Mesenchymal stem cell (MSC)-based therapies have attracted much attention and show promising clinical application prospects in COPD treatment. Understanding the communication between injured alveolar cells and MSCs will help us improve the efficiency of MSC-based therapies. Here, we showed that exosomes secreted by injured alveolar epithelial type II (AEC-II) cells could promote the proliferation and migration of MSCs, accompanied with increased expression levels of genes related to mitochondrial synthesis and transfer. Moreover, we identified 21 significantly dysregulated exosomal lncRNAs (16 upregulated and 5 downregulated) using lncRNA sequencing. In addition, we found that lncRNA TCONS_00064356-overexpressing MSCs showed increased proliferation and migration capacities and upregulated expression levels of the genes related to mitochondrial synthesis and transfer. Together, our study uncovers a new potential exosome-mediated communication pathway between injured AEC-II cells and MSCs and provides new targets and ideas for improving the efficiency of MSC-based therapies for COPD.

Alveolar epithelial cell fate is maintained in a spatially restricted manner to promote lung regeneration after acute injury.

SUMMARYAlveolar epithelial type 2 (AT2) cells integrate signals from multiple molecular pathways to proliferate and differentiate to drive regeneration of the lung alveolus. Utilizing in vivo genetic and ex vivo organoid models, we investigated the role of Fgfr2 signaling in AT2 cells across the lifespan and during adult regeneration after influenza infection. We show that, although dispensable for adult homeostasis, Fgfr2 restricts AT2 cell fate during postnatal lung development. Using an unbiased computational imaging approach, we demonstrate that Fgfr2 promotes AT2 cell proliferation and restrains differentiation in actively regenerating areas after injury. Organoid assays reveal that Fgfr2-deficient AT2 cells remain competent to respond to multiple parallel proliferative inputs. Moreover, genetic blockade of AT2 cell cytokinesis demonstrates that cell division and differentiation are uncoupled during alveolar regeneration. These data reveal that Fgfr2 maintains AT2 cell fate, balancing proliferation and differentiation during lung alveolar regeneration.

Warburg and Scars? Alveolar Type II Cell Glycolytic Reprogramming and Differential Mitochondrial Dynamics Observed in a Murine Model of Pulmonary Fibrosis

Our emerging conceptual understanding of Idiopathic Pulmonary Fibrosis (IPF) highlights the significant role of alveolar epithelial type II cell (AT2) cell dysfunction in underlying susceptibility, disease severity, and disease progression. This multifunctional cell is solely responsible for the synthesis and secretion of pulmonary surfactant, a highly metabolically taxing process. Though mutations in surfactant component genes are one of the recognized etiological causes of IPF, the relationship between the metabolic program of the AT2 cell and IPF is poorly understood. Leveraging a novel preclinical model of IPF, the IER-SP-CI73T mouse, we aimed to characterize the role of altered AT2 metabolism in IPF fibrogenesis and remodeling. Using this model, we characterized the metabolic profile of SftpcI73T AT2 cells at 3 key time points (7,14, and 28 days) post tamoxifen induction corresponding to an initiation phase (0-7 days), development of a multicellular alveolitis (7-14 days), and transition to spontaneous fibrotic remodeling (14-28 days). Endpoints included assessment of intracellular ATP levels, TCA cycle intermediates, lactate, mitochondrial copy number, mitochondrial function, and mitochondrial dynamics. Transcriptional changes underlying the acquired metabolic program were assessed via bulk AT2 RNA-sequencing with key proteins validated by immunoblotting. We identified significant alterations to AT2 metabolism and mitochondrial function beginning in the early inflammatory phase and sustained though fibrogenesis which included: (i) early and significant transcriptional changes reflective of glycolytic reprograming and alterations in PPARg/PGC1a signaling; (ii) a novel metabolic profile marked by increased cellular ATP and accumulate TCA intermediates supporting the glycolytic phenotype; (iii) genomic, biochemical, and morphological changes indicative of impaired mitochondrial biogenesis and network dynamics suggesting sustained mitochondrial dysfunction during injury and fibrotic remodeling; (iv) transcriptional and histochemical evidence of a persistent, highly proliferative AT2 phenotype. In this preclinical model of IPF we observe an AT2 hyperproliferative state supported by glycolytic reprogramming and high ATP levels with features that recapitulate the classical Warburg effect observed in tumorigenesis. Together with observations of altered mitochondrial dynamics and function, these data suggest a significant role for metabolic dysregulation of the AT2 cell in IPF pathogenesis and present novel pathways for disease intervention.

Rosiglitazone restores pulmonary surfactant and lipid homeostasis in mice exposed to ozone

Ozone is a ubiquitous urban air pollutant that causes airway inflammation and hyperresponsiveness. Evidence indicates that alterations in lung lipid homeostasis due to dysregulated catabolism by alveolar macrophages and/or disrupted recycling by alveolar epithelial type II cells drives inflammation and chronic lung injury. RNA-seq analysis of alveolar macrophages revealed that exposure of mice to ozone resulted in alterations in inflammatory and lipid metabolism pathways, and down-regulation of PPARγ, a nuclear transcription factor important in controlling lipid metabolism, macrophage activation, and inflammation resolution. In the present studies, we analyzed the role of PPARγ in ozone-induced alterations in lung lipid homeostasis. Female C57BL/6J mice were treated with the PPARγ agonist, rosiglitazone or vehicle control by daily intraperitoneal injection beginning 24 hr prior to exposure to ozone (0.8 ppm, 3 hr) or air; mice were euthanized 72 hr post ozone. Bronchoalveolar lavage fluid (BAL) was collected and analyzed for total protein and phospholipid content, and expression of surfactant proteins. Effects on pulmonary function were assessed using a SciReq Flexivent. Ozone exposure resulted in increases in total protein and phospholipid content in BAL, consistent with lung injury and dysregulation of lipid metabolism. An increase in levels of the hydrophilic collectin, surfactant protein D (SP-D) and a decrease in the hydrophobic surfactant protein B (SP-B) were also observed in BAL after ozone exposure. SP-B and phospholipids regulate pulmonary function by maintaining lung surface tension and hysteresivity. Ozone exposure reduced lung hysteresivity at positive end-expiratory pressures of 1 and 3 cm H20 indicating reduced viscoelasticity of the lungs. Rosiglitazone administration significantly reduced BAL phospholipid content and restored SP-B to control levels. Consistent with these findings, rosiglitazone rescued ozone-induced impairment of lung function. Collectively, these results suggest that ozone exposure disrupts SP-B and phospholipid homeostasis which impairs pulmonary function; PPARγ improves lung function by restoring surfactant and lipid balance.

Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.

Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.

Segmentation of epithelial human type 2 cell images for the indirect immune fluorescence based on modified quantum entropy

The autoimmune disorders such as rheumatoid, arthritis, and scleroderma are connective tissue diseases (CTD). Autoimmune diseases are generally diagnosed using the antinuclear antibody (ANA) blood test. This test uses indirect immune fluorescence (IIf) image analysis to detect the presence of liquid substance antibodies at intervals the blood, which is responsible for CTDs. Typically human alveolar epithelial cells type 2 (HEp2) are utilized as the substrate for the microscope slides. The various fluorescence antibody patterns on HEp-2 cells permits the differential designation-diagnosis. The segmentation of HEp-2 cells of IIf images is therefore a crucial step in the ANA test. However, not only this task is extremely challenging, but physicians also often have a considerable number of IIf images to examine.In this study, we propose a new methodology for HEp2 segmentation from IIf images by maximum modified quantum entropy. Besides, we have used a new criterion with a flexible representation of the quantum image(FRQI). The proposed methodology determines the optimum threshold based on the quantum entropy measure, by maximizing the measure of class separability for the obtained classes over all the gray levels. We tested the suggested algorithm over all images of the MIVIA HEp 2 image data set.To objectively assess the proposed methodology, segmentation accuracy (SA), Jaccard similarity (JS), the F1-measure,the Matthews correlation coefficient(MCC), and the peak signal-to-noise ratio (PSNR) were used to evaluate performance. We have compared the proposed methodology with quantum entropy, Kapur and Otsu algorithms, respectively.The results show that the proposed algorithm is better than quantum entropy and Kapur methods. In addition, it overcomes the limitations of the Otsu method concerning the images which has positive skew histogram.This study can contribute to create a computer-aided decision (CAD) framework for the diagnosis of immune system diseases

The Intervention of the Pirfenidone with Pericyst Layer Building of the Hydatid Cyst

Hydatid disease is a zoonosis caused by Echinococcus granulosus larval stage, characterized by developing a fluid-filled hydatid cyst in any part in the body, with a particular liver and lung localization. The released hydatid cyst fluid (HCF) antigens actively induce epithelial to mesenchymal transition (EMT) in the epithelial cells surrounding the mass of the hydatid cyst, result in pericyst layer construction, which plays a vital role in preventing the antiparasitic medications from being reached to the parasite. Therefore, the current study attempted to employ the antifibrotic effect of the Pirfenidone to hinder the induced EMT by HCF antigens. For this reason, the adenocarcinomic human alveolar basal epithelial cells (A549 cell line) was used as a model system to human alveolar epithelial cells type II (AEC II) to assess the effect of the Pirfenidone on the induced EMT by HCF antigens. Different assays used to investigate the EMT induction, then Pirfenidone effect assessed using immunofluorescence assay to detect cytoskeletal markers expression prior to and after treatment. The results reached that the Pirfenidone interrupting the induced EMT in A549 cells, and thus, it may interrupt the EMT and pericyst fibrosis in the same way in the infected tissues, which would facilitate the antiparasitic drugs delivery across the cyst wall and help in parasite killing.

TLR3-Activated Monocyte-Derived Dendritic Cells Trigger Progression from Acute Viral Infection to Chronic Disease in the Lung.

Acute infection is implicated as a trigger for chronic inflammatory disease, but the full basis for this switch is uncertain. In this study, we examine this issue using a mouse model of chronic lung disease that develops after respiratory infection with a natural pathogen (Sendai virus). We investigate this model using a combination of TLR3-deficient mice and adoptive transfer of immune cells into these mice versus the comparable responses in wild-type mice. We found that acute and transient expression of TLR3 on monocyte-derived dendritic cells (moDCs) was selectively required to induce long-term expression of IL-33 and consequent type 2 immune-driven lung disease. Unexpectedly, moDC participation was not based on canonical TLR3 signaling and relied instead on a trophic effect to expand the alveolar epithelial type 2 cell population beyond repair of tissue injury and thereby provide an enriched and persistent cell source of IL-33 required for progression to a disease phenotype that includes lung inflammation, hyperreactivity, excess mucus production, and remodeling. The findings thereby provide a framework wherein viral infection activates TLR3 in moDCs as a front-line immune cell niche upstream of lung epithelial cells to drive the type 2 immune response, leading to chronic inflammatory diseases of the lung (such as asthma and chronic obstructive pulmonary disease in humans) and perhaps progressive and long-term postviral disease in general.

Synthetic Lung Surfactant Treatment for COVID-19 Pneumonia

COVID-19 has led to morbidity in millions of patients, ranging from mild flu-like symptoms to severe respiratory failure, necessitating oxygen supplementation and mechanical ventilation, and ultimately death. The SARS-CoV-2 virus reacts with angiotensin-converting enzyme 2 (ACE2) molecules that are especially found in alveolar epithelial type 2 cells in the lungs and thereby causes a loss in lung surfactant, a protein-lipid mixture that is crucial for both native immunity and reduction of surface tension in the lung alveoli. Lung surfactant insufficiency results in atelectasis and loss of functional lung tissue amid an inflammatory storm and may be countered by treating COVID-19 pneumonia patients with exogenous lung surfactant, preferably by aerosol delivery of a novel dry powder synthetic lung surfactant. More research on timing, dosing, and delivery of synthetic lung surfactant in patients with COVID-19 pneumonia is of crucial importance to implement this approach in clinical practice.

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung.

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air–liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.

SARS-CoV-2 Infection and Disease Modelling Using Stem Cell Technology and Organoids.

In this Review, we briefly describe the basic virology and pathogenesis of SARS-CoV-2, highlighting how stem cell technology and organoids can contribute to the understanding of SARS-CoV-2 cell tropisms and the mechanism of disease in the human host, supporting and clarifying findings from clinical studies in infected individuals. We summarize here the results of studies, which used these technologies to investigate SARS-CoV-2 pathogenesis in different organs. Studies with in vitro models of lung epithelia showed that alveolar epithelial type II cells, but not differentiated lung alveolar epithelial type I cells, are key targets of SARS-CoV-2, which triggers cell apoptosis and inflammation, while impairing surfactant production. Experiments with human small intestinal organoids and colonic organoids showed that the gastrointestinal tract is another relevant target for SARS-CoV-2. The virus can infect and replicate in enterocytes and cholangiocytes, inducing cell damage and inflammation. Direct viral damage was also demonstrated in in vitro models of human cardiomyocytes and choroid plexus epithelial cells. At variance, endothelial cells and neurons are poorly susceptible to viral infection, thus supporting the hypothesis that neurological symptoms and vascular damage result from the indirect effects of systemic inflammatory and immunological hyper-responses to SARS-CoV-2 infection.

Pulmonary alveolar stem cells undergo senescence, apoptosis and differentiation by p53-dependent and -independent mechanisms in telomerase deficient mice.

Pulmonary senescence and fibrosis occur with deoxyribonucleic acid (DNA) damage response in the lungs deficient of telomerase. The molecular mechanism mediating pulmonary alveolar cell fates remains to be investigated. The present study shows that pulmonary alveolar epithelial type 2 cells (AEC2) (alveolar stem cells) undergo not only replicative senescence, but also apoptosis and differentiation in association with increased innate immune natural killer (NK) cells in telomerase knockout (KO) mice. Telomerase ribonucleic acid (RNA) component (TERC) deficiency results in increased senescence-associated heterochromatin foci marker HP1γ, p21, p16 and apoptosis-associated cleaved caspase-3 in AEC2. However, p53 deficiency in the Trp53-/- allele of the late generation of TERC KO mice attenuates the increased senescent and apoptotic markers significantly. Moreover, p53 deficiency has no significant effect on the increased gene expression of T1α (a marker of terminal differentiated alveolar epithelial type 1 cells [AEC1]) in AEC2 of the late generation of TERC KO mice. Collectively, our findings suggest that pulmonary senescence takes place in deficiency of telomerase RNA component with the alveolar stem cells undergoing p53-dependent senescence and apoptosis as well as p53-independent differentiation.

Cigarette smoke-inactivated SIRT1 promotes autophagy-dependent senescence of alveolar epithelial type 2 cells to induce pulmonary fibrosis

AimsThe senescence of alveolar epithelial type 2 (AT2) cells is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Cigarette smoke (CS) is a strong risk factor for IPF and it is also a pro-senescent factor. Here we aimed to investigate whether and how CS induces AT2 cells senescence via a SIRT1/autophagy dependent pathway. Our results showed that CS extract (CSE) reduced autophagy and mitophagy and increased mitochondrial reactive oxygen species (mitoROS) in MLE-12 cells, an AT2 cell line. The autophagy inducer rapamycin (RAPA) and the mitochondria-targeted antioxidant mitoquinone (mitoQ) inhibited CSE-related senescence and decreased mitoROS. Next, we found that CSE promoted DNA damage, downregulated the nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide (NADH) ratio and suppressed SIRT1 activity. Activating SIRT1 with its activator SRT1720 attenuated senescence through an autophagy-dependent pathway. The NAD+ precursor nicotinamide mononucleotide and the poly ADP-ribose polymerase (PARP1) inhibitor olaparib also exerted anti-senescent effects by activating SIRT1. Moreover, the results showed that mitoQ and RAPA, in turn, elevated SIRT1 activity by inhibiting DNA damage. Consistent with these results, SRT1720 and mitoQ mitigated CS-induced AT2 cells senescence and lung fibrosis in vivo. Moreover, autophagy in AT2 cells was rescued by SRT1720. Taken together, our results suggested that CS-induced senescence of AT2 cells was due to decreased autophagy mediated by SIRT1 inactivation, which was attributed to competitive consumption of NAD+ caused by DNA damage-induced PARP1 activation. The reduction in autophagy, in turn, decreased SIRT1 activity by promoting mitochondrial oxidative stress-related DNA damage, thereby establishing a positive feedback loop between SIRT1 and autophagy in CS-induced AT2 cells senescence. Consequently, CS-inactivated SIRT1 promoted autophagy-dependent senescence of AT2 cells to induce pulmonary fibrosis.

Improved Alveolar Dynamics and Structure After Alveolar Epithelial Type II Cell Transplantation in Bleomycin Induced Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressively and ultimately fatal lung disease. Previously it has been shown that intratracheal administration of alveolar epithelial type II cells (AE2C) in the animal model of bleomycin-induced pulmonary fibrosis is able to reverse fibrosis and restore surfactant protein levels. However, to date, it has not been evaluated whether these changes involve any improvement in alveolar dynamics. Consequently, the aim of the present work was to study lung physiology after AE2C transplantation at different time points during the development of injury and fibrosis. Lung fibrosis was induced by intratracheal instillation of bleomycin (4U/kg) in rat lungs. The animals were transplanted with AE2C (2.5 × 106 cells/animal) 3 or 7 days after bleomycin instillation. Assessments were done at day 7 and 14 after the induction of fibrosis to plot time dependent changes in lung physiology and mechanics. To assess the pressures and rates at which closed alveoli reopens invasive pulmonary tests using a small-animal mechanical ventilator (Flexivent®, Scireq, Canada) including de-recruitability tests and forced oscillation technique as well as quasi-static pressure volume loops were performed. Afterwards lungs were fixed by vascular perfusion and subjected to design-based stereological evaluation at light and electron microscopy level. AE2C delivered during the lung injury phase (3 days) of the disease are only able to slightly recover the volume of AE2C and volume fraction of LB in AE2C. However, it did not show either positive effects regarding ventilated alveolar surface nor any increase of lung compliance. On the other hand, when AE2C are delivered at the beginning of the fibrotic phase (7 days after bleomycin instillation), an increased ventilated alveolar surface to control levels and reduced septal wall thickness can be observed. Moreover, transplanted animals showed better lung performance, with increased inspiratory capacity and compliance. In addition, a detailed analysis of surfactant active forms [mainly tubular myelin, lamellar body (LB)-like structures and multilamellar vesicles (MLV)], showed an effective recovery during the pro-fibrotic phase due to the healthy AE2C transplantation. In conclusion, AE2C transplantation during fibrogenic phases of the disease improves lung performance, structure and surfactant ultrastructure in bleomycin-induced lung fibrosis.

Versatile, High-Resolution, and Large Field-of-View Single-Molecule Imaging by Oblique Plane Microscopy

Building on recent advancements in oblique plane microscopy (OPM), also known as single objective light sheet, we demonstrate a versatile, high resolution, and large field-of-view OPM with single-molecule resolution. We achieve 300-nm lateral resolution and 800-nm axial resolution, without deconvolution, through the use of a bespoke solid immersion objective for remote focusing. We extend the imaging volume by rapidly translating the sample through the oblique light sheet using optimized stage scanning and digital control of the light sheet to eliminate motion blur. Because OPM requires only one objective at the sample, we can perform high-resolution light sheet imaging using standard sample mounting methods, including microfluidics for flow cytometry and fluidic chambers for iterative multiplexed labeling. We quantitatively evaluated and verified the instrument's volume performance using calibration samples and multiple imaging-based resolution metrics. As constructed, we can image 1-cm x 1-cm x 60-micron (x,y,z) per color per hour, with single-molecule resolution, in real samples. To demonstrate the instrument's capability, we quantified the co-expression of RNA and protein markers for the SARS-CoV-2 entry genes ACE2 and TMPRSS2 in alveolar epithelial type II cells across centimeter-scale fixed human lung tissue slices. In contrast to currently used imaging methods for spatial transcriptomics, our OPM approach provides the correct spatial sampling required to perform rigorous 3D single-molecule localization. Importantly, we achieve this high sampling rate and light collection efficiency at volume imaging rates that are orders of magnitude faster than existing approaches. Beyond spatial -omics, we additionally performed 3D single-molecule localization microscopy in fixed cells and live-cell imaging with with no modifications to the instrument. We envision the increase in resolution, sampling, and speed of high numerical aperture OPM versus traditional high numerical aperture microscopes will greatly aid high-throughput single-molecule imaging.

The Obesity Paradox Predicts the Second Wave of COVID-19 to Be Severe in Western Countries.

While COVID-19 infection and mortality rates are soaring in Western countries, Southeast Asian countries have successfully avoided the second wave of the SARS-CoV-2 pandemic despite high population density. We provide a biochemical hypothesis for the connection between low COVID-19 incidence, mortality rates, and high visceral adiposity in Southeast Asian populations. The SARS-CoV-2 virus uses angiotensin-converting enzyme 2 (ACE2) as a gateway into the human body. Although the highest expression levels of ACE2 are found in people’s visceral adipose tissue in Southeast Asia, this does not necessarily make them vulnerable to COVID-19. Hypothetically, high levels of visceral adiposity cause systemic inflammation, thus decreasing the ACE2 amount on the surface of both visceral adipocytes and alveolar epithelial type 2 cells in the lungs. Extra weight gained during the pandemic is expected to increase visceral adipose tissue in Southeast Asians, further decreasing the ACE2 pool. In contrast, weight gain can increase local inflammation in fat depots in Western people, leading to worse COVID-related outcomes. Because of the biological mechanisms associated with fat accumulation, inflammation, and their differential expression in Southeast Asian and Western populations, the second wave of the pandemic may be more severe in Western countries, while Southeast Asians may benefit from their higher visceral fat depots.

Hydroxypropyl Cyclodextrin Improves Amiodarone-induced Aberrant Lipid Homeostasis of Alveolar Cells.

Alveolar epithelial type II (AT2) cells secrete pulmonary surfactant via lamellar bodies (LBs). Abnormalities in LBs are associated with pulmonary disorders, including fibrosis. However, high-content screening (HCS) for LB abnormalities is limited by the lack of understanding of AT2 cell functions. In the present study, we have developed LB cells harboring LB-like organelles that secrete surfactant proteins. These cells were more similar to AT2 cells than to parental A549 cells. LB cells recapitulated amiodarone (AMD)-induced LB enlargement, similar to AT2 cells of patients exposed to AMD. To reverse AMD-induced LB abnormalities, we performed HCS of approved drugs and identified 2-hydroxypropyl-β-cyclodextrin (HPβCD), a cyclic oligosaccharide, as a potential therapeutic agent. A transcriptome analysis revealed that HPβCD modulates lipid homeostasis. In addition, HPβCD inhibited AMD-induced LB abnormalities in human induced pluripotent stem cell-derived AT2 cells. Our results demonstrate that LB cells are useful for HCS and suggest that HPβCD is a candidate therapeutic agent for AMD-induced interstitial pneumonia.

Dexmedetomidine targets miR-146a and participates in the progress of chronic obstructive pulmonary disease in vivo and in vitro.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease and the third leading cause of death in the world. Dexmedetomidine has been reported to effectively inhibit histamine-induced bronchoconstriction. However, the molecular mechanism of dexmedetomidine in COPD has not been found. To explore the role and mechanism of dexmedetomidine in COPD, and to provide theoretical basis for clinical treatment of COPD. The expression of miR-146a was regulated by mimics or inhibitor and the relative expression of apoptotic proteins p53, Bax and Bcl-2 in human bronchial epithelial 16HBE cells was determined by real-time PCR and Western blot. Dexmedetomidine was treated for 16HBE cells and alveolar epithelial type II cells (AEC2), the cell apoptosis was detected by TUNEL and Hoechst33342 staining. A COPD rat model was established by smoking to test the effects of dexmedetomidine on the progression of COPD. The levels of IL-6, IL-1β and TNF-α in serum were measured by ELISA and the protein concentration of bronchoalveolar lavage fluid (BALF) was also detected in dexmedetomidine treated COPD rat model. miR-146a promoted 16HBE cell apoptosis and reduced cell proliferation. Additionally, dexmedetomidine was showed to reduce the 16HBEL cell apoptosis through reducing the expression of miR-146a. Moreover, dexmedetomidine regulated cell apoptosis and cell apoptosis through miR-146a in AEC2 cells. More importantly, dexmedetomidine attenuated the morphology and pathology of COPD rat model. Dexmedetomidine reduced 16HBE cells and AEC2 cell apoptosis and attenuated COPD by down-regulating miR-146a.