Alveolar Epithelial Type Research Articles

Andrographolide Suppresses Expressions of Coagulation and Fibrinolytic Inhibition-Related Factors in LPS-Induced Alveolar Epithelial Cell Type II via NF-κB Signal Pathway In Vitro

BackgroundAndrographolide (Andro) has been confirmed to ameliorate alveolar hypercoagulation and fibrinolysis inhibition via NF-κB pathway in acute respiratory distress syndrome (ARDS), but the specific target of Andro is unknown.PurposeOur aim is to explore the specific target of Andro through which the drug exerted its effects on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS.MethodsAECII was treated with different doses of Andro for 1 h, and then stimulated with LPS for 24 h. Expressions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) were detected. Concentrations of thrombin-antithrombin complex (TAT), pro-collagen type III peptide (PIIIP), antithrombin III (ATIII) and activated protein C (APC) in cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). NF-κB signaling pathways activation was simultaneously determined. AECII with p65 down-/over-expression were used as control.ResultsAndro effectively inhibited TF and PAI-1 and promoted TFPI expressions on AECII induced by LPS stimulation. Andro also significantly suppressed the productions of TAT and PIIIP but promoted ATIII and APC secretions from the LPS-treated cell. Furthermore, Andro application obviously inhibited NF-κB signaling pathway activation provoked by LPS, as shown by decreased level of phosphorylation (p‑)-IKKβ/IKKβ, p-p65/p65 and p65 DNA binding activity. The effects of Andro on those factors were obviously strengthened by down- but were weakened by up-regulation of p65 gene in AECII cell.ConclusionsOur data demonstrates that targeting AECII is the mechanism by which Andro ameliorates alveolar hypercoagulaiton and fibrinolytic inhibition via NF-κB pathway in ARDS. Andro is worth to be clinically further studied in ARDS treatment.

Klf5 defines alveolar epithelial type 1 cell lineage commitment during lung development and regeneration.

Alveolar epithelial cell fate decisions drive lung development and regeneration. Using transcriptomic and epigenetic profiling coupled with genetic mouse and organoid models, we identified the transcription factor Klf5 as an essential determinant of alveolar epithelial cell fate across the lifespan. We show that although dispensable for both adult alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell homeostasis, Klf5 enforces AT1 cell lineage fidelity during development. Using infectious and non-infectious models of acute respiratory distress syndrome, we demonstrate that Klf5 represses AT2 cell proliferation and enhances AT2-AT1 cell differentiation in a spatially restricted manner during lung regeneration. Moreover, exvivo organoid assays identify that Klf5 reduces AT2 cell sensitivity to inflammatory signaling to drive AT2-AT1 cell differentiation. These data define the roll of a major transcriptional regulator of AT1 cell lineage commitment and of the AT2 cell response to inflammatory crosstalk during lung regeneration.

MicroRNA-20b carried by mesenchymal stem cell-derived extracellular vesicles protects alveolar epithelial type II cells from Mycobacterium tuberculosis infection in vitro.

Mesenchymal stem cells (MSCs) have been largely used for their immunomodulatory and regenerative properties in the treatment of immune-based disorders and bacterial infections. This study explores the function of MSC-derived extracellular vesicles (MSC-EVs) in alveolar epithelial type II cells (AECII) against Mycobacterium tuberculosis (MTB) infection. EVs were extracted from the acquired MSCs. AECII-like MLE-15 and A549 cells were treated with MSC-EVs and then subjected to MTB infection. MSC-EVs treatment significantly prevented the increase in bacterial load, and it prevented the production of proinflammatory cytokines in cells induced by MTB infection. MicroRNA-20b (miR-20b) was upregulated in cells after MSC-EVs treatment. Artificial inhibition of miR-20b blocked the protective effects of MSC-EVs against MTB infection. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to analyze the key molecules involved in the immune regulation in cells mediated by miR-20b. miR-20b directly targeted nuclear factor of activated T cells 5 (NFAT5) and inactivated the Toll-Like Receptor (TLR) signaling pathway by reducing the formation of TLR2-TLR4 dimer after MTB infection. In conclusion, this study suggests that MSC-EVs carry miR-20b to inhibit NFAT5 and inactivate the TLR signaling pathway, thus mediating innate immune response and preventing AECII from MTB infection-induced damage.

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Alveolar Type II Cell Proliferation and Attenuate Lung Inflammation in a Rat Model of Bronchopulmonary Dysplasia.

Although it is known that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) alleviate hyperoxic lung injury of bronchopulmonary dysplasia (BPD) in animal models, the role of microvesicles (MVs) derived from hUCMSCs in BPD is poorly defined. Furthermore, antenatal inflammation has been linked to high risk of BPD in preterm infants. The purpose of this study was to explore whether MVs derived from hUCMSCs can preserve lung structure and function in an antenatal lipopolysaccharide- (LPS-) induced BPD rat model and to clarify the underlying mechanism. We demonstrate that antenatal LPS induced alveolar simplification, altered lung function, and dysregulated pulmonary vasculature, which restored by hUCMSCs and MVs treatment. Furthermore, MVs were large vesicles with a diameter of 100-900 nanometers and mostly uptaken by alveolar epithelial type II cells (AT2) and macrophages. Compared with the LPS-exposed group, MVs restored the AT2 cell number and SP-C expression in vivo and promoted the proliferation of AT2 cells in vitro. MVs also restored the level of IL-6 and IL-10 in lung homogenate. Additionally, PTEN/AKT and MAPK pathways were associated with the protection of MVs. Taken together, this study suggests MVs derived from hUCMSCs improve lung architecture and function in an antenatal LPS-induced BPD rat model by promoting AT2 cell proliferation and attenuating lung inflammation; thus, MVs provide a promising therapeutic vehicle for BPD treatment.

Activating transcription factor 3 protects alveolar epithelial type II cells from Mycobacterium tuberculosis infection-induced inflammation

Activating transcription factor 3 (ATF3) is a stress-inducible gene reported with anti-inflammatory response effects against bacterial infections. This study focuses on the function of ATF3 in alveolar epithelial type II cells (A549) following Mycobacterium tuberculosis (MTB) infection. First, RT-qPCR results detected reduced ATF3 expression in broncho-alveolar lavage fluid (BALF) of MTB-infected patients, whereas the ATF3 level was upregulated in A549 cells at early stages after MTB infection but decreased later. The binding relationship between ATF3 and TIMP metallopeptidase inhibitor 2 (TIMP2) promoter was predicted via bioinformatic prediction and validated by ChIP and luciferase assays. ATF3 bound to TIMP2 promoter for transcriptional activation. Overexpression of ATF3 or TIMP2 enhanced autophagy activity, elevated p62 levels and the LC3BII/LC3BI ratio, and decreased IL-6 and TNF-α levels in A549 cells. The ATF3/TIMP2 axis suppressed the NF-κB pathway to alleviate inflammatory responses in A549 cells. Mice were exposed to MTB aerosol for in vivo experiments. Increased ATF3 expression was correlated with increased autophagy activity, clearance of bacteria as well as inflammation resolution in mouse lung tissues. In conclusion, this study demonstrates that ATF3 promotes cell autophagy and suppresses inflammatory response in MTB-infected A549 cells via TIMP2 activation and NF-κB suppression.

Revealing the pathogenic and ageing-related mechanisms of the enigmatic idiopathic pulmonary fibrosis (and chronic obstructive pulmonary disease).

Growing evidence suggests that ageing-associated alterations occur in both idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Here, we review the most recent literature on dysregulated ageing pathways in IPF and COPD and discuss how they may contribute to disease pathogenesis. Recent studies have shown that alveolar epithelial type II (ATII) cells undergo premature senescence under stress and that senescent ATII cells promote lung fibrogenesis. Some studies have explored the role of mitochondrial dysfunction in IPF. They have provided evidence that dysfunctional mitochondria are important contributors to fibrogenesis through release of damaged DNA and excessive formation of reactive oxygen species, whereas restoration of mitochondrial homeostasis may attenuate lung fibrosis. Insufficient autophagy has been shown to promote epithelial-to-mesenchymal transition and aberrant epithelial-fibroblast crosstalk, suggesting that autophagy augmentation may represent a potential therapeutic strategy. A number of studies have also explored the role of cellular senescence, mitochondrial homeostasis and autophagy in COPD. Several ageing mechanisms are dysregulated in the lungs of patients with IPF and COPD, although how they contribute to disease development and progression remains elusive. Genetic or pharmacologic attenuation of senescence-related pathways and elimination of senescent cells may represent a promising therapeutic strategy.

Regeneration-Associated Transitional State Cells in Pulmonary Fibrosis.

Pulmonary fibrosis is a chronic, progressive fibrosing interstitial disease. It is characterized by fibroblast proliferation, myofibroblast activation, and massive extracellular matrix deposition. These processes result in loss of lung parenchyma function. The transdifferentiation of alveolar epithelial type II (AEC2) to alveolar epithelial type I cells (AEC1) plays an important role in the epithelial repair after lung injury. Pulmonary fibrosis begins when this transdifferentiation process is blocked. Several recent studies have found that novel transitional state cells (intermediate states in the transdifferentiation of AEC2 to AEC1) can potentially regenerate the alveolar epithelium surface and promote a repair process. During the AEC2 to AEC1 trans-differentiation process after injury, AEC2 lose their specific markers and become transitional state cells. Furthermore, transdifferentiation of transitional state cells into AEC1 is the critical step for lung repair. However, transitional cells stagnate in the intermediate states in which failure of transdifferentiation to AEC1 may induce an inadequate repair process and pulmonary fibrosis. In this review, we focus on the traits, origins, functions, and activation of signaling pathways of the transitional state cell and its communication with other cells. We also provide a new opinion on pulmonary fibrosis pathogenesis mechanisms and novel therapeutic targets.

Abstract 267: Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition

Abstract Background: Circulating tumor cells (CTCs) represent micrometastatic disease and may offer unique insights into future recurrences in lethal malignancies, including non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs in this potentially curable patient group. Methods: Surgically resected NSCLC primary tumor tissues were implanted in immunodeficient mice to establish ten patient-derived xenografts (PDXs). CTCs from 2/10 PDX models led to generation of two stable metastatic models that were studied by single cell sequencing. Results: Single cell analysis revealed an additional alveolar epithelial type II (AT2) population in metastatic tumors, besides a common AT2 cluster in PDX/metastatic tumors. This was consistent with an external validation set analysis in primary and metastatic NSCLC patient tumors. Further, AT2 clusters of metastatic tumors expressed higher cancer stemness genes versus primary PDX tumor that was recapitulated in patients primary and metastatic tumors. Conclusions: Stable metastatic models from early stage NSCLC patients can be generated with CTCs from PDX models. The distinct AT2 population identified in CDX tumors with cancer stemness features might be critical mediator of metastasis that deserves further study to discover personalized strategies against NSCLC micrometastases. Citation Format: Kanve Nagaraj Suvilesh, Yulia I. Nussbaum, Vijay Radhakrishnan, Yariswamy Manjunath, Diego M. Avella, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Aadel A. Chaudhuri, Chi-Ren Shyu, Guangfu Li, Klaus Pantel, Wesley C. Warren, Jonathan B. Mitchem, Jussuf T. Kaifi. Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 267.

Fyn-kinase and caveolin-1 in the alveolar epithelial junctional adherence complex contribute to the early stages of pulmonary fibrosis

Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1β. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-β damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-β and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-β and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-β/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.

MiR-199a-3p in mouse bone marrow mesenchymal stem cell exosomes increases epithelial sodium channel expression in lung injury.

Acute lung injury (ALI) causes significant morbidity and mortality in critically ill patients, which often presents with extensive accumulation of activated inflammatory cells and diffused alveolar damage accompanied by oxidative stress. Exosomes are nanovesicles, which have notable anti-inflammatory and repair properties, thus alleviating the symptoms of ALI. Epithelial sodium channel (ENaC) is essential for the transepithelial absorption of Na+ and fluid from alveolar spaces. We studied the effects of bone marrow mesenchymal stem cell exosomes (BMSC-exo) on the apoptosis and protein expression of ENaC in primary mouse alveolar epithelial type 2 cells (AT 2 cells). Moreover, the change of miR-199a-3p in AT 2 cells was detected by qRT-PCR, and we studied the regulation of miR-199a-3p on ENaC protein expression. Our results demonstrated that BMSC-exo could not only improve viability and reduce apoptosis in AT 2 cells, but also enhance the expression of ENaC protein and miR-199a-3p. Meanwhile, the upregulation of miR-199a-3p resulted in increased expression of ENaC protein. In summary, the BMSC-exo could participate in the regulation of ENaC through miR-199a-3p originated from BMSC-exo, thereby providing a new pharmacological tool for the treatment of ALI.

Impact of e-cigarette aerosol on primary human alveolar epithelial type 2 cells.

Electronic cigarettes (e-cigarettes) are designed to simulate combustible cigarette smoking and to aid in smoking cessation. Although the number of e-cigarette users has been increasing, the potential health impacts and biological effects of e-cigarettes are still not fully understood. Previous research has focused on the biological effects of e-cigarettes on lung cancer cell lines and distal airway epithelial cells; however, there have been few published studies on the effect of e-cigarettes on primary lung alveolar epithelial cells. The primary purpose of this study was to investigate the direct effect of e-cigarette aerosol on primary human lung alveolar epithelial type 2 (AT2) cells, both alone and in the presence of viral infection. The Melo-3 atomizer caused direct AT2 cell toxicity, whereas the more popular Juul pod's aerosol did not have a detectable cytotoxic effect on AT2 cells. Juul nicotine aerosol also did not increase short-term susceptibility to viral infection. However, 3 days of exposure upregulated genes central to the generation of reactive oxygen species, lipid peroxidation, and carcinogen metabolism and downregulated key innate immune system genes related to cytokine and chemokine signaling. These findings have implications for the potentially injurious impact of long-term use of popular low-power e-cigarette pods on the human alveolar epithelium. Gene expression data might be an important endpoint for evaluating the potential harmful effects of vaping devices that do not cause overt toxicity.

THE WNK4/SPAK PATHWAY STIMULATES ALVEOLAR FLUID CLEARANCE BY UPREGULATION OF EPITHELIAL SODIUM CHANNEL IN MICE WITH LIPOPOLYSACCHARIDE-INDUCED ACUTE RESPIRATORY DISTRESS SYNDROME.

ABSTRACTWith-No lysine Kinases (WNKs) have been newly implicated in alveolar fluid clearance (AFC). Epithelial sodium channels (ENaCs) serve a vital role in AFC. The potential protective effect of WNK4 in acute respiratory distress syndrome (ARDS), mediated by ENaC-associated AFC was investigated in the study. A model of lipopolysaccharide (LPS)-induced ARDS was established in C57BL/6 mice. WNK4, Sterile 20-related proline-alanine-rich kinase (SPAK), small interfering RNA (siRNA)-WNK4 or siRNA-SPAK were transfected into mouse lung or primary alveolar epithelial type II (ATII) cells. AFC, bronchoalveolar lavage fluid and lung histomorphology were determined. The expression of ENaC was determined to investigate the regulation of AFC by WNK4-SPAK signaling pathway. Activation of WNK4-SPAK signaling improved lung injury and survival rate, with enhanced AFC and reduced pulmonary edema via the upregulation of ENaC in ARDS. In primary rat ATII cells, gene-silencing by siRNA transfection reduced ENaC expression and the level of WNK4-associated SPAK phosphorylation. Immunoprecipitation revealed that the level of neural precursor cell-expressed developmentally downregulated gene 4 (Nedd4-2) binding to ENaC was decreased as a result of WNK4-SPAK signaling. The present study demonstrated that the WNK4/SPAK pathway improved AFC during LPS-induced ARDS, which is mainly dependent on the upregulation of ENaC with Nedd4-2-mediated ubiquitination.

Imbalance of alveolar epithelial type I and type Ⅱ cells in lipopolysaccharide-induced chronic lung injury young mouse model

Children are susceptible to pneumonia, which affects their growth and development. Immune disorders and unrepaired alveolar mucosal epithelium following pneumonia cause chronic lung injury. The mechanism of chronic lung injury is unknown and lacks animal models for reference. Therefore, we developed a chronic lung injury young mouse model to simulate the pathological process of children. 3-week-old mice were intratracheal instillation of lipopolysaccharide (LPS) every other day for six weeks. Consequently, the histopathology showed damaged integrity of lung tissue, fibrosis, and abnormally distributed alveolar epithelial cells. The total protein concentration in bronchoalveolar lavage fluid (BALF) was increased, alveolar epithelial type (AT) I cells were abnormal distribution, and AT II cells were reduced. The phosphorylation levels of IKBα and the expression levels of NF-κB p65 in lung tissue were up-regulated. In serum and BALF, the IL-6 was oversecretion, nitric oxide (NO) and superoxide dismutase (SOD) were perturbed secretion, oxidative stress imbalance. In addition, blood viscosity, plasma viscosity, and erythrocyte sedimentation rate (ESR) indexes in hemorheology were increased. In conclusion, it is feasible to construct the mouse model of chronic lung injury, and AT I and AT Ⅱ cells were imbalanced, which paves the way for further investigations on the pathogenesis of chronic lung injury and the efficacy of novel treatments.

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; Fgfr2bflox/flox; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called “injury activated alveolar progenitors” or IAAPs. Upon continuous tamoxifen exposure for either 1 or 2 weeks to delete Fgfr2b, a shrinking of the AT2 population is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature, suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a 1-week tamoxifen exposure followed by an 8-week chase period. Although mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung during homeostasis and suggest that the IAAPs could represent a new population of AT2 progenitors.

Distinct Epithelial Cell Profiles in Normal Versus Induced-Congenital Diaphragmatic Hernia Fetal Lungs.

BackgroundRecent studies identified a great diversity of cell types in precise number and position to create the architectural features of the lung that ventilation and respiration at birth depend on. With damaged respiratory function at birth, congenital diaphragmatic hernia (CDH) is one of the more severe causes of fetal lung hypoplasia with unspecified cellular dynamics.Objectives To characterize the epithelial cell tissue in hypoplastic lungs, a careful analysis regarding pulmonary morphology and epithelial cell profile was conducted from pseudoglandular-to-saccular phases in normal versus nitrofen-induced CDH rat lungs.DesignOur analysis comprises three experimental groups, control, nitrofen (NF) and CDH, in which the relative expression levels (western blot) by group and developmental stage were analyzed in whole lung. Spatiotemporal distribution (immunohistochemistry) was revealed by pulmonary structure during normal and hypoplastic fetal lung development. Surfactant protein-C (SP-C), calcitonin gene-related peptide (CGRP), clara cell secretory protein (CCSP), and forkhead box J1 (FOXJ1) were the used molecular markers for alveolar epithelial cell type 2 (AEC2), pulmonary neuroendocrine, clara, and ciliated cell profiles, respectively.ResultsGenerally, we identified an aberrant expression of SP-C, CGRP, CCSP, and FOXJ1 in nitrofen-exposed lungs. For instance, the overexpression of FOXJ1 and CGRP in primordia of bronchiole defined the pseudoglandular stage in CDH lungs, whereas the increased expression of CGRP in bronchi; FOXJ1 and CGRP in terminal bronchiole; and SP-C in BADJ classified the canalicular and saccular stages in hypoplastic lungs. We also described higher expression levels in NF than CDH or control groups for both FOXJ1 in bronchi, terminal bronchiole and BADJ at canalicular stage, and SP-C in bronchi and terminal bronchiole at canalicular and saccular stages. Finally, we report an unexpected expression of FOXJ1 in BADJ at canalicular and saccular stages, whereas the multi cilia observed in bronchi were notably absent at embryonic day 21.5 in induced-CDH lungs.ConclusionThe recognized alterations in the epithelial cell profile contribute to a better understanding of neonatal respiratory insufficiency in induced-CDH lungs and indicate a problem in the epithelial cell differentiation in hypoplastic lungs.

Mechanical Tension‐induced Vascular and Alveolar Morphogenesis during Lung Regeneration

Mechanical forces altered after unilateral pneumonectomy (PNX) control post‐PNX vascular and alveolar morphogenesis. This study aims to understand the mechanosensitive mechanism of lung vascular and alveolar regeneration in the post‐PNX mouse lungs. Vascular formation is stimulated at the subpleural region in the remaining mouse lung after unilateral PNX, where mechanical stretch is enhanced, while inhibition of mechanical tension by insertion of prosthesis attenuates these effects. The focal adhesion protein, paxillin senses mechanical stretch after PNX and the levels of paxillin are upregulated in SPB‐positive alveolar epithelial type II (ATII) cells and endothelial cells (ECs), which stimulates the expression of angiogenic factor angiopoietin‐1 (Ang1) in ATII cells and Ang2 in ECs to induce EC directional motility to the subpleural region. Cellular stretching increases the expression of paxillin, which induces expression of Ang2 in ECs and Ang1 in ATII cells in vitro. Post‐PNX lung growth and vascular formation toward the subpleural region are suppressed in Pxnfl/fl‐Sftpc‐CreERT2 mouse lungs, in which paxillin expression is attenuated in ATII cells. Structure of lamellar bodies that control surfactant homeostasis is also disrupted in post‐PNX Pxnfl/fl‐Sftpc‐CreERT2 mouse lung ATII cells. These results suggest that cooperative action of mechanosensitive paxillin signaling in ECs and ATII cells mediates vascular and alveolar epithelial formation during post‐PNX lung growth.

Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis

Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial “transitional state” enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown.We previously reported the in vivo modeling of PF utilizing inducible, knock‐in expression of a clinical Surfactant Protein C (SP‐C) mutation in alveolar type 2 cells (AEC2). Expression of the SP‐C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury.Using two clinical SP‐C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis.We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro‐fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

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

In 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 the 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 in 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.

Immunophenotyping of Acute Inflammatory Exacerbations of Lung Injury Driven by Mutant Surfactant Protein-C: A Role for Inflammatory Eosinophils.

Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. Among the several inflammatory populations mobilizing during AIEs, eosinophils have been identified as promising indicators of an active inflammatory exacerbation. To better study the eosinophil-parenchymal crosstalk during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by inducible expression of a mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. Unbiased single-cell sequencing analysis of controls and SP-CI73T mutants at a time coordinated with peak eosinophilia (14 days) defined heightened inflammatory activation, chemotaxis, and survival signaling (IL-6, IL-4/13, STAT3, Glucocorticoid Receptor, mTOR, and MYC) in eosinophils. To study the impact of eosinophils in inflammatory exacerbations, the SP-CI73T line was crossed with eosinophil lineage deficient mice (GATA1Δdbl) to produce the SP-CI73TGATA1KO line. Time course analysis (7–42 days) demonstrated improved lung histology, survival, and reduced inflammation in SP-CI73TGATA1KO cohorts. Spectral flow cytometry of tissue digests confirmed eosinophil depletion in GATA1KO mice and the absence of a compensatory shift in neutrophils and immature monocyte recruitment. Eosinophil deletion resulted in progressive monocyte-derived macrophage accumulation (14 days post-injury), combined with declines in CD3+CD4+ lymphocyte and B220+ B cell abundance. Histochemical analysis revealed atypical inflammatory cell activation in SP-CI73TGATA1KO mice, with reduced numbers of Arg-1+ and iNOS+ cells, but increases in tgfb1 mRNA expression in bronchoalveolar lavage cells and tissue. Dexamethasone treatment (1 mg/kg daily, i.p.) was utilized to investigate corticosteroid efficacy in highly eosinophilic exacerbations induced by mutant SP-CI73T. Dexamethasone successfully reduced total and eosinophil (CD11b+SigF+CD11c−) counts at 14 days and was linked to reduced evidence of structural damage and perivascular infiltrate. Together, these results illustrate the deleterious role of eosinophils in inflammatory events preceding lung fibrosis and demonstrate the efficacy of corticosteroid treatment in highly eosinophilic exacerbations induced by mutant SP-CI73T.

Increased Serum SP-D Level, Neutrophils and Lymphocytes Sputum in Malang Splendid Bird Market Workers

Background: Workers in the bird market have a high risk of exposure in the form of air pollution, pollutant particles including debris / organic dust, loose feathers, insects/ticks, aerosol particles in food, bird excreta (ammonia), various kinds of gram bacteria, fungi and virus. Exposure to particles will stimulate the immune system against harmful pathogens in the form of inflammatory response. An infection or injury will stimulate the secretion of Surfactant protein-D (SP-D) which is a group of collectin (collagen-lectin) with subgroups of the C-type lectin superfamily, together with bovine coglutinin, mannose-binding lectin (MBL), and CL43 protein. This aim of this study was to determine serum SP-D and neutrophil and sputum lymphocyte levels in workers at splendid bird market.Methods: A cross sectional analytic observational study on 35 subjects, analyzed the characteristics of the workers, calculated neutrophil types, lymphocytes on induced sputum and serum SP-D levels using sandwich ELISA.Results: Mean SP-D serum levels in workers in the bird market environment increased (mean SD: 81.39 + 47.656 ng/ml) from normal levels (60 ± 3 ng/ml). There was a significant positive correlation between length of exposure and serum SP-D levels (r: 0.693; p <0.001). There was an average increase in the percentage of neutrophils and sputum lymphocytes (90.71% + 4.04% and 9.17% + 4.42%) compared to normal limit (50.3% + 23.5% and 2.6% + 5.2%).Conclusion: Inhalation exposure in the Bird Market environment can cause an increase in the percentage of neutrophils, sputum lymphocytes and serum SP-D levels in workers that indicate an airway inflammation process, as well as an alleged increase in alveolar wall permeability, damage and regeneration of alveolar epithelial cell type II cells (AEC type II).