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The mortality associated with acute lung injury (ALI) increases with age. Alveolar epithelial type II (AEII) cells are the progenitor cells of the alveolar epithelium and are crucial for repair after injury. We hypothesize that telomere dysfunction-mediated AEII cell senescence impairs regeneration and promotes the development of ALI. To discriminate between the impact of old age and AEII cell senescence in ALI, young (3 mo) and old (18 mo) Sftpc-Ai9 mice with surfactant protein c mediated tdTomato expression, and young Sftpc-Ai9-Trf1 mice with additional telomeric repeat-binding factor 1 (Trf1) knockout-mediated senescence in AEII cells were treated with 1 μg LPS per gram body weight ( n  = 9-11). Control mice received saline solution ( n  = 7). Mice were killed 4 or 7 days later. Lung mechanics, pulmonary inflammation, and proteomes were analyzed, and parenchymal injury, AEII cell proliferation and AEI cell differentiation rate were quantified using stereology. Old mice showed 55% mortality by Day 4, whereas all young mice survived. Pulmonary inflammation was most severe in old Sftpc-Ai9 mice, followed by Sftpc-Ai9-Trf1 mice. Young Sftpc-Ai9 mice recovered almost completely by Day 7, whereas Sftpc-Ai9-Trf1 mice still showed mild signs of injury. An expansion of AEII cells was measured only in young Sftpc-Ai9 mice at Day 7. Aging and telomere dysfunction-mediated senescence had no impact on AEI differentiation rate in controls, but the reduced number of AEII cells in Sftpc-Ai9-Trf1 mice also affected de novo differentiation after injury. In conclusion, telomere dysfunction- mediated AEII cell senescence promoted parenchymal inflammation in ALI, but did not enhance mortality like old age. Although the differentiation rate remained functional with old age and AEII cell senescence, AEII cell proliferative capacity was impaired in ALI, affecting the regenerative ability.

Alveolar type II (ATII) cells are essential for the maintenance of the alveolar homeostasis. However, knowledge of the expression of the miRNAs and miRNA-regulated networks which control homeostasis and coordinate diverse functions of murine ATII cells is limited. Therefore, we asked how miRNAs expressed in ATII cells might contribute to the regulation of signaling pathways. We purified "untouched by antibodies" ATII cells using a flow cytometric sorting method with a highly autofluorescent population of lung cells. TaqMan ® miRNA low-density arrays were performed on sorted cells and intersected with miRNA profiles of ATII cells isolated according to a previously published protocol. Of 293 miRNAs expressed in both ATII preparations, 111 showed equal abundances. The target mRNAs of bona fide ATII miRNAs were used for pathway enrichment analysis. This analysis identified nine signaling pathways with known functions in fibrosis and/or epithelial-to-mesenchymal transition (EMT). In particular, a subset of 19 miRNAs was found to target 21 components of the TGF-β signaling pathway. Three of these miRNAs (miR-16-5p, -17-5p and -30c-5p) were down-modulated by TGF-β1 stimulation in human A549 cells, and concomitant up-regulation of associated mRNA targets (BMPR2, JUN, RUNX2) was observed. These results suggest an important role for miRNAs in maintaining the homeostasis of the TGF-β signaling pathway in ATII cells under physiological conditions.

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., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar dysplasia and pulmonary microvascular remodeling. In the present study, we have investigated the functional roles of ubiquitin proteasome pathway (UPP) in BPD, and its relationship with endoplasmic reticulum stress (ERS) mediated type II alveolar epithelial cell (AECII) apoptosis., Methods: A hyperoxia-induced BPD rat model was constructed and the pathologic changes of lung tissues were evaluated by hematoxylin-eosin staining. Cell apoptosis and protein expression were determined by TUNEL assay and Western blotting, respectively. Further reagent kit with specific fluorescent substrate was utilized to measure the activity of 20 s proteasome. Meanwhile, AECII were cultured in vitro and exposed to hyperoxia. AECII apoptosis were measured by flow cytometry. In contrast, MG132 treatment was induced to explore UPP during hyperoxia exposure on AECII apoptosis and ERS sensors expression., Results: A significant increase in apoptosis and total ubiquitinated proteins expression were observed in BPD rats and AECII culture, and the change of UPP was associated with ERS. In order to confirm the role of UPP in AECII apoptosis of BPD, AECII cells were treated by MG132 with the concentration of 10 μmol/L under hyperoxia exposure. We found that the proteins expression of glucose-regulated protein 78 (GRP-78), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), as well as AECII apoptosis were increased following MG132 treatment. Furthermore, the relatively up-regulated in the levels of total ubiquitinated proteins expression and 20 s proteasome activity were correlated with increased ERS sensors expression., Conclusions: Our findings indicate that UPP may participate in the ERS-induced AECII apoptosis under hyperoxia condition., (© 2021. The Author(s).)

Arrested alveolar development is the main pathological characteristic of neonatal bronchopulmonary dysplasia (BPD); however, a number of studies aiming to improve alveolarization have focused on alveolar epithelial cell damage and impairment. Previously, the authors reported that the Wnt signaling plays a key role in alveolar injury and repair by regulating alveolar epithelial type II cell (AECII) proliferation and differentiation. In the present study, the authors wished to investigate whether Yes‑associated protein (YAP), a transcriptional coactivator in the Hippo signaling pathway, affects AECII proliferation and differentiation via the Wnt/β‑catenin pathway in BPD. It was found that YAP regulated AECII proliferation and differentiation. A decreased expression of YAP, Wnt3a and nuclear β‑catenin was observed in lung tissues affected by BPD. In vitro, YAP and Wnt3a overexpression in BPD promoted AECII proliferation and differentiation into AECIs by increasing the nuclear transfer of β‑catenin and vice versa. The effects of a decreased Wnt3a expression in primary AECIIs in BPD were compensated by YAP overexpression, as were the effects of Wnt3a knockdown in primary AECIIs. On the whole, the findings of the present study demonstrate that YAP and Wnt3a independently promote AECII proliferation and differentiation in experimental BPD by increasing the nuclear β‑catenin levels. Therefore, Wnt3a or YAP may be candidate regulatory targets for improving the outcomes of BPD.

Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 μg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.

Alveolar epithelial type II cells (ATII cells) are the main target cells being damaged and releasing the inflammatory mediators during acute respiratory distress syndrome (ARDS). Extensive apoptosis of epithelial cells leads to the breakdown of the alveolar-epithelial barrier in ARDS. Cyclooxygenase-2 (COX-2) plays an important role in pulmonary inflammatory response. Dexmedetomidine (DEX), a potent selective α2 adrenergic receptor (α2-AR) agonist, presents sedative, anxiolytic, and analgesic effects for anesthetic procedures. DEX has anti-apoptotic and anti-inflammatory properties. Our study demonstrated that DEX exerted anti-apoptotic effect on primary human epithelial cells with the inhibition of caspase activation, which was partly via the α2AR/PI3K/AKT pathway. Moreover, DEX significantly reduced the expression of COX-2 as well as prostaglandinE 2 (PGE 2 ) and tumor necrosis factor-α (TNF-α) production induced by lipopolysaccharide (LPS). Our next step is to determine whether DEX can regulate apoptosis in animal models. These results suggest DEX may be a promising therapy for preventing and treating ARDS as well as chronic diseases by directly targeting epithelial cell actions., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute lung injury (ALI) is characterized by alveolar epithelial damage and uncontrolled pulmonary inflammation. Mitochondrial damage-associated molecular patterns (DAMPs), including mitochondrial peptides [ N-formyl peptides (NFPs)], are released during cell injury and death and induce inflammation by unclear mechanisms. In this study, we have investigated the role of mitochondrial DAMPs (MTDs), especially NFPs, in alveolar epithelial injury and lung inflammation. In murine models of ALI, high levels of mitochondrial NADH dehydrogenase 1 in bronchoalveolar lavage fluid (BALF) were associated with lung injury scores and increased formyl peptide receptor (FPR)-1 expression in the alveolar epithelium. Cyclosporin H (CsH), a specific inhibitor of FPR1, inhibited lung inflammation in the ALI models. Both MTDs and NFPs upon intratracheal challenge caused accumulation of neutrophils into the alveolar space with elevated BALF levels of mouse chemokine KC, interleukin-1β, and nitric oxide and increased pulmonary FPR-1 levels. CsH significantly attenuated MTDs or NFP-induced inflammatory lung injury and activation of MAPK and AKT pathways. FPR1 expression was present in rat primary alveolar epithelial type II cells (AECIIs) and was increased by MTDs. CsH inhibited MTDs or NFP-induced CINC-1/IL-8 release and phosphorylation of p38, JNK, and AKT in rat AECII and human cell line A549. Inhibitors of MAPKs and AKT also suppressed MTD-induced IL-8 release and NF-κB activation. Collectively, our data indicate an important role of the alveolar epithelium in initiating immune responses to MTDs released during ALI. The potential mechanism may involve increase of IL-8 production in MTD-activated AECII through FPR-1 and its downstream MAPKs, AKT, and NF-κB pathways.

Inhaled drugs are critical for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). To develop better therapeutics for pulmonary disease it is of potential importance to understand molecular mechanisms of local biotransformation in the lung. Alveolar epithelial type II (ATII) cells have a key role in homeostasis in the lung, but little is known about expression patterns of genes encoding cytochrome P450 (CYP) enzymes in ATII cells. In addition, alteration of CYP gene expression has not been fully defined in COPD. We previously established a method to purify ATII cells from the adult human lung using fluorescence-activated cell sorting. By employing this technique we determined gene expression patterns of 14 CYP enzymes in ATII cells from nonsmokers (n = 4) and smokers (n = 4), both having normal pulmonary function. Although most CYP genes are highly expressed in primary hepatocytes, we found that CYP1B1 mRNA expression was 7.2-fold higher in ATII compared to hepatocytes ( P  = .0275). Additionally we noted a 3.0-fold upregulation of CYP2C19 and 50% reduction in CYP2J2 mRNA expressions in ATII cells isolated from patients with COPD (n = 3) compared to smokers without COPD (n = 4). These data, for the first time, detail a comprehensive set of genes encoding CYP enzymes in human ATII cells and highlights differentially expressed CYP mRNAs of patients with COPD. Such understanding may have important implications for the development of novel inhaled drugs.