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Impaired interaction of fibroblasts with pneumocytes contributes to the progression of chronic lung disease such as idiopathic pulmonary fibrosis (IPF). Mucin 5B (MUC5B) is associated with IPF. Here we analyzed the interaction of primary fibroblasts and alveolar type 2 (AT2) pneumocytes in the organoid model. Single-cell analysis, histology, and qRT-PCR revealed that fibroblasts expressing high levels of fibrosis markers regulate STAT3 signaling in AT2 cells, which is accompanied by cystic organoid growth and MUC5B expression. Cystic growth and MUC5B expression were also caused by the cytokine IL-6. The PI3K-Akt signaling pathway was activated in fibroblasts. The drug dasatinib prevented the formation of MUC5B-expressing cystic organoids. MUC5B associated with AT2 cells in samples obtained from IPF patients. Our model shows that fibrotic primary fibroblasts induce impaired differentiation of AT2 cells via STAT3 signaling pathways, as observed in IPF patients. It can be used for mechanistic studies and drug development., Competing Interests: Declarations. Ethics approval and consent to participate: The protocol for isolation of human cells was approved by the Institutional Review Board (ethics committee, Nr-34/18) of the Saarland State Medical Association and informed consent was obtained from the patients. Consent for publication: Not applicable. Statistics: Graph Prism software (GraphPad Software (version 8.0), San Diego, CA) was used for statistical analysis. Comparisons between groups were analyzed by one-way ANOVA, unpaired t-test or Mann-Whitney test as indicated in the figure legends. The results were considered statistically significant for p < 0.05. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Alveolar type II (ATII) cells have a stem cell potential in the adult lung and repair the epithelium after injury induced by harmful factors. Their damage contributes to emphysema development, characterized by alveolar wall destruction. Cigarette smoke is the main risk factor for this disease development., Methods: ATII cells were obtained from control non-smoker and smoker organ donors and emphysema patients. Isolated cells were used to study the role of PICT1 in this disease. Also, a cigarette smoke-induced murine model of emphysema was applied to define its function in disease progression further., Results: Decreased PICT1 expression was observed in human and murine ATII cells in emphysema. PICT1 was immunoprecipitated, followed by mass spectrometry analysis. We identified MRE11, which is involved in DNA damage repair, as its novel interactor. PICT1 and MRE11 protein levels were decreased in ATII cells in this disease. Moreover, cells with PICT1 deletion were exposed to cigarette smoke extract. This treatment induced cellular and mitochondrial ROS, cell cycle arrest, nuclear and mitochondrial DNA damage, decreased mitochondrial respiration, and impaired DNA damage repair., Conclusions: This study indicates that PICT1 dysfunction can negatively affect genome stability and mitochondrial activity in ATII cells, contributing to emphysema development. Targeting PICT1 can lead to novel therapeutic approaches for this disease., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Alveolar epithelial type I (AE1) cells with their wide spatial expansion form approximately 95% of the outer surface area of the air-blood barrier inside the lung. Serial block-face scanning electron microscopy (SBF-SEM) investigations led to the hypothesis that AE1 cell mitochondria are preferentially distributed as aggregates in those parts of AE1 cells that are located above connective tissue pillars between capillaries, thus not increasing the thickness of the diffusion distance for oxygen and carbon dioxide. Furthermore, it was hypothesised that postnatal development requires adapting the amount and distribution of mitochondria in AE1 cells. Human lung samples from three infant (26 and 30 days, 6 months) and three adult (20, 39 and 40 years) samples were investigated by light microscopy, transmission electron microscopy and stereology. The volume fraction of mitochondria was similar in infant and adult lungs with a mean value of 6.3%. The ratio between mitochondrial profiles on top of capillaries or above connective tissue pillars was approximately 3:1 in infants and adults. However, regarding the volume of both cytoplasmic compartments, infants showed a higher number of mitochondrial profiles on top of capillaries while adults showed a higher number above connective tissue pillars. Samples of three additional adult lungs were analysed by confocal laser scanning microscopy. Again, mitochondria were not preferentially found as aggregates above connective tissue pillars. In conclusion, AE1 cell mitochondria were not preferentially found as aggregates, showed the same volume density in infants and adults but differed in distribution between the age groups., Competing Interests: Declarations Conflict of interest The authors declare that they have no conflict of interest. Ethical approval Human lung tissue for TEM investigations and subsequent stereology (C1, C2, C3, A2, A4, A8) was approved by bioethical regulations of the University of Bern during late 1970s and 1980s and reapproved by the ethics committee of Hannover Medical School (Permission No. 2263-2014). Usage of archived human lung tissue for CLSM was approved by the ethics board of Hannover Medical School (ethics vote number: 1741-2013 and 3381-2016)., (© 2024. The Author(s).)

Pulmonary fibrosis (PF) is a chronic, progressive lung disease characterized by fibroblast proliferation, extensive extracellular matrix and collagen deposition, accompanied by inflammatory damage, ultimately leading to death due to respiratory failure. Endoplasmic reticulum (ER) stress in pulmonary fibrotic tissue is indeed recognized as a significant factor exacerbating PF development. Emerging evidences indicated a potential association between ER stress induced by lactate and cellular apoptosis in PF. However, the mechanisms in this process need further elucidation. In this paper, pulmonary fibrosis model was induced by bleomycin (BLM) intratracheally in mice. In the cellular model, type II epithelial cells were treated by lactate and TGF-β to detect ER stress and apoptosis markers. Lactate could promote ER stress response and apoptosis. Mechanically, lactate activated Caspase-12 via ATF4-Chop axis to induce cell apoptosis and promote fibrosis. ER stress inhibitor could effectively suppress alveolar epithelial cells apoptosis and pulmonary fibrosis. We concluded that pro-fibrotic properties of lactate are associated with alveolar epithelial cells apoptosis by causing ER stress and thus provide new potential therapeutic targets for pulmonary fibrosis., (© 2024. The Author(s).)

Background and Purposes: Liver X receptors (LXRs) are specialized nuclear receptors essential for maintaining cholesterol homeostasis, modulating LXR activity could have therapeutic potential in lung diseases. Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by impaired alveolar development, in which apoptosis of alveolar epithelial cells is a key contributing factor. The current research focuses on exploring the potential mechanism by which the LXR pathway regulating alveolar epithelial type II cell apoptosis in response to hyperoxia exposure., Methods: BPD infants and non-BPD preterm infants were enrolled to measure serum total cholesterol (TC) levels. To further investigate the role of cholesterol metabolism in BPD, a neonatal rat model of BPD was established, and in vitro studies were conducted using mouse lung epithelial cells (MLE12). These experiments aimed to explore the impact of hyperoxia on cholesterol metabolism and assess the effects of LXR agonist intervention., Results: Elevated serum TC levels in BPD infants were observed, accompanied by lung cholesterol overload in BPD rats. Hyperoxia exposure also led to intracellular cholesterol accumulation in MLE12 cells, which may be attributed to the downregulated LXR signaling pathway. Activation of the LXR pathway prevented apoptosis and mitochondrial dysfunction in MLE12 cell. In BPD rats, intervention with the LXR agonist restored alveolar architecture and reduced alveolar epithelial type II cell apoptosis, which was associated with decreased oxidative stress and lung cholesterol accumulation., Conclusions: Disrupted cholesterol metabolism and impaired homeostasis in premature infants may contribute to the development of BPD. Targeting LXR signaling may provide potential therapeutic targets in BPD., Clinical Trial Number: Not applicable., (© 2024. The Author(s).)

Sepsis-induced acute lung injury (SI-ALI) leads to significant deaths in critically ill patients worldwide. This study explores the mechanism of EZH2 regulating ferroptosis of alveolar epithelial cells (AECs) in SI-ALI. In vitro cell model and in vivo mouse lung injury model of sepsis were established. EZH2 expression in lung tissues was intervened by sh-EZH2, followed by H&E staining observation of lung tissue pathological changes. EZH2, H3K27me3, USP10, GPX4, and ACSL4 expressions were determined by qRT-PCR or Western blot. ROS, GSH, and iron ion levels were detected using fluorescent labeling and reagent kits, respectively. ChIP analyzed the enrichment of EZH2 and H3K27me3 on USP10 promoter. The binding between USP10 and GPX4, and the ubiquitination level of GPX4 were detected using Co-IP. EZH2 was highly expressed in lung tissues of SI-ALI mice. EZH2 silencing alleviated ALI and ferroptosis of AECs; EZH2 increased the H3K27me3 level on USP10 promoter through histone methylation. USP10 stabilized GPX4 protein expression through ubiquitination; inhibition of USP10 partially reversed the inhibitory effect of EZH2 silencing on ferroptosis of AECs. In conclusion, EZH2 depresses USP10 expression by promoting histone H3K27me3 modification on USP10 promoter, thereby enhancing ubiquitination degradation of GPX4 and ultimately facilitating ferroptosis of AECs in sepsis., (© 2024 Wiley Periodicals LLC.)

Mitochondrial abnormalities in lung epithelial cells have been associated with chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke (CS) can induce alterations in the molecular pathways regulating mitochondrial function in lung epithelial cells. Recently, heated tobacco products (HTPs) have been marketed as harm reduction products compared with regular cigarettes. However, the effects of HTP emissions on human alveolar epithelial cell metabolism and on the molecular mechanisms regulating mitochondrial content and function are unclear. In this study, human alveolar epithelial cells (A549) were exposed to cigarette or HTP emissions in the form of liquid extracts. The oxygen consumption rate of differently exposed cells was measured, and mRNA and protein abundancy of key molecules involved in the molecular regulation of mitochondrial metabolism were assessed. Furthermore, we used a mitophagy detection probe to visualize mitochondrial breakdown over time in response to the extracts. Both types of extracts induced increases in basal-, maximal- and spare respiratory capacity, as well as in cellular ATP production. Moreover, we observed alterations in the abundancy of regulatory molecules controlling mitochondrial biogenesis and mitophagy. Mitophagy was not significantly altered in response to the extracts, as no significant differences compared to vehicle-treated cells were observed., Competing Interests: Declaration of Competing Interest The authors of the manuscript: ‘Alterations in the molecular regulation of mitochondrial metabolism in human alveolar epithelial cells in response to cigarette- and heated tobacco product emissions’ have declared no conflict of interest and no present or past affiliation with the tobacco industry., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no cure except transplantation. Abnormal alveolar epithelial regeneration is a key driver of IPF development. The function of Yes1 Associated Transcriptional Regulator (YAP) in alveolar regeneration and IPF pathogenesis remains elusive. Here, we first revealed the activation of YAP in alveolar epithelium 2 cells (AEC2s) from human IPF lungs and fibrotic mouse lungs. Notably, conditional deletion of YAP in mouse AEC2s exacerbated bleomycin-induced pulmonary fibrosis. Intriguingly, we showed in both conditional knockout mice and alveolar organoids that YAP deficiency impaired AEC2 proliferation and differentiation into alveolar epithelium 1 cells (AEC1s). Mechanistically, YAP regulated expression levels of genes associated with cell cycle progression and AEC1 differentiation. Furthermore, overexpression of YAP in vitro promoted cell proliferation. These results indicate the critical role of YAP in alveolar regeneration and IPF pathogenesis. Our findings provide new insights into the regulation of alveolar regeneration and IPF pathogenesis, paving the road for developing novel treatment strategies.

Oxygen therapy is a crucial medical intervention, but it is undeniable that it can lead to lung damage. The mTOR pathway plays a pivotal role in governing cell survival, including autophagy and apoptosis, two phenomena deeply entwined with the evolution of diseases. However, it is unclarified whether the mTOR pathway is involved in hyperoxic acute lung injury (HALI). The current study aims to clarify the molecular mechanism underlying the pathogenesis of HALI by constructing in vitro and in vivo models using H 2 O 2 and hyperoxia exposure, respectively. To investigate the role of mTOR, the experiment was divided into five groups, including normal group, injury group, mTOR inhibitor group, mTOR activator group, and DMSO control group. Western blotting, Autophagy double labeling, TUNEL staining, and HE staining were applied to evaluate protein expression, autophagy activity, cell apoptosis, and pathological changes in lung tissues. Our data revealed that hyperoxia can induce autophagy and apoptosis in Type II alveolar epithelial cell (AECII) isolated from the treated rats, as well as injuries in the rat lung tissues; also, H 2 O 2 stimulation increased autophagy and apoptosis in MLE-12 cells. Noticeably, the experiments performed in both in vitro and in vivo models proved that the mTOR inhibitor Rapamycin (Rapa) functioned synergistically with hyperoxia or H 2 O 2 to promote AECII autophagy, which led to increased apoptosis and exacerbated lung injury. On the contrary, activation of mTOR with MHY1485 suppressed autophagy activity, consequently resulting in reduced apoptosis and lung injury in H 2 O 2 -challenged MLE-12 cells and hyperoxia-exposed rats. In conclusion, hyperoxia caused lung injury via mTOR-mediated AECII autophagy., (© 2023. The Author(s).)

Tuberculosis-induced pulmonary fibrosis (PF) is a chronic, irreversible interstitial lung disease, which severely affects lung ventilation and air exchange, leading to respiratory distress, impaired lung function, and ultimately death. As previously reported, epithelial-mesenchymal transition (EMT) and fibrosis in type II alveolar epithelial cells (AEC II) are two critical processes that contributes to the initiation and progression of tuberculosis-related PF, but the underlying pathological mechanisms remain unclear. In this study, through performing Real-Time quantitative PCR (RT-qPCR), Western blot, immunohistochemistry, and immunofluorescence staining assay, we confirmed that the expression levels of EMT and fibrosis-related biomarkers were significantly increased in lung tissues with tuberculosis-associated PF in vivo and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strain-infected AEC II cells in vitro. Besides, we noticed that the mitogen-activated protein kinase 19 (MAP3K19) was aberrantly overexpressed in PF models, and silencing of MAP3K19 significantly reduced the expression levels of fibronectin, collagen type I, and alpha-smooth muscle actin to decrease fibrosis, and upregulated E-cadherin and downregulated vimentin to suppress EMT in BCG-treated AEC II cells. Then, we uncovered the underlying mechanisms and found that BCG synergized with MAP3K19 to activate the pro-inflammatory transforming growth factor-beta (TGF-β)/Smad2 signal pathway in AEC II cells, and BCG-induced EMT process and fibrosis in AEC II cells were all abrogated by co-treating cells with TGF-β/Smad2 signal pathway inhibitor LY2109761. In summary, our results uncovered the underlying mechanisms by which the MAP3K19/TGF-β/Smad2 signaling pathway regulated EMT and fibrotic phenotypes of AEC II cells to facilitate the development of tuberculosis-associated PF, and these findings will provide new ideas and biomarkers to ameliorate tuberculosis-induced PF in clinic., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acute respiratory distress syndrome (ARDS) is a severe lung condition without targeted therapy that is characterized by the disruption of epithelial and endothelial barriers. The role of the tight junction protein occludin in the pathogenesis of this disease is unknown, although it has previously been deemed redundant in some tissues. The aim of the present study is to determine whether occludin is required for lung function by controlling alveolar barrier integrity in mouse models. Immunofluorescence staining of lungs from ARDS patients revealed a significant decrease in occludin expression compared to controls. Gene delivery of shRNA against occludin in the mouse lung reduced occludin levels and induced lung injury, as assessed by wet-to-dry-ratio, histology, and cellularity and protein content of bronchial alveolar lavage fluid. Conversely, gene delivery of an occludin-expressing plasmid increased occludin expression and dampened endotoxin-induced lung injury. In primary rat alveolar epithelial cells, occludin levels were positively correlated with barrier integrity, as well as membrane localization of claudin-18, another tight junction protein. Collectively, our data demonstrate that occludin plays a significant role in alveolar barrier function and that targeting occludin may provide a new therapeutic approach for ARDS.

Cell plasticity, changes in cell fate, is crucial for tissue regeneration. In the lung, failure of regeneration leads to diseases, including fibrosis. However, the mechanisms governing alveolar cell plasticity during lung repair remain elusive. We previously showed that PCLAF remodels the DREAM complex, shifting the balance from cell quiescence towards cell proliferation. Here, we find that PCLAF expression is specific to proliferating lung progenitor cells, along with the DREAM target genes transactivated by lung injury. Genetic ablation of Pclaf impairs AT1 cell repopulation from AT2 cells, leading to lung fibrosis. Mechanistically, the PCLAF-DREAM complex transactivates CLIC4, triggering TGF-β signaling activation, which promotes AT1 cell generation from AT2 cells. Furthermore, phenelzine that mimics the PCLAF-DREAM transcriptional signature increases AT2 cell plasticity, preventing lung fibrosis in organoids and mice. Our study reveals the unexpected role of the PCLAF-DREAM axis in promoting alveolar cell plasticity, beyond cell proliferation control, proposing a potential therapeutic avenue for lung fibrosis prevention., (© 2024. The Author(s).)

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive fibrotic lung disease. The development of IPF involves different molecular and cellular processes, and recent studies indicate that lactate plays a significant role in promoting the progression of the disease. Nevertheless, the mechanism by which lactate metabolism is regulated and the downstream effects remain unclear. The molecular chaperone CCT6A performs multiple functions in a variety of biological processes. Our research has identified a potential association between CCT6A and serum lactate levels in IPF patients. Herein, we found that CCT6A was highly expressed in type 2 alveolar epithelial cells (AEC2s) of fibrotic lung tissues and correlated with disease severity. Lactate increases the accumulation of lipid droplets in epithelial cells. CCT6A inhibits lipid synthesis by blocking the production of lactate in AEC2s and alleviates bleomycin-induced pulmonary fibrosis in mice. In addition, our results revealed that CCT6A blocks HIF-1α-mediated lactate production by driving the VHL-dependent ubiquitination and degradation of HIF-1α and further inhibits lipid accumulation in fibrotic lungs. In conclusion, we propose that there is a pivotal regulatory role of CCT6A in lactate metabolism in pulmonary fibrosis, and strategies aimed at targeting these key molecules could represent potential therapeutic approaches for pulmonary fibrosis., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Hermansky-Pudlak syndrome (HPS)-associated pulmonary fibrosis (HPS-PF) is a progressive lung disease that is a major cause of morbidity and mortality in HPS patients. Previous studies have demonstrated that the HPS proteins play an essential role in the biogenesis and function of lysosome-related organelles (LROs) in alveolar epithelial type II (AT2) cells and found that HPS-PF is associated with dysfunction of AT2 cells and abnormal immune reactions. Despite recent advances in research on HPS and the pathology of HPS-PF, the pathological mechanisms underlying HPS-PF remain poorly understood, and no effective treatment has been established. Therefore, it is necessary to refresh the progress in the pathogenesis of HPS-PF to increase our understanding of the pathogenic mechanism of HPS-PF and develop targeted therapeutic strategies. This review summarizes the recent progress in the pathogenesis of HPS-PF provides information about the current treatment strategies for HPS-PF, and hopefully increases our understanding of the pathogenesis of HPS-PF and offers thoughts for new therapeutic interventions.

Transient receptor potential vanilloid 4 (TRPV4) channels have been associated with numerous pulmonary pathologies, including hypertension, asthma, and acute lung injury. However, their role in the alveolar epithelium remains unclear. We performed impedance-based resistance measurements in primary differentiated alveolar epithelial type I (AT1) cells from wild-type (WT) and TRPV4-deficient (TRPV4-/-) C57/BL6J mice to detect changes in AT1 barrier integrity upon TRPV4 activation. Both pharmacological (GSK1016790A) and a low pH-driven activation of TRPV4 were quantified, and the downstream effects on adherens junctions were assessed through the Western blotting of epithelial cadherin (E-cadherin) protein levels. Importantly, a drop in pH caused a rapid decrease in AT1 barrier resistance and increased the formation of a ~35 kDa E-cadherin C-terminal fragment, with both effects significantly reduced in TRPV4-/- AT1 cells. Similarly, the pharmacological activation of TRPV4 in AT1 cells triggered an immediate transient loss of barrier resistance and the formation of the same E-cadherin fragment, which was again diminished by TRPV4 deficiency. Moreover, TRPV4-mediated E-cadherin cleavage was significantly reduced by GI254023X, an antagonist of a disintegrin and metalloprotease 10 (ADAM10). Our results confirm the role of TRPV4 in regulating alveolar epithelial barrier permeability and provide insight into a novel signaling pathway by which TRPV4-induced Ca 2+ influx stimulates metalloprotease-driven ectodomain shedding.

Aberrant repair underlies the pathogenesis of pulmonary fibrosis while effective strategies to convert fibrosis to normal regeneration are scarce. Here, we found that thyroid hormone is decreased in multiple models of lung injury but is essential for lung regeneration. Moreover, thyroid hormone receptor α (TRα) promotes cell proliferation, while TRβ fuels cell maturation in lung regeneration. Using a specific TRβ agonist, sobetirome, we demonstrate that the anti-fibrotic effects of thyroid hormone mainly rely on TRβ in mice. Cellularly, TRβ activation enhances alveolar type-2 (AT2) cell differentiation into AT1 cell and constrains AT2 cell hyperplasia. Molecularly, TRβ activation directly regulates the expression of KLF2 and CEBPA, both of which further synergistically drive the differentiation program of AT1 cells and benefit regeneration and anti-fibrosis. Our findings elucidate the modulation function of the TRβ-KLF2/CEBPA axis on AT2 cell fate and provide a potential treatment strategy to facilitate lung regeneration and anti-fibrosis., (© 2024. The Author(s).)

In lung disease, persistence of KRT8-expressing aberrant basaloid cells in the alveolar epithelium is associated with impaired tissue regeneration and pathological tissue remodeling. We analyzed single cell RNA sequencing datasets of human interstitial lung disease and found the profibrotic Interleukin-11 (IL11) cytokine to be highly and specifically expressed in aberrant KRT8 + basaloid cells. IL11 is similarly expressed by KRT8 + alveolar epithelial cells lining fibrotic lesions in a mouse model of interstitial lung disease. Stimulation of alveolar epithelial cells with IL11 causes epithelial-to-mesenchymal transition and promotes a KRT8-high state, which stalls the beneficial differentiation of alveolar type 2 (AT2)-to-AT1 cells. Inhibition of IL11-signaling in AT2 cells in vivo prevents the accumulation of KRT8 + cells, enhances AT1 cell differentiation and blocks fibrogenesis, which is replicated by anti-IL11 therapy. These data show that IL11 inhibits reparative AT2-to-AT1 differentiation in the damaged lung to limit endogenous alveolar regeneration, resulting in fibrotic lung disease., (© 2024. The Author(s).)

Background: Interstitial lung disease (ILD) poses a serious threat in patients with rheumatoid arthritis (RA). However, the impact of cornerstone drugs, including methotrexate (MTX) and TNF inhibitor, on RA-associated ILD (RA-ILD) remains controversial., Methods: Using an SKG mouse model and single-cell transcriptomics, we investigated the effects of MTX and TNF blockade on ILD., Findings: Our study revealed that MTX exacerbates pulmonary inflammation by promoting immune cell infiltration, Th17 activation, and fibrosis. In contrast, TNF inhibitor ameliorates these features and inhibits ILD progression. Analysis of data from a human RA-ILD cohort revealed that patients with ILD progression had persistently higher systemic inflammation than those without progression, particularly among the subgroup undergoing MTX treatment., Interpretation: These findings highlight the need for personalized therapeutic approaches in RA-ILD, given the divergent outcomes of MTX and TNF inhibitor., Funding: This work was funded by GENINUS Inc., and the National Research Foundation of Korea, and Seoul National University Hospital., Competing Interests: Declaration of interests Dr. Sparks has received research support from Bristol Myers Squibb and Boehringer Ingelheim unrelated to this work. He has performed consultancy for AbbVie, Amgen, Boehringer Ingelheim, Bristol Myers Squibb, Gilead, Inova Diagnostics, Janssen, Optum, Pfizer, ReCor, Sobi, and UCB unrelated to this work., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner., Methods: ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-ILD group, for anti-CarP levels. Male C57BL/6 mice were used for the pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5) and patient serum-derived or commercialized anti-CarP was used for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and co-immunoprecipitation followed by mass spectrometry (MS) analysis. Quantitative RT-PCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. A native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites., Results: A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and microfibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5., Conclusions: Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progression., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The apoptosis-prone property of alveolar epithelial cells plays a crucial role in pulmonary fibrosis(PF), but the role of pyroptosis in it is still unclear. Toll-like receptor 9(TLR9) has been reported to play a vital role in the pathogenesis of many diseases. However, the effect of TLR9 on alveolar epithelial cells in PF has not been fully elucidated. Gene expression microarray related to Idiopathic pulmonary fibrosis(IPF) was obtained from the Gene Expression Omnibus(GEO) database. In the mouse model of bleomycin-induced PF, adeno-associated virus(AAV6) was used to interfere with TLR9 to construct TLR9 knockdown mice to study the role of TLR9 in PF, and the specific mechanism was studied by intratracheal instillation of NLR family pyrin domain containing 3(NLRP3) activator. In vitro experiments were performed using A549 cells. Bleomycin-induced pyroptosis in the lung tissue of PF mice increased, and TLR9 protein levels also increased, especially in alveolar epithelial cells. The levels of fibrosis and pyroptosis in lung tissue of TLR9 knockdown mice were improved. We found that TLR9 can bind to the NLRP3, thereby increasing the activation of the NLRP3/caspase-1 inflammasome pathway. When we use the NLRP3 activator, the levels of fibrosis and pyroptosis in lung tissue of TLR9 knockout mice can be counteracted. Pyroptosis of alveolar epithelial cells plays a vital role in PF, and TLR9 can promote NLRP3-mediated pyroptosis of alveolar epithelial cells to aggravate the progression of PF and may become a feasible target for the prevention and treatment of PF., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Excessive autophagy may lead to degradation and damage of alveolar epithelial cells after lung transplantation, eventually leading to alveolar epithelial cell loss, affecting the structural integrity and function of alveoli. Glutamine (Gln), a nutritional supplement, regulates autophagy through multiple signaling pathways. In this study, we explored the protective role of Gln on alveolar epithelial cells by inhibiting autophagy. In vivo, a rat orthotopic lung transplant model was carried out to evaluate the therapeutic effect of glutamine. Ischemia/reperfusion (I/R) induced alveolar collapse, edema, epithelial cell apoptosis, and inflammation, which led to a reduction of alveolar physiological function, such as an increase in peak airway pressure, and a decrease in lung compliance and oxygenation index. In comparison, Gln preserved alveolar structure and function by reducing alveolar apoptosis, inflammation, and edema. In vitro, a hypoxia/reoxygenation (H/R) cell model was performed to simulate IR injury on mouse lung epithelial (MLE) cells and human lung bronchus epithelial (Beas-2B) cells. H/R impaired the proliferation of epithelial cells and triggered cell apoptosis. In contrast, Gln normalized cell proliferation and suppressed I/R-induced cell apoptosis. The activation of mTOR and the downregulation of autophagy-related proteins (LC3, Atg5, Beclin1) were observed in Gln-treated lung tissues and alveolar epithelial cells. Both in vivo and in vitro, rapamycin, a classical mTOR inhibitor, reversed the beneficial effects of Gln on alveolar structure and function. Taken together, Glnpreserved alveolar structure and function after lung transplantation by inhibiting autophagy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Treating Idiopathic pulmonary fibrosis (IPF) remains challenging owing to its relentless progression, grim prognosis, and the scarcity of effective treatment options. Emerging evidence strongly supports the critical role of accelerated senescence in alveolar epithelial cells (AECs) in driving the progression of IPF. Consequently, targeting senescent AECs emerges as a promising therapeutic strategy for IPF., Purpose: Curcumin analogue EF24 is a derivative of curcumin and shows heightened bioactivity encompassing anti-inflammatory, anti-tumor and anti-aging properties. The objective of this study was to elucidate the therapeutic potential and underlying molecular mechanisms of EF24 in the treatment of IPF., Methods: A549 and ATII cells were induced to become senescent using bleomycin. Senescence markers were examined using different methods including senescence-associated β-galactosidase (SA-β-gal) staining, western blotting, and q-PCR. Mice were intratracheally administrated with bleomycin to induce pulmonary fibrosis. This was validated by micro-computed tomography (CT), masson trichrome staining, and transmission electron microscope (TEM). The role and underlying mechanisms of EF24 in IPF were determined in vitro and in vivo by evaluating the expressions of PTEN, AKT/mTOR/NF-κB signaling pathway, and mitophagy using western blotting or flow cytometry., Results: We identified that the curcumin analogue EF24 was the most promising candidate among 12 compounds against IPF. EF24 treatment significantly reduced senescence biomarkers in bleomycin-induced senescent AECs, including SA-β-Gal, PAI-1, P21, and the senescence-associated secretory phenotype (SASP). EF24 also effectively inhibited fibroblast activation which was induced by senescent AECs or TGF-β. We revealed that PTEN activation was integral for EF24 to inhibit AECs senescence by suppressing the AKT/mTOR/NF-κB signaling pathway. Additionally, EF24 improved mitochondrial dysfunction through induction of mitophagy. Furthermore, EF24 administration significantly reduced the senescent phenotype induced by bleomycin in the lung tissues of mice. Notably, EF24 mitigates fibrosis and promotes overall health benefits in both the acute and chronic phases of IPF, suggesting its therapeutic potential in IPF treatment., Conclusion: These findings collectively highlight EF24 as a new and effective therapeutic agent against IPF by inhibiting senescence in AECs., Competing Interests: Declaration of competing interest Y.H.H, J.l, Y.H.Z, and J.H.L filed a patent application for the use of EF24 as an anti-pulmonary fibrosis agent. Ohter authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H + -ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H + -ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

Studies on in vitro-in vivo correlations of inflammatory and genotoxic responses are needed to advance new approach methodologies. Here, we assessed pro-inflammatory and genotoxic responses by 13 nanosized metal oxides (nMeOx) and quartz (DQ12) in alveolar epithelial cells (A549) and macrophages (THP-1a) exposed in submerged conditions, and in A549:THP-1a co-cultures in air-liquid interface (ALI) system. Soluble nMeOx produced the highest IL-8 expression in A549 and THP-1a cells in submerged conditions (≥2-fold, p < 0.05), whereas only CuO caused a strong response in co-cultures exposed in the ALI system (13-fold, p < 0.05). IL-8 expression in A549 cells with concentrations as nMeOx specific surface area (SSA) correlated with neutrophil influx in mice (r = 0.89-0.98, p < 0.05). Similarly, IL-8 expression in THP-1a cell with concentrations as mass and SSA (when excluding soluble nMeOx) correlated with neutrophil influx in mice (r = 0.81-0.84, p < 0.05). DNA strand breaks (SB) was measured by the comet assay. We used a scoring system that categorizes effects in standard deviation units for comparison of genotoxicity in different models. Concordant genotoxicity was observed between SB levels in vitro (A549 and co-culture) and in vivo (broncho-alveolar lavage fluid cells and lung tissue). In conclusion, this study shows in vitro-in vivo correlations of nMeOx-induced inflammatory and genotoxic responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Early-life (EL) respiratory infections increase pulmonary disease risk, especially EL-Respiratory Syncytial Virus (EL-RSV) infections linked to asthma. Mechanisms underlying asthma predisposition remain unknown. In this study, we examined the long-term effects on the lung after four weeks post EL-RSV infection. We identified alterations in the lung epithelial cell, with a rise in the percentage of alveolar type 2 epithelial cells (AT2) and a decreased percentage of cells in the AT1 and AT2-AT1 subclusters, as well as upregulation of Bmp2 and Krt8 genes that are associated with AT2-AT1 trans-differentiation, suggesting potential defects in lung repair processes. We identified persistent upregulation of asthma-associated genes, including Il33. EL-RSV-infected mice allergen-challenged exhibited exacerbated allergic response, with significant upregulation of Il33 in the lung and AT2 cells. Similar long-term effects were observed in mice exposed to EL-IL-1β. Notably, treatment with IL-1ra during acute EL-RSV infection mitigated the long-term alveolar alterations and the allergen-exacerbated response. Finally, epigenetic modifications in the promoter of the Il33 gene were detected in AT2 cells harvested from EL-RSV and EL-IL1β groups, suggesting that long-term alteration in the epithelium after RSV infection is dependent on the IL-1β pathway. This study provides insight into the molecular mechanisms of asthma predisposition after RSV infection., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The complex cellular interactions supporting metastasis formation remain incompletely understood. In this issue of Developmental Cell, Rodrigues et al. describe a positive feedback loop within the lung metastatic niche, whereby disseminated cancer cells promote the multilineage potential of alveolar type 2 cells, favoring cancer cell stemness and metastasis initiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A key step for metastatic outgrowth involves the generation of a deeply altered microenvironment (niche) that supports the malignant behavior of cancer cells. The complexity of the metastatic niche has posed a significant challenge in elucidating the underlying programs driving its origin. Here, by focusing on early stages of breast cancer metastasis to the lung in mice, we describe a cancer-dependent chromatin remodeling and activation of developmental programs in alveolar type 2 (AT2) cells within the niche. We show that metastatic cells can prime AT2 cells into a reprogrammed multilineage state. In turn, this cancer-induced reprogramming of AT2 cells promoted stem-like features in cancer cells and enhanced their initiation capacity. In conclusion, we propose the concept of "reflected stemness" as an early phenomenon during metastatic niche initiation, wherein metastatic cells reprogram the local tissue into a stem-like state that enhances intrinsic cancer-initiating potential, creating a positive feedback loop where tumorigenic programs are amplified., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Micro/nanoplastics (MNPs), which are widely spread in the environment, have gained attention because of their ability to enter the human body mainly through ingestion, inhalation, and skin contact, thus representing a serious health threat. Several studies have reported the presence of MNPs in lung tissue and the potential role of MNP inhalation in triggering lung fibrosis and tumorigenesis. However, there is a paucity of knowledge regarding the cellular response to MNPs composed of polyethylene (PE), one of the most common plastic pollutants in the biosphere. In this study, we investigated the effects of low/high concentrations of PE MNPs on respiratory epithelial cell viability and migration/invasion abilities, using MTT, scratch, and transwell assays. Morphological and molecular changes were assessed via immunofluorescence, Western blot, and qRT-PCR. We demonstrated that acute exposure to PE MNPs does not induce cellular toxicity. Instead, cells displayed visible morphological changes also involving actin cytoskeleton reorganization. Our data underlined the role of epithelial-mesenchymal transition (EMT) in triggering this process. Moreover, a remarkable increase in migration potential was noticed, in absence of a significant alteration of the cell's invasive capacity. The present study highlights the potential impact of PE MNPs inhalation on the human respiratory epithelium, suggesting a possible role in carcinogenesis.

Molecular and cellular mechanisms of human lung alveolar development are poorly understood due to a lack of in vitro model systems. This protocol details the isolation, derivation, and genetic modification of lung tip epithelial progenitors from human fetal lungs. It includes steps for isolating distal lung epithelial cells, expanding tip progenitor organoids, culturing tip organoids in vitro, and differentiating them into alveolar type 2 cells. This will aid in understanding alveolar differentiation mechanisms and neonatal diseases. For complete details on the use and execution of this protocol, please refer to Lim et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq, bulk ATAC-seq, and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16., (© 2024. The Author(s).)

Background: Ventilator-induced lung injury (VILI) is one of the severe complications in the clinic concerning mechanical ventilation (MV). Capsaicin (CAP) has anti-inflammatory and inhibitory effects on oxidative stress, which is a significant element causing cellular ferroptosis. Nevertheless, the specific role and potential mechanistic pathways through which CAP modulates ferroptosis in VILI remain elusive., Methods: VILI was established in vivo, and the pulmonary epithelial cell injury model induced by circulation stretching (CS) was established in vitro. Both mice and cells were pretreated with CAP. Transmission electron microscopy, ELISA, Western blot, immunofluorescence, RT-PCR, fluorescent probes, and other experimental methods were used to clarify the relationship between iron death and VILI in alveolar epithelial cells, and whether capsaicin alleviates VILI by inhibiting iron death and its specific mechanism., Results: Ferroptosis was involved in VILI by utilizing in vivo models. CAP inhibited ferroptosis and alleviated VILI's lung damage and inflammation, and this protective effect of CAP was dependent on maintaining mitochondrial redox system through SITR3 signaling. In the CS-caused lung epithelial cell injury models, CAP reduced pathological CS-caused ferroptosis and cell injury. Knockdown SIRT3 reversed the role of CAP on the maintaining mitochondria dysfunction under pathological CS and eliminated its subsequent advantageous impacts for ferroptosis against overstretching cells., Conclusion: The outcomes showed that CAP alleviated ferroptosis in VILI via improving the activity of SITR3 to suppressing mitochondrial oxidative damage and maintaining mitochondrial redox homeostasis, illustrating its possibility as a novel therapeutic goal for VILI., (© 2024. The Author(s).)

Background: Pulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF., Methods: Firstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways., Results: The aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins., Conclusions: IL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF., (© 2024. The Author(s).)

Background: Idiopathic pulmonary fibrosis (PF) is a chronic progressive interstitial lung disease characterized by alveolar epithelial cell (AEC) injury and fibroblast activation. Inadequate autophagy in AECs may result from the activation of several signaling pathways following AEC injury, with glycoproteins serving as key receptor proteins. The core fucosylation (CF) modification in glycoproteins is crucial. Mesenchymal stem cells derived from bone marrow (BMSCs) have the ability to regenerate damaged tissue and treat PF. This study aimed to elucidate the relationship and mechanism of interaction between BMSCs, CF modification, and autophagy in PF., Methods: C57BL/6 male mice, AEC-specific FUT8 conditional knockout (CKO) mice, and MLE12 cells were administered bleomycin (BLM), FUT8 siRNA, and mouse BMSCs, respectively. Experimental techniques including tissue staining, Western blotting, immunofluorescence, autophagic flux detection, and flow cytometry were used in this study., Results: First, we found that autophagy was inhibited while FUT8 expression was elevated in PF mice and BLM-induced AEC injury models. Subsequently, CKO mice and MLE12 cells transfected with FUT8 siRNA were used to demonstrate that inhibition of CF modification induces autophagy in AECs and mitigates PF. Finally, mouse BMSCs were used to demonstrate that they alleviate the detrimental autophagy of AECs by inhibiting CF modification and decreasing PF., Conclusions: Suppression of CF modification enhanced the suppression of AEC autophagy and reduced PF in mice. Additionally, through the prevention of CF modification, BMSCs can assist AECs deficient in autophagy and partially alleviate PF., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Acute lung injury (ALI) is manifested by increased blood vessel permeability within the lungs and subsequent impairment of alveolar gas exchange. Methylprednisolone (MP) is commonly used as a treatment for ALI to reduce inflammation, yet its molecular mechanism remains unclear. This study aims to explore the underlying mechanisms of MP on ALI in a model induced by lipopolysaccharide (LPS)., Material and Methods: The proliferation, viability, apoptosis, and miR-151-5p expression of alveolar type II epithelial cells (AECII) were detected using the cell EdU assay, Annexin V/PI Apoptosis Kit, counting kit-8 (CCK-8) assay, and RT-qPCR. Western blot analysis was used to detect the Usp38 protein level. IL-6 and TNF-α were measured by ELISA. The combination of miR-151-5p and USP38 was determined by chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase reporter assay., Results: MP greatly improved pulmonary function in vivo, reduced inflammation, and promoted the proliferation of the alveolar type II epithelial cells (AECII) in vitro. By comparing the alterations of microRNAs in lung tissues between MP treatment and control groups, we found that miR-151-5p exhibited a significant increase after LPS-treated AECII, but decreased after MP treatment. Confirmed by a luciferase reporter assay, USP38, identified as a downstream target of miR-151-5p, was found to increase after MP administration. Inhibition of miR-151-5p or overexpression of USP38 in AECII significantly improved the anti-inflammatory, anti-apoptotic, and proliferation-promotive effects of MP., Conclusion: In summary, our data demonstrated that MP alleviates the inflammation and apoptosis of AECII induced by LPS, and promotes the proliferation of AECII partially via miR-151-5p suppression and subsequent USP38 activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic disease that significantly impacts patient quality of life, and its incidence is on the rise. The pathogenesis of IPF remains poorly understood. Alveolar type 2 (AT2) cells are crucial in the onset and progression of IPF, yet the specific mechanisms involved are not well defined. Lon protease 1 (LONP1), known for its critical roles in various diseases, has an unclear function in IPF. Our research investigated the impact of Lonp1 gene deletion on AT2 cell functionality and its subsequent effect on IPF development. We generated a bleomycin-induced pulmonary fibrosis mouse model with a targeted Lonp1 knockout in AT2 cells and assessed the consequences on AT2 cell function and fibrosis progression. Additionally, we constructed the MLE12 cells with stable Lonp1 knockdown and utilized transcriptome sequencing to identify pathways altered by the Lonp1 knockdown. Our results indicated that mice with AT2 cell-specific Lonp1 knockout exhibited more severe fibrosis compared to controls. These mice exhibited a reduction in AT2 and AT1 cell populations, along with an increase in p53- and p21-positive AT2 cells. Lonp1 knockdown in MLE12 cells led to the upregulation of aging-associated pathways, with fibroblast growth factor 2 (Fgf2) gene emerging as a central gene interconnecting these pathways. Therefore, loss of Lonp1 appears to promote AT2 cell aging and exacerbate bleomycin-induced pulmonary fibrosis. Fgf2 emerges as a pivotal downstream gene associated with cellular senescence. This study uncovers the role of the Lonp1 gene in pulmonary fibrosis, presenting a novel target for investigating the pathological mechanisms and potential therapeutic approaches for IPF.

An earlier study found that respiratory cadmium chloride (CdCl 2 ) exposure caused COPD-like lung injury. This study aimed to explore whether mitochondrial dysfunction-mediated alveolar epithelial senescence is involved in CdCl 2 -induced COPD-like lung injury. Adult C57BL/6 mice were exposed to CdCl 2 (10 mg/L) aerosol for six months. Beta-galactosidase-positive cells, p21 and p16 were increased in CdCl 2 -exposed mouse lungs. The in vitro experiments showed that γ-H 2 AX was elevated in CdCl 2 -exposed alveolar epithelial cells. The cGAS-STING pathway was activated in CdCl 2 -exposed alveolar epithelial cells and mouse lungs. Cxcl1, Cxcl9, Il-10, Il-1β and Mmp2, several senescence-associated secretory phenotypes (SASP), were upregulated in CdCl 2 -exposed alveolar epithelial cells. Mechanistically, CdCl 2 exposure caused SIRT3 reduction and mitochondrial dysfunction in mouse lungs and alveolar epithelial cells. The in vitro experiment found that Sirt3 overexpression attenuated CdCl 2 -induced alveolar epithelial senescence and SASP. The in vivo experiments showed that Sirt3 gene knockout exacerbated CdCl 2 -induced alveolar epithelial senescence, alveolar structure damage, airway inflammation and pulmonary function decline. NMN, an NAD + precursor, attenuated CdCl 2 -induced alveolar epithelial senescence and SASP in mouse lungs. Moreover, NMN supplementation prevented CdCl 2 -induced COPD-like alveolar structure damage, epithelial-mesenchymal transition and pulmonary function decline. These results suggest that mitochondrial dysfunction-associated alveolar epithelial senescence is involved in CdCl 2 -induced COPD-like lung injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic disease. Recent studies have highlighted the persistence of an intermediate state of alveolar stem cells in IPF lungs. In this study, we discovered a close correlation between the distribution pattern of intermediate alveolar stem cells and the progression of fibrotic changes. We showed that amphiregulin (AREG) expression is significantly elevated in intermediate alveolar stem cells of mouse fibrotic lungs and IPF patients. High levels of serum AREG correlate significantly with profound deteriorations in lung function in IPF patients. We demonstrated that AREG in alveolar stem cells is both required and sufficient for activating EGFR in fibroblasts, thereby driving lung fibrosis. Moreover, pharmacological inhibition of AREG using a neutralizing antibody effectively blocked the initiation and progression of lung fibrosis in mice. Our study underscores the therapeutic potential of anti-AREG antibodies in attenuating IPF progression, offering a promising strategy for treating fibrotic diseases., Competing Interests: Declaration of interests N.T. and H.W. are inventors of a patent application (US application no. US17/614,673) related to this research., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Extensive loss of alveolar epithelial cells (AECs) undergoing necroptosis is a crucial mechanism of acute lung injury (ALI), but its triggering mechanism needs to be thoroughly investigated. Neutrophil extracellular traps (NETs) play a significant role in ALI. However, the effect of NETs on AECs' death has not been clarified. Our study found that intratracheal instillation of NETs disrupted lung tissue structure, suggesting that NETs could induce ALI in mice. Moreover, we observed that NETs could trigger necroptosis of AECs in vivo and in vitro . The phosphorylation levels of RIPK3 and MLKL were increased in MLE12 cells after NETs treatment ( P < 0.05). Mechanistically, NETs taken up by AECs through endocytosis activated the cGAS-STING pathway and triggered AECs necroptosis. The expression of cGAS, STING, TBK1 and IRF3 were increased in MLE12 cells treated with NETs ( P < 0.05). Furthermore, the cGAS inhibitor RU.521 inhibited NETs-triggered AECs necroptosis and alleviated the pulmonary damage induced by NETs in mice. In conclusion, our study demonstrates that NETs taken up by AECs via endocytosis can activate the cGAS-STING pathway and trigger AECs necroptosis to promote ALI in mice. Our findings indicate that targeting the NETs/cGAS-STING/necroptosis pathway in AECs is an effective strategy for treating ALI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pulmonary sclerosing pneumocytoma (PSP) is a rare, distinctive benign lung adenoma of pneumocyte origin. Despite its rarity, the tumor's unique cellular morphology has sparked ongoing debates regarding the origin of its constituent cells. This study aimed to elucidate the molecular features of PSP tumor cells and enhance our understanding of the cellular processes contributing to PSP formation and biological behavior. Tissue samples from PSP and corresponding normal lung tissues (n = 4) were collected. We employed single-cell RNA sequencing and microarray-based spatial transcriptomic analyses to identify cell types and investigate their transcriptomes, with a focus on transcription factors, enriched gene expression, and single-cell trajectory evaluations. Our analysis identified 2 types of tumor cells: mesenchymal-epithelial dual-phenotype (MEDP) cells and a distinct subpopulation of type II alveolar epithelial cells exhibiting characteristics slightly reminiscent of type I alveolar epithelial cells (AT2Cs) corresponding to histologic round stromal cells and surface cuboidal cells, respectively. MEDP cells displayed weak alveolar epithelial differentiation but strong collagen production capabilities, as indicated by the expression of both TTF-1 and vimentin. These cells played a pivotal role in forming the solid and sclerotic areas of PSP. Moreover, MEDP cells exhibited a pronounced propensity for epithelial-mesenchymal transition, suggesting a greater potential for metastasis compared with AT2Cs. The capillary endothelial cells of PSP displayed notable diversity. Overall, this study provides, for the first time, a comprehensive mapping of the single-cell transcriptome profile of PSP. Our findings delineate 2 distinct subtypes of tumor cells, MEDP cells and AT2Cs, each with its own biological characteristics and spatial distribution. A deeper understanding of these cell types promises insights into the histology and biological behaviors of this rare tumor., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

There are limited number of studies investigating the role of microRNAs (miRNAs) in Aspergillus infections. In this study, we designed an in vitro aspergillosis model to identify differentially expressed Aspergillus-related miRNAs. For this purpose, carcinoma cell lines "A549" and "Calu-3" were infected with Aspergillus fumigatus. Total miRNA was isolated at 0, 1, 6, and 24 h post-infection. Quantitative real-time PCR assay was conducted to screen 31 human miRNAs that were possibly related to aspergillosis. Up- and downregulated miRNAs were detected in the infected cells. Highest level of miRNA expression was detected at 6 h post-infection. miR-21, hsa-miR-186-5p, hsa-miR-490-5p, miR-26a-5p, miR-26b-5p, hsa-miR-424-5p, hsa-miR-548d-3p, hsa-miR-196a-5p, miR-150-5p, miR-17-5p, and hsa-miR-99b-5p were found to be significantly upregulated (p < 0.001) at 6 h after A. fumigatus infection compared with the controls. Among the screened miRNAs, hsa-miR-145-5p (p < 0.001); hsa-miR-583 and hsa-miR-3978 (p < 0.01); and miR-21-5p, hsa-miR-4488, and hsa-miR-4454 (p < 0.05) were found to be downregulated compared with the controls. In conclusion, screening the identified miRNAs may reveal the personal predisposition to aspergillosis, which might be valuable from the perspective of personalized medicine., (© 2024. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.)

The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-β-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: Alveolar epithelial cell (AEC) ferroptosis contributes to the progression of acute lung injury (ALI). Esketamine (ESK) is a new clinical sedative, anesthetic, and analgesic drug that has attracted substantial attention in mental health research because of its antidepressant effects. However, the effects of ESK on ferroptosis-mediated ALI remain unclear., Objective: This study aimed to explore the protective effect of ESK on AEC ferroptosis in ALI and its potential molecular mechanism in vivo and in vitro., Methods: The antiferroptotic and anti-inflammatory effects of ESK were assessed in a mouse model of lipopolysaccharide (LPS)-induced ALI. In vitro, the epithelial cell lines MLE-12 and A549 were used to examine the underlying mechanism by which ESK regulates inflammation and ferroptosis., Results: ESK protected mice against LPS-induced ALI, significantly attenuated pathological changes in the lungs and decreased inflammation and ferroptosis. In vitro, ESK inhibited LPS-induced inflammation and ferroptosis in MLE-12 and A549 cells. Moreover, ferroptosis mediated inflammation in LPS-induced ALI in vivo and in vitro, and ESK decreased the LPS-induced inflammatory response by suppressing ferroptosis. ESK promoted the HIF-1α/HO-1 pathway in LPS-treated AECs and in the lung tissues of mice with LPS-induced ALI. Moreover, pretreatment with ESK and the HIF-1α stabilizer dimethyloxaloylglycine (DMOG) substantially attenuated lung injury and prevented changes in ferroptosis-related biochemical indicators, including glutathione (GSH) depletion, malondialdehyde (MDA) production and glutathione peroxidase 4 (GPX4) downregulation, in untreated LPS-induced mice but not in LPS-induced mice treated with the HO-1 inhibitor zinc protoporphyrin (ZNPP). Similar effects were observed in vitro in HO-1 siRNA-transfected A549 cells after LPS incubation but not in control siRNA-transfected cells., Conclusion: ESK can inhibit ferroptosis-mediated lipid peroxidation by increasing the expression of HIF-1α/HO-1 pathway, highlighting the potential of ESK to treat LPS-induced ALI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The alveolar-capillary barrier includes microvascular endothelial and alveolar epithelial cells and their matrices, and its disruption is a critical driver of lung injury during development of acute respiratory distress syndrome. In this review, we provide an overview of the structure and function of the alveolar-capillary barrier during health and highlight several important signaling mechanisms that underlie endothelial and epithelial injury during critical illness, emphasizing areas with potential for development of therapeutic strategies targeting alveolar-capillary leak. We also emphasize the importance of biomarker and preclinical studies in developing novel therapies and highlight important areas warranting future investigation., Competing Interests: Disclosure The authors declare no conflicts of interest., (Published by Elsevier Inc.)

Purpose: Tripartite motif-containing protein 13 (TRIM13) directly or indirectly participates in autophagy and apoptosis. However, it remains unclear whether TRIM13 participates in chronic obstructive pulmonary disease (COPD) progression. This study aimed to reveal the molecular mechanisms through which TRIM13 regulates alveolar epithelial cell injury in COPD to provide new molecular targets for COPD treatment., Methods: The TRIM13 expression levels were determined in clinical COPD patients and a rat emphysema model. A cigarette smoke-induced model of endoplasmic reticulum stress (ERS) and endoplasmic reticulum autophagy (ER-phagy) was developed using A549 cells, and the effects of TRIM13 gene overexpression/knockdown on ERS, ER-phagy, and cell apoptosis were assessed in these cells., Results: TRIM13 expression was significantly decreased in the lung tissues of COPD patients and rats with emphysema. Moreover, the apoptosis level was significantly increased in the lung tissues of rats with emphysema. TRIM13 gene overexpression reduced the expression levels of ERS-related molecules (GRP78, GRP94, XBP-1, and eIF2a) in the COPD model; it also lowered the ER-phagy level, as evidenced by decreased number of autolysosomes observed by transmission electron microscopy, improved endoplasmic reticulum structure, reduced LC3-II/LC3-I and Beclin1 expression levels, and increased expression level of the autophagy inhibitory molecule Bcl-2. TRIM13 gene knockdown, however, led to opposite results., Conclusion: TRIM13 expression attenuated alveolar epithelial cell injury in COPD by inhibiting ERS-induced ER-phagy., (© 2024. The Author(s).)

COVID-19, caused by SARS-CoV-2 infection, results in irreversible or fatal lung injury. We assumed that necroptosis of virus-infected alveolar epithelial cells (AEC) could promote local inflammation and further lung injury in COVID-19. Since CD8+ lymphocytes induced AEC cell death via cytotoxic molecules such as FAS ligands, we examined the involvement of FAS-mediated cell death in COVID-19 patients and murine COVID-19 model. We identified the occurrence of necroptosis and subsequent release of HMGB1 in the admitted patients with COVID-19. In the mouse model of COVID-19, lung inflammation and injury were attenuated in Fas-deficient mice compared to Fas-intact mice. The infection enhanced Type I interferon-inducible genes in both groups, while inflammasome-associated genes were specifically upregulated in Fas-intact mice. The treatment with necroptosis inhibitor, Nec1s, improved survival rate, lung injury, and systemic inflammation. SARS-CoV-2 induced necroptosis causes cytokine induction and lung damage, and its inhibition could be a novel therapeutic strategy for COVID-19., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shinsuke Yasuda reports a relationship with AbbVie Inc. that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with Asahi Kasei Pharma Corporation that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with Chugai Pharmaceutical Co Ltd. that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with CSL Behring that includes: funding grants. Shinsuke Yasuda reports a relationship with Eisai Inc. that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with ImmunoForge that includes: funding grants. Shinsuke Yasuda reports a relationship with Mitsubishi Tanabe Pharma Corporation that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with Ono Pharmaceutical Co Ltd. that includes: funding grants and speaking and lecture fees. Shinsuke Yasuda reports a relationship with Eli Lilly that includes: speaking and lecture fees. Shinsuke Yasuda reports a relationship with GlaxoSmithKline that includes: speaking and lecture fees. Shinsuke Yasuda reports a relationship with Pfizer Inc. that includes: speaking and lecture fees. Tadashi Hosoya reports a relationship with Sony Corporation that includes: funding grants. Tadashi Hosoya reports a relationship with Terumo life science foundation that includes: funding grants. Mari Kamiya reports a relationship with GlaxoSmithKline that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Deficiency of TOM5, a mitochondrial protein, causes organizing pneumonia (OP) in mice. The clinical significance and mechanisms of TOM5 in the pathogenesis of OP remain elusive. We demonstrated that TOM5 was significantly increased in the lung tissues of OP patients, which was positively correlated with the collagen deposition. In a bleomycin-induced murine model of chronic OP, increased TOM5 was in line with lung fibrosis. In vitro, TOM5 regulated the mitochondrial membrane potential in alveolar epithelial cells. TOM5 reduced the proportion of early apoptotic cells and promoted cell proliferation. Our study shed light on the roles of TOM5 in OP.

Backgroud: Bronchopulmonary dysplasia (BPD) is one of the most important complications plaguing neonates and can lead to a variety of sequelae. the ability of the HIF-1α/VEGF signaling pathway to promote angiogenesis has an important role in neonatal lung development., Method: Newborn rats were exposed to 85% oxygen. The effects of hyperoxia exposure on Pleomorphic Adenoma Gene like-2 (PLAGL2) and the HIF-1α/VEGF pathway in rats lung tissue were assessed through immunofluorescence and Western Blot analysis. In cell experiments, PLAGL2 was upregulated, and the effects of hyperoxia and PLAGL2 on cell viability were evaluated using scratch assays, CCK-8 assays, and EDU staining. The role of upregulated PLAGL2 in the HIF-1α/VEGF pathway was determined by Western Blot and RT-PCR. Apoptosis and ferroptosis effects were determined through flow cytometry and viability assays., Results: Compared with the control group, the expression levels of PLAGL2, HIF-1α, VEGF, and SPC in lung tissues after 3, 7, and 14 days of hyperoxia exposure were all decreased. Furthermore, hyperoxia also inhibited the proliferation and motility of type II alveolar epithelial cells (AECII) and induced apoptosis in AECII. Upregulation of PLAGL2 restored the proliferation and motility of AECII and suppressed cell apoptosis and ferroptosis, while the HIF-1α/VEGF signaling pathway was also revived., Conclusions: We confirmed the positive role of PLAGL2 and HIF-1α/VEGF signaling pathway in promoting BPD in hyperoxia conditions, and provided a promising therapeutic targets.

Lung cancer remains a major contributor to cancer fatalities, with cigarette smoking known to be responsible for up to 80% of cases. Based on the ability of cigarette smoke to induce inflammation in the lungs and increased lung cancer incidence in smokers with inflammatory conditions such as COPD, we hypothesized that inflammation plays an important role in the carcinogenicity of cigarette smoke. To test this hypothesis, we performed multi-omic analyses of Type II pneumocytes of A/J mice exposed to cigarette smoke for various time periods. We found that cigarette smoke exposure resulted in significant changes in DNA methylation and hydroxymethylation, gene expression patterns, and protein abundance that were partially reversible and contributed to an inflammatory and potentially oncogenic phenotype.

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress., (© 2024. The Author(s).)