Alveolar Type II Cells Research Articles

Ssc-miR-101-3p inhibits hypoxia-induced apoptosis and inflammatory response in alveolar type-II epithelial cells of Tibetan pigs via targeting FOXO3

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.

Airway derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD.

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

Abstract 2762: Spatial proteomics of lung adenocarcinoma mouse models with divergent propensities for metastasis

Abstract Introduction: Lung cancer is the leading cause of cancer deaths worldwide with most deaths attributed to metastasis. Metastases in existing mouse models of lung adenocarcinoma (LUAD) is sporadic and often requires several months of aging. These factors limit these models for basic and pre-clinical research aimed at identifying mechanisms of metastatic disease. We have recently developed two novel mouse models of LUAD that feature constitutively active Kras, loss of Trp53 function and truncated Dicer1 in different cell types. The observed survival and metastatic phenotypes of the models are dependent on the cell type in which the genetic alterations are made. We applied spatial proteomics and metabolomics to investigate molecular drivers underlying the phenotypic differences in our models. Methods and Results: Mouse models were generated by adding mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway, to a mouse model of Kras-driven LUAD. For both models, tumorigenesis was induced by conditional expression of an oncogenic allele of Kras, deletion of both alleles of Trp53 and deletion of one allele of Dicer1 in one cell type and the expression of a truncated Dicer1 allele in a different cell type. Mice expressing KrasG12D and deleted Trp53 in club cells without Dicer1 truncation have a reported median survival of 28.6 weeks after tumor induction. We observed accelerated development of LUAD and lymph node metastasis (12.1 weeks) only when we induced tumorigenesis in club cells and truncate Dicer1 in alveolar type II (ATII) cells. Induction of tumorigenesis in ATII cells and truncation of Dicer1 in club cells did not accelerate tumorigenesis or lead to detectable metastasis. Prelminary spatial proteomic analysis revealed significant upregulation of proteins associated with mitochondrial respiratory chain complex I in the tumors of both models which is consistent with processes of bioenergetic adaptation of tumor cells to hypoxic conditions. Gene Ontology annotation term enrichment revealed that the tumors of the two models have distinctive molecular function profiles. Both models show significant disruption of amino acid metabolism but only the model with increased metastatic potential showed apparent disruption in metabolites associated with steroidogenesis. Conclusions: Through cell type specific truncation/deletion of Dicer1 we have generated a mouse model that rapidly develops Kras/Trp53 driven LUAD and metastatic disease. Our findings suggest that tumorigenesis and metastasis are influenced by miRNA-mediated communication between different cell types. Additional studies are underway to link miRNA expression in these models to the results reported here for spatial proteomics and metabolomics. We will continue to develop this model to investigate the molecular mechanisms underlying metastasis. Citation Format: Paige Ramkissoon, Naomi Mitchell-Hutchinson, Julie Wells, Rick Maser, Tim Stodola, Brian Hoffmann, Anne Marchini, Elaine Bechtel, Rosalinda Doty, Carol J. Bult. Spatial proteomics of lung adenocarcinoma mouse models with divergent propensities for metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2762.

Inhalation of particulate matter containing environmentally persistent free radicals induces endothelial dysfunction mediated via AhR activation at the air-blood interface.

Particulate matter (PM) containing environmentally persistent free radicals (EPFR) is formed by the incomplete combustion of organic wastes, resulting in the chemisorption of pollutants to the surface of PM containing redox-active transition metals. In prior studies in mice, EPFR inhalation impaired endothelium-dependent vasodilation. These findings were associated with aryl hydrocarbon receptor (AhR) activation in the alveolar type-II (AT-II) cells that form the air-blood interface in the lung. We thus hypothesized that AhR activation in AT-II cells promotes the systemic release of mediators that promote endothelium dysfunction peripheral to the lung. To test our hypothesis, we knocked down AhR in AT-II cells of male and female mice and exposed them to 280 µg/m3 EPFR lo (2.7e + 16 radicals/g) or EPFR (5.5e + 17 radicals/g) compared with filtered air for 4 h/day for 1 day or 5 days. AT-II-AhR activation-induced EPFR-mediated endothelial dysfunction, reducing endothelium-dependent vasorelaxation by 59%, and eNOS expression by 50%. It also increased endothelin-1 mRNA levels in the lungs and peptide levels in the plasma in a paracrine fashion, along with soluble vascular cell adhesion molecule-1 and iNOS mRNA expression, possibly via NF-kB activation. Finally, AhR-dependent increases in antioxidant response signaling, coupled to increased levels of 3-nitrotyrosine in the lungs of EPFR-exposed littermate control but not AT-II AhR KO mice suggested that ATII-specific AhR activation promotes oxidative and nitrative stress. Thus, AhR activation at the air-blood interface mediates endothelial dysfunction observed peripheral to the lung, potentially via release of systemic mediators.

The Regulation of Fatty Acid Synthase by Exosomal miR-143-5p and miR-342-5p in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease caused by an aberrant repair of injured alveolar epithelial cells. The maintenance of the alveolar epithelium and its regeneration after the damage is fueled by alveolar type II (ATII) cells. Injured cells release exosomes containing microRNAs (miRNAs), which can alter the recipient cells' function. Lung tissue, ATII cells, fibroblasts, plasma, and exosomes were obtained from naive patients with IPF, patients with IPF taking pirfenidone or nintedanib, and control organ donors. miRNA expression was analyzed to study their impact on exosome-mediated effects in IPF. High miR-143-5p and miR-342-5p levels were detected in ATII cells, lung tissue, plasma, and exosomes in naive patients with IPF. Decreased FASN (fatty acid synthase) and ACSL-4 (acyl-CoA-synthetase long-chain family member 4) expression was found in ATII cells. miR-143-5p and miR-342-5p overexpression or ATII cell treatment with IPF-derived exosomes containing these miRNAs lowered FASN and ACSL-4 levels. Also, this contributed to ATII cell injury and senescence. However, exosomes isolated from patients with IPF taking nintedanib or pirfenidone increased FASN expression in ATII cells compared with naive patients with IPF. Furthermore, fibroblast treatment with exosomes obtained from naive patients with IPF increased SMAD3, CTGF, COL3A1, and TGFβ1 expression. Our results suggest that IPF-derived exosomes containing miR-143-5p and miR-342-5p inhibited the de novo fatty acid synthesis pathway in ATII cells. They also induced the profibrotic response in fibroblasts. Pirfenidone and nintedanib improved ATII cell function and inhibited fibrogenesis. This study highlights the importance of exosomes in IPF pathophysiology.

Role of mitochondrial fusion proteins MFN2 and OPA1 on lung cellular senescence in chronic obstructive pulmonary disease

BackgroundMitochondrial dysfunction and lung cellular senescence are significant features involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) stands as the primary contributing factor to COPD. This study examined mitochondrial dynamics, mitophagy and lung cellular senescence in COPD patients and investigated the effects of modulation of mitochondrial fusion [mitofusin2 (MFN2) and Optic atrophy 1 (OPA1)] on CS extract (CSE)-induced lung cellular senescence.MethodsSenescence-associated secretory phenotype (SASP) component mRNAs (IL-1β, IL-6, CXCL1 and CXCL8), mitochondrial morphology, mitophagy and mitochondria-related proteins (including phosphorylated-DRP1(p-DRP1), DRP1, MFF, MNF2, OPA1, PINK1, PARK2, SQSTM1/p62 and LC3b) and senescence-related proteins (including P16, H2A.X and Klotho) were measured in lung tissues or primary alveolar type II (ATII) cells of non-smokers, smokers and COPD patients. Alveolar epithelial (A549) cells were exposed to CSE with either pharmacologic inducer (leflunomide and BGP15) or genetic induction of MFN2 and OPA1 respectively.ResultsThere were increases in mitochondrial number, and decreases in mitochondrial size and activity in lung tissues from COPD patients. SASP-related mRNAs, DRP1 phosphorylation, DRP1, MFF, PARK2, SQSTM1/p62, LC3B II/LC3B I, P16 and H2A.X protein levels were increased, while MFN2, OPA1, PINK1 and Klotho protein levels were decreased in lung tissues from COPD patients. Some similar results were identified in primary ATII cells of COPD patients. CSE induced increases in oxidative stress, SASP-related mRNAs, mitochondrial damage and dysfunction, mitophagy and cellular senescence in A549 cells, which were ameliorated by both pharmacological inducers and genetic overexpression of MFN2 and OPA1.ConclusionsImpaired mitochondrial fusion, enhanced mitophagy and lung cellular senescence are observed in the lung of COPD patients. Up-regulation of MFN2 and OPA1 attenuates oxidative stress, mitophagy and lung cellular senescence, offering potential innovative therapeutic targets for COPD therapy.Graphical

LIRAGLUTIDE ALLEVIATES ACUTE LUNG INJURY AND MORTALITY IN PNEUMONIA-INDUCED SEPSIS THROUGH REGULATING SURFACTANT PROTEIN EXPRESSION AND SECRETION.

Glucagon-like peptide 1 (GLP-1) analogs are used to treat type 2 diabetes, and they can regulate insulin secretion, energy homeostasis, inflammation, and immune cell function. This study sought to determine whether the GLP-1 analog liraglutide exerts a beneficial action in an acute lung injury model of pneumonia-induced sepsis. Methods: Wild-type FVB/NJ mice (n = 114) were infected by intratracheal injection with Pseudomonas aeruginosa Xen5 (4 × 10 4 CFU/mouse) or an equal volume (50 μL) of saline (control) with or without a subcutaneous injection of liraglutide (2 mg/kg, 30 min after infection). Mice were killed 24 h after infection. Lung tissues and BALF were analyzed. In separate experiments, the dynamic growth of bacteria and animal mortality was monitored using in vivo imaging system within 48 h after infection. In addition, primary lung alveolar type II cells isolated from mice were used to study the mechanism of liraglutide action. Result: Liraglutide improved survival ( P < 0.05), decreased bacterial loads in vivo , and reduced lung injury scores ( P < 0.01) in septic mice. Liraglutide-treated mice showed decreased levels of inflammatory cells ( P < 0.01) and proinflammatory cytokines (TNF-α and IL-6) ( P < 0.01) in the lung compared with septic controls. Liraglutide significantly increased pulmonary surfactant proteins (SP-A and SP-B) expression/secretion ( P < 0.01) and phospholipid secretion ( P < 0.01) in vivo . Primary alveolar type II cells pretreated with liraglutide improved SP-A and SP-B expression after LPS exposure ( P < 0.01). Conclusion: Liraglutide attenuates mortality and lung inflammation/injury in pneumonia-induced sepsis. The increased surfactant expression/secretion and anti-inflammatory effects of liraglutide represent potential mechanisms by GLP-1 agonists potentiate host defense and maintain alveolar respiratory function in acute lung injury.

Sevoflurane with Low Concentration Decrease DNA Methylation on Chronic Obstructive Pulmonary Disease (COPD)-Related Gene Promoter in COPD Rat

Chronic obstructive pulmonary disease (COPD) is a difficult-to-cure disease that mainly affects the respiratory system. Inhaled anesthetic drug such as sevoflurane plays a controversial role in COPD by different concentration, but the underlying epigenetic mechanism remains unclear. Here, we prepared lipopolysaccharide (LPS)-induced COPD rat model, and isolated Alveolar type II (ATII) cells. We mainly focused DNA methylation on the promoter of COPD-related genes including Sftpa1, Napsa, Ca2, Sfta2, Lamp3, Wif1, Pgc, and Etv5. We observed COPD rat treated by sevoflurane with low (0.5%) and high (2%) concentrations displayed an opposite DNA methylation pattern. These six genes' promoter were all hypomethylated by 0.5% sevoflurane whereas hypermethylated by 2% sevoflurane, accompanied with the opposite transcriptional activity. We further verified that the DNMT1 binding ability contributed to DNA methylation these six genes' promoter. Moreover, we also captured DNMT1 and identified REC8 meiotic recombination protein (REC8) as the specific binding protein only existed in ATII cells treated with 0.5% sevoflurane rather than 2% and control. The binding ability of REC8 on these target genes' promoter showed highly positive correlation with DNMT1. In summary, we uncovered a potential epigenetic role of sevoflurane with low concentration in ATII cells of COPD that may help us deeply understand the pathogenesis and treatment mechanism of inhaled anesthesia drugs in COPD via a dose-dependent manner.

IL-1β mediates the induction of immune checkpoint regulators IDO1 and PD-L1 in lung adenocarcinoma cells

IntroductionInflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators.MethodsTo determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry.ResultsUpon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells.ConclusionsOur studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.AWNWNjEXZEagXxNY6iY8VPVideo

The deubiquitinase UCHL3 mediates p300-dependent chemokine signaling in alveolar type II cells to promote pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, fatal, fibrotic, interstitial lung disease of unknown cause. Despite extensive studies, the underlying mechanisms of IPF development remain unknown. Here, we found that p300 was upregulated in multiple epithelial cells in lung samples from patients with IPF and mouse models of lung fibrosis. Lung fibrosis was significantly diminished by the alveolar type II (ATII) cell–specific deletion of the p300 gene. Moreover, we found that ubiquitin C-terminal hydrolase L3 (UCHL3)-mediated deubiquitination of p300 led to the transcriptional activation of the chemokines Ccl2, Ccl7, and Ccl12 through the cooperative action of p300 and C/EBPβ, which consequently promoted M2 macrophage polarization. Selective blockade of p300 activity in ATII cells resulted in the reprogramming of M2 macrophages into antifibrotic macrophages. These findings demonstrate a pivotal role for p300 in the development of lung fibrosis and suggest that p300 could serve as a promising target for IPF treatment.

Short telomeres in alveolar type II cells associate with lung fibrosis in post COVID-19 patients with cancer.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic. The severity of COVID-19 increases with each decade of life, a phenomenon that suggest that organismal aging contributes to the fatality of the disease. In this regard, we and others have previously shown that COVID-19 severity correlates with shorter telomeres, a molecular determinant of aging, in patient's leukocytes. Lung injury is a predominant feature of acute SARS-CoV-2 infection that can further progress to lung fibrosis in post-COVID-19 patients. Short or dysfunctional telomeres in Alveolar type II (ATII) cells are sufficient to induce pulmonary fibrosis in mouse and humans. Here, we analyze telomere length and the histopathology of lung biopsies from a cohort of alive post-COVID-19 patients and a cohort of age-matched controls with lung cancer. We found loss of ATII cellularity and shorter telomeres in ATII cells concomitant with a marked increase in fibrotic lung parenchyma remodeling in post- COVID-19 patients compared to controls. These findings reveal a link between presence of short telomeres in ATII cells and long-term lung fibrosis sequel in Post-COVID-19 patients.

119 Differential Alveolar Toxicity of Exhaust and Non-Exhaust Fine Particulate Matter

Abstract Introduction Airborne fine particulate matter (diameter &amp;lt;2.5μm; PM2.5) is a leading risk factor for ill health and premature death. Road vehicle technology is decreasing exhaust-derived PM emissions but poorly characterised, unregulated non-exhaust PM persists. This study aimed to characterise the toxicology of brakewear PM2.5 (BWPM) from 4 brakepad types, and roadwear PM, compared to diesel exhaust PM2.5 (DEP). Methods An alveolar type-II epithelial cell line (ATIIER:KRASv12) was exposed to 3.9-31.6µg/cm2 PM2.5 generated from 4 brake pad types (low-metallic/semi-metallic/non-asbestos organic [NAO]/ceramic), roadwear, or DEP for 2-24h. PM composition was determined by ICP-MS, cytotoxicity by LDH and MTT assays, cytokine release by ELISA, gene expression by RT-qPCR and RNA-Seq, and protein expression by western blot. Results NAO and ceramic BWPM, enriched in copper and zirconium, exerted greatest increases in cytotoxicity, cytokine release (IL-6: NAO 26-fold, ceramic 20-fold; p&amp;lt;0.05), and oxidative stress-related (HMOX1 – NAO 28-fold, ceramic 26-fold, p&amp;lt;0.001) and metal homeostasis genes (MT1G: NAO 197-fold, ceramic 183-fold; p&amp;lt;0.001), consistently greater than roadwear PM or DEP. Amelioration by copper-chelating tetraethylenepentamine implicated BWPM copper. RNA-Seq showed NAO and ceramic BWPM induced most differentially expressed genes vs. control, related to inflammatory, oxidative stress, heat-shock, and hypoxia responses. Effects on hypoxia signalling were confirmed through HIF1α western blot and HIF-signalling reporter assay. Conclusions NAO and ceramic BWPM were the most potent across multiple endpoints. Future work will study the relevance of HIF-related responses, which have been implicated in fibrotic lung disease. Understanding source-dependent toxicity may facilitate manufacturing- and healthcare-related mitigation of PM toxicity.

55 Investigating the co-Exposure of Micro(nano)Plastics and Indoor/Outdoor Particulate Matter Using an Alveolar BarrierIn Vitro Model

Abstract The impact of micro-, nanoplastics (MNPs) and particulate matter (PM) upon human health are escalating concerns worldwide. MNPs released from surgical face masks, and PM from ambient air pollution, can both be inhaled and consequently have potentially significant negative impacts upon human health, particularly on our lung epithelial cell structure and function. Currently, the impact of the realistic co-exposure of these samples is unknown. Therefore, the purpose of this study was to determine the potential inhaled hazardous health effects of the co-exposure of MNPs and ambient (indoor and outdoor) air PM. Using an advanced in vitro testing model combined with a specific mechanistic toxicology approach, it is hypothesised that the co-exposure approach will elucidate important information regarding the differential toxicology of ambient (indoor and outdoor) air PM. The in vitro system will consist of alveolar epithelial type-II cells (-NCI-H441) combined with differentiated monocytes (dTHP-1), cultured at the relevant in vivo ratio and at the air-liquid interface. Cell cultures will be exposed for both 4 and 24hrs to MNP (commercially available polypropylene (PP) and blue face mask extracted PP), PM (NIST 2584 and NIST 1649a respectively) and then subsequent MNP+PM (both NIST 2584 and NIST 1649a respectively) via aerosol (VITROCELL Cloud12), using alternative dosimetry patterns. The biochemical endpoints of cytotoxicity, oxidative stress, (pro-)inflammatory response and genotoxicity, will then be assessed to provide the basis for future global analytical techniques to examine the currently unknown human hazard of inhaled MNPs (+/- PM).

Abstract 1321: New mouse models of metastatic lung cancer

Abstract INTRODUCTION: Lung cancer is the leading cause of cancer deaths worldwide and greater than 75% of human lung cancer deaths can be attributed to metastasis. Metastatic disease in most existing mouse models of pulmonary adenocarcinoma is typically sporadic and often requires aging mice for several months. These factors limit the usefulness of most current mouse models for basic and pre-clinical research aimed at identifying mechanisms and effective treatments of metastatic disease. We have developed two novel mouse models of metastatic lung cancer and report here on changes in protein expression and immune cell recruitment in the lungs of these models. METHODS AND RESULTS: Mouse models were generated by adding mutations in Dicer1, an RNAse III enzyme within the microRNA (miRNA) biosynthesis pathway, to a mouse model of Kras-driven pulmonary adenocarcinoma. For both models, tumorigenesis was induced by conditional expression of an oncogenic allele of Kras (KrasG12D), deletion of both alleles of Trp53 and deletion of one allele of Dicer1 in one cell type and the expression of a truncated Dicer1 allele in a different cell type. In the absence of Dicer1 truncation, mice expressing KrasG12D and deleting Trp53 in club cells have a reported median survival of 28.6 weeks after tumor induction. We detected accelerated development of pulmonary adenocarcinoma and lymph node metastasis (12.1 weeks) only when we induced tumorigenesis in club cells and truncated DICER1 in alveolar type II (ATII) cells. Induction of tumorigenesis in ATII cells and truncation of DICER1 in club cells did not accelerate tumorigenesis or metastasis. To evaluate the molecular and cellular changes in the different phenotypes of these models we have characterized protein expression, using spatial proteomics, and immune cell recruitment, using flow cytometry. CONCLUSIONS: Through cell type specific truncation/deletion of Dicer1 we have generated a new mouse model that rapidly develops pulmonary adenocarcinomas and metastatic disease. Our findings support our hypothesis that tumorigenesis and metastasis are influenced by miRNA regulated communication between different cell types. Preliminary flow cytometry and spatial proteomic analyses have suggested cellular targets underlying phenotypic differences between our two mouse models. These models have potential for both understanding the basic processes of metastasis and for pre-clinical studies aimed at preventing and/or treating metastatic lung cancer. Citation Format: Paige Ramkissoon, Julie Wells, Richard S. Maser, Jiayuan Shi, Zheng Gong, Qing Li, Brian Hoffmann, Anne Marchini, Elaine Bechtel, Rosalinda Doty, Gary Ren, Carol J. Bult. New mouse models of metastatic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1321.

Abstract 861: Modelling the role of EGFR mutation in the initiation of never-smoker lung cancer

Abstract Genetic mutations associated with never-smoker lung cancer frequently appear in normal lung tissue, in both cancer and non-cancer patients. It is unclear what circumstances are required for a mutation to fix in healthy tissue and eventually lead to a tumor. To investigate this, alveolar type-II murine cells were grown in a 3D nutrient system, forming spheroidal clusters. The volumes of these clusters were measured at 7 and 14 days and a mathematical model was devised to describe their growth. In the absence of an EGFR mutation, variations in growth between clusters can be explained by a skew-normal distribution of initial division rates. These rates decline exponentially as the clusters age, and the decline rate is the same across clusters. When cells possess an epidermal growth factor receptor (EGFR) mutation or are grown in the presence of interleukin-1 beta (il-1b) growth factor, clusters form more complex shapes, which we believe may be driven by branching processes and cell-cell interactions. Accordingly, more complex models are needed to explain their development. This work lays the groundwork for new, parameterized models of mutational spread in lung tissue, and a deeper understanding of the role of mutation in carcinogenesis. Citation Format: Helena M. Coggan, Clare E. Weeden, Mohit Dalwadi, Charles Swanton, Philip Pearce, Karen M. Page. Modelling the role of EGFR mutation in the initiation of never-smoker lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 861.

Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1.

The IFN system constitutes a powerful antiviral defense machinery. Consequently, effective IFN responses protect against severe COVID-19 and exogenous IFNs inhibit SARS-CoV-2 in vitro. However, emerging SARS-CoV-2 variants of concern (VOCs) may have evolved reduced IFN sensitivity. Here, we determined differences in replication and IFN susceptibility of an early SARS-CoV-2 isolate (NL-02-2020) and the Alpha, Beta, Gamma, Delta, and Omicron VOCs in Calu-3 cells, iPSC-derived alveolar type-II cells (iAT2) and air-liquid interface (ALI) cultures of primary human airway epithelial cells. Our data show that Alpha, Beta, and Gamma replicated to similar levels as NL-02-2020. In comparison, Delta consistently yielded higher viral RNA levels, whereas Omicron was attenuated. All viruses were inhibited by type-I, -II, and -III IFNs, albeit to varying extend. Overall, Alpha was slightly less sensitive to IFNs than NL-02-2020, whereas Beta, Gamma, and Delta remained fully sensitive. Strikingly, Omicron BA.1 was least restricted by exogenous IFNs in all cell models. Our results suggest that enhanced innate immune evasion rather than higher replication capacity contributed to the effective spread of Omicron BA.1.

The Defenders of the Alveolus Succumb in COVID-19 Pneumonia to SARS-CoV-2 and Necroptosis, Pyroptosis, and PANoptosis.

Alveolar type II (ATII) pneumocytes as defenders of the alveolus are critical to repairing lung injury. We investigated the ATII reparative response in coronavirus disease 2019 (COVID-19) pneumonia, because the initial proliferation of ATII cells in this reparative process should provide large numbers of target cells to amplify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus production and cytopathological effects to compromise lung repair. We show that both infected and uninfected ATII cells succumb to tumor necrosis factor-α (TNF)-induced necroptosis, Bruton tyrosine kinase (BTK)-induced pyroptosis, and a new PANoptotic hybrid form of inflammatory cell death mediated by a PANoptosomal latticework that generates distinctive COVID-19 pathologies in contiguous ATII cells. Identifying TNF and BTK as the initiators of programmed cell death and SARS-CoV-2 cytopathic effects provides a rationale for early antiviral treatment combined with inhibitors of TNF and BTK to preserve ATII cell populations, reduce programmed cell death and associated hyperinflammation, and restore functioning alveoli in COVID-19 pneumonia.

Effect of Hyaluronic Acid and Mesenchymal Stem Cells Secretome Combination in Promoting Alveolar Regeneration.

Pharmacological therapies in lung diseases are nowadays useful in reducing the symptomatology of lung injury. However, they have not yet been translated to effective treatment options able to restore the lung tissue damage. Cell-therapy based on Mesenchymal Stem Cells (MSCs) is an attractive, as well as new therapeutic approach, although some limitations can be ascribed for therapeutic use, such as tumorigenicity and immune rejection. However, MSCs have the capacity to secrete multiple paracrine factors, namely secretome, capable of regulating endothelial and epithelial permeability, decrease inflammation, enhancing tissue repair, and inhibiting bacterial growth. Furthermore, Hyaluronic acid (HA) has been demonstrated to have particularly efficacy in promoting the differentiation of MSCs in Alveolar type II (ATII) cells. In this frame, the combination of HA and secretome to achieve the lung tissue regeneration has been investigated for the first time in this work. Overall results showed how the combination of HA (low and medium molecular weight HA) plus secretome could enhance MSCs differentiation in ATII cells (SPC marker expression of about 5 ng/mL) compared to the only HA or secretome solutions alone (SPC about 3 ng/mL, respectively). Likewise, cell viability and cell rate of migration were reported to be improved for HA and secretome blends, indicating an interesting potentiality of such systems for lung tissue repair. Moreover, an anti-inflammatory profile has been revealed when dealing with HA and secretome mixtures. Therefore, these promising results can allow important advance in the accomplishment of the future therapeutic approach in respiratory diseases, up to date still missing.

The Role of Pulmonary Surfactant Phospholipids in Fibrotic Lung Diseases

Diffuse parenchymal lung diseases (DPLD) or Interstitial lung diseases (ILD) are a heterogeneous group of lung conditions with common characteristics that can progress to fibrosis. Within this group of pneumonias, idiopathic pulmonary fibrosis (IPF) is considered the most common. This disease has no known cause, is devastating and has no cure. Chronic lesion of alveolar type II (ATII) cells represents a key mechanism for the development of IPF. ATII cells are specialized in the biosynthesis and secretion of pulmonary surfactant (PS), a lipid-protein complex that reduces surface tension and minimizes breathing effort. Some differences in PS composition have been reported between patients with idiopathic pulmonary disease and healthy individuals, especially regarding some specific proteins in the PS; however, few reports have been conducted on the lipid components. This review focuses on the mechanisms by which phospholipids (PLs) could be involved in the development of the fibroproliferative response.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture

BackgroundMultiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7’s carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities.Results We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group.ConclusionsOur findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.