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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-1ß. 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-IL1ß groups, suggesting that long-term alteration in the epithelium after RSV infection is dependent on the IL-1ß pathway. This study provides insight into the molecular mechanisms of asthma predisposition after RSV infection.

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The concurrence of chronic obstructive pulmonary disease (COPD) and lung cancer has been widely reported and extensively addressed by pulmonologists and oncologists. However, most studies have focused on shared risk factors, DNA damage pathways, immune microenvironments, inflammation, and imbalanced proteases/antiproteases. In the present review, we explored the association between COPD and lung cancer in terms of airway pluripotent cell fate determination and discussed the various cell types and signaling pathways involved in the maintenance of lung epithelium homeostasis, and their involvement in the pathogenesis of co-occurrence of COPD and lung cancer.

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A cell is a dynamic system in which various processes occur simultaneously. In particular, intra- and intercellular signaling pathway crosstalk has a significant impact on a cell's life cycle, differentiation, proliferation, growth, regeneration, and, consequently, on the normal functioning of an entire organ. Hippo signaling and YAP/TAZ nucleocytoplasmic shuttling play a pivotal role in normal development, homeostasis, and tissue regeneration, particularly in lung cells. Intersignaling communication has a significant impact on the core components of the Hippo pathway and on YAP/TAZ localization. This review describes the crosstalk between Hippo signaling and key lung signaling pathways (WNT, SHH, TGFß, Notch, Rho, and mTOR) using lung cells as an example and highlights the remaining unanswered questions.

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The surface spike (S) glycoprotein mediates cell entry of SARS-CoV-2 into the host through fusion at the plasma membrane or endocytosis. Omicron lineages/sublineages have acquired extensive mutations in S to gain transmissibility advantages and altered antigenicity. The fusogenicity, antigenicity, and evasion of Omicron subvariants have been extensively investigated at unprecedented speed to align with the mutation rate of S. Cells that overexpress receptors/cofactors are mostly used as hosts to amplify infection sensitivity to tested variants. However, systematic cell entry comparisons of most prior dominant Omicron subvariants using human lung epithelium cells are yet to be well-studied. Here, with human bronchial epithelium BEAS-2B cells as the host, we compared single-round virus-to-cell entry and cell-to-cell fusion of Omicron BA.1, BA.5, BQ.1.1, CH.1.1, XBB.1.5, and XBB.1.16 based upon split NanoLuc fusion readout assays and the S-pseudotyped lentivirus system. Virus-to-cell entry of tested S variants exhibited cell-type dependence. The parental Omicron BA.1 required more time to develop full entry to HEK293T-ACE2-TMPRSS2 than BEAS-2B cells. Compared to unchanged P681, S-cleavage constructs of P681H/R did not have any noticeable advantages in cell entry. Omicron BA.1 and its descendants entered BEAS-2B cells more efficiently than D614G, and it was slightly less or comparable to that of Delta. Serine protease-pretreated Omicron subvariants enhanced virus-to-cell entry in a dose-dependent manner, suggesting fusion at the plasma membrane persists as a productive cell entry route. Spike-mediated cell-to-cell fusion and total S1/S2 processing of Omicron descendants were similar. Our results indicate no obvious entry or fusion advantages of recent Omicron descendants over preceding variants since Delta, thus supporting immune evasion conferred by antigenicity shifts due to altered S sequences as probably the primary viral fitness driver.

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The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

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Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.

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The global burden of respiratory diseases is very high and still on the rise, prompting the need for accurate models for basic and translational research. Several model systems are currently available ranging from simple airway cell cultures to complex tissue-engineered lungs. In recent years, human lung organoids have been established as highly transferrable three-dimensional in vitro model systems for lung research. For acute infectious and chronic inflammatory diseases as well as lung cancer, human lung organoids have opened possibilities for precise in vitro research and a deeper understanding of mechanisms underlying lung injury and regeneration. Human lung organoids from induced pluripotent stem cells or from adult stem cells of patients' samples introduce tools for understanding developmental processes and personalized medicine approaches. When further state-of-the-art technologies and protocols come into use, the full potential of human lung organoids can be harnessed. High-throughput assays in drug development, gene therapy, and organoid transplantation are current applications of organoids in translational research. In this review, we emphasize novel approaches in translational and personalized medicine in lung research focusing on the use of human lung organoids.

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Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon in the air, triggers pulmonary inflammation. This study focused on BaP-induced inflammation in the alveolar epithelium. A549 cells were stimulated with BaP for four days. BaP treatment markedly increased NLRP1 expression but slightly decreased NLRP3. Furthermore, aryl hydrocarbon receptor (AhR) knockdown displayed no increase in BaP-induced NLRP1 expression. Similar results were also observed by blocking reactive oxygen species (ROS), which is mediated through AhR, suggesting that the AhR-ROS axis operates in BaP-induced NLRP1 expression. p53 involvement in ROS-mediated NLRP1 induction has also been implied. When we confirmed inflammasome activation in cells treated with BaP for four days, while BaP transiently activated NLRP3, it predominantly activated the NLRP1 inflammasome. These findings have led to the conclusion that BaP could be a potential ligand for the NLRP1 inflammasome persistently observed in the lung epithelium. Our study may provide additional evidence for the sustained pulmonary inflammation caused by environmental air pollution.

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The respiratory system is composed of a multitude of cells that organize to form complex branched airways that end in alveoli, which respectively function to guide air flow and mediate gas exchange with the bloodstream. The organization of the respiratory sytem relies on distinct forms of cell polarity, which guide lung morphogenesis and patterning in development and provide homeostatic barrier protection from microbes and toxins. The stability of lung alveoli, the luminal secretion of surfactants and mucus in the airways, and the coordinated motion of multiciliated cells that generate proximal fluid flow, are all critical functions regulated by cell polarity, with defects in polarity contributing to respiratory disease etiology. Here, we summarize the current knowledge of cell polarity in lung development and homeostasis, highlighting key roles for polarity in alveolar and airway epithelial function and outlining relationships with microbial infections and diseases, such as cancer.

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Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E2. The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O2) or normoxia (21% O2) during 24 h, with or without transforming growth factor (TGF)-ß1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-ß1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE2, IL-6 and IL-8, whereas TGF-ß1 stimulus significantly increased the release of PGE2 and IL-6. TGF-ß1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-ß1 stimulated hAELVi cells showed a decreased release of PGE2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders.

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Chronic cigarette smoke condensate (CSC) exposure is one of the preventable risk factors in the CS-induced lung cancer. However, understanding the mechanism of cellular transformation induced by CS in the lung remains limited. We investigated the effect of long term exposure of CSC in human normal lung epithelial Beas-2b cells, and chemopreventive mechanism of organosulphur garlic compounds, diallyl sulphide (DAS) and diallyl disulphide (DADS) using Next Generation Sequencing (NGS) transcriptomic analysis. CSC regulated 1077 genes and of these 36 genes are modulated by DAS while 101 genes by DADS. DAS modulated genes like IL1RL1 (interleukin-1 receptor like-1), HSPA-6 (heat shock protein family A, member 6) while DADS demonstrating ADTRP (Androgen-Dependent TFPI Regulating Protein), ANGPT4 (Angiopoietin 4), GFI1 (Growth Factor-Independent 1 Transcriptional Repressor), TBX2 (T-Box Transcription Factor 2), with some common genes like NEURL-1 (Neuralized E3-Ubiquitin Protein Ligase 1), suggesting differential effects between these two garlic compounds. They regulate genes by influencing pathways including HIF-1alpha, STAT-3 and matrix metalloproteases, contributing to the chemoprotective ability of organosulfur garlic compounds against CSC-induced cellular transformation. Taken together, we demonstrated CSC induced global gene expression changes pertaining to cellular transformation which potentially can be delayed with dietary chemopreventive phytochemicals like DS and DADS influencing alterations at the transcriptomic level.

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The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our laboratory has demonstrated that an inhaled combination of a Toll-like receptor (TLR) 2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust protection against otherwise lethal pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are required for inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse-phase protein array analysis demonstrated robust STAT3 (signal transducer and activator of transcription 3) phosphorylation following treatment of lung epithelial cells. We show here that Pam2ODN-induced STAT3 phosphorylation is IL-6-independent. We further found that therapeutic epithelial STAT3 activation is required for inducible protection against Pseudomonas aeruginosa pneumonia. Additional studies showed that inhibiting epithelial dual oxidases or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these mechanisms, we analyzed the contributions of redox-sensitive kinases and found that Pam2ODN activated epithelial growth factor receptor in an ROS-dependent manner that is required for therapeutically inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN's function and describe a novel redox-based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.

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The lungs are an immunologically unique environment; they are exposed to innumerable pathogens and particulate matter daily. Appropriate clearance of pathogens and response to pollutants is required to prevent overwhelming infection, while preventing tissue damage and maintaining efficient gas exchange. Broadly, the innate immune system is the collection of immediate, intrinsic immune responses to pathogen or tissue injury. In this review, we will examine the innate immune responses of the lung, with a particular focus on their role in pneumonia. We will discuss the anatomic barriers and antimicrobial proteins of the lung, pathogen and injury recognition, and the role of leukocytes (macrophages, neutrophils, and innate lymphocytes) and lung stromal cells in innate immunity. Throughout the review, we will focus on new findings in innate immunity as well as features that are unique to the lung.

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Silver (Ag) is known to possess antimicrobial properties which is commonly attributed to soluble Ag ions. Here, we showed that Ag nanoparticles (NPs) potently inhibited SARS-CoV-2 infection using two different pseudovirus neutralization assays. We also evaluated a set of Ag nanoparticles of different sizes with varying surface properties, including polyvinylpyrrolidone (PVP)-coated and poly (ethylene glycol) (PEG)-modified Ag nanoparticles, and found that only the bare (unmodified) nanoparticles were able to prevent virus infection. For comparison, TiO2 nanoparticles failed to intercept the virus. Proteins and lipids may adsorb to nanoparticles forming a so-called bio-corona; however, Ag nanoparticles pre-incubated with pulmonary surfactant retained their ability to block virus infection in the present model. Furthermore, the secondary structure of the spike protein of SARS-CoV-2 was perturbed by the Ag nanoparticles, but not by the ionic control (AgNO3) nor by the TiO2 nanoparticles. Finally, Ag nanoparticles were shown to be non-cytotoxic towards the human lung epithelial cell line BEAS-2B and this was confirmed by using primary human nasal epithelial cells. These results further support that Ag nanoparticles may find use as anti-viral agents.

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The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.

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With the aim of expediting drug target discovery and validation for respiratory diseases, we developed an optimized method for in situ somatic gene disruption in murine lung epithelial cells via AAV6-mediated CRISPR-Cas9 delivery. Efficient gene editing was observed in lung type II alveolar epithelial cells and distal airway cells following assessment of single- or dual-guide AAV vector formats, Cas9 variants, and a sequential dosing strategy with combinatorial guide RNA expression cassettes. In particular, we were able to demonstrate population-wide gene disruption within distinct epithelial cell types for separate targets in Cas9 transgenic animals, with minimal to no associated inflammation. We also observed and characterized AAV vector integration events that occurred within directed double-stranded DNA break sites in lung cells, highlighting a complicating factor with AAV-mediated delivery of DNA nucleases. Taken together, we demonstrate a uniquely effective approach for somatic engineering of the murine lung, which will greatly facilitate the modeling of disease and therapeutic intervention.

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One of the causes of death of patients infected by SARS-CoV-2 is the induced respiratory failure caused by excessive activation of the immune system, the so-called "cytokine storm", leading to damage to lung tissue. In vitro models reproducing various stages of the disease can be used to explore the pathogenetic mechanisms and therapeutic approaches to treating the consequences of a cytokine storm. We have developed an in vitro test system for simulating damage to the pulmonary epithelium as a result of the development of a hyperinflammatory reaction based on the co-cultivation of pulmonary epithelial cells (A549 cells) and human peripheral blood mononuclear cells (PBMC) primed with lipopolysaccharide (LPS). In this model, after 24 h of co-cultivation, a sharp decrease in the rate of proliferation of A549 cells associated with the intrinsic development of oxidative stress and, ultimately, with the induction of PANoptotic death were observed. There was a significant increase in the concentration of 40 cytokines/chemokines in a conditioned medium, including TNF-α, IFN-α, IL-6, and IL-1a, which corresponded to the cytokine profile in patients with severe manifestation of COVID-19. In order to verify the model, the analysis of the anti-inflammatory effects of well-known substances (dexamethasone, LPS from Rhodobacter sphaeroides (LPS-RS), polymyxin B), as well as multipotent mesenchymal stem cells (MSC) and MSC-derived extracellular vesicles (EVs) was carried out. Dexamethasone and polymyxin B restored the proliferative activity of A549 cells and reduced the concentration of proinflammatory cytokines. MSC demonstrated an ambivalent effect through stimulated production of both pro-inflammatory cytokines and growth factors that regenerate lung tissue. LPS-RS and EVs showed no significant effect. The developed test system can be used to study molecular and cellular pathological processes and to evaluate the effectiveness of various therapeutic approaches for the correction of hyperinflammatory response in COVID-19 patients.

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Advances in adeno-associated virus (AAV) engineering have provided exciting new tools for research and potential solutions for gene therapy. However, the lung has not received the same tailored engineering as other major targets of debilitating genetic disorders. To address this, here we engineered the surface-exposed residues AA452-458 of AAV9 capsid proteins at the three-fold axis of symmetry and employed a Cre-transgenic-based screening platform to identify AAV capsids targeted to the lung after intravenous delivery in mice. Using a custom image processing pipeline to quantify transgene expression across whole tissue images, we found that one engineered variant, AAV9.452sub.LUNG1, displays dramatically improved transgene expression in lung tissue after systemic delivery in mice. This improved transduction extends to alveolar epithelial type II cells, expanding the toolbox for gene therapy research for diseases specific to the lung.

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Lung epithelium is constantly exposed to the environment and is critically important for the orchestration of initial responses to infectious organisms, toxins, and allergic stimuli, and maintenance of normal gaseous exchange and pulmonary function. The integrity of lung epithelium, fluid balance, and transport of molecules is dictated by the tight junctions (TJs). The TJs are formed between adjacent cells. We have focused on the topic of the TJ structure and function in lung epithelial cells. This review includes a summary of the last twenty years of literature reports published on the disrupted TJs and epithelial barrier in various lung conditions and expression and regulation of specific TJ proteins against pathogenic stimuli. We discuss the molecular signaling and crosstalk among signaling pathways that control the TJ structure and function. The Toll-like receptor-4 (TLR4) recognizes the pathogen- and damage-associated molecular patterns released during lung injury and inflammation and coordinates cellular responses. The molecular aspects of TLR4 signaling in the context of TJs or the epithelial barrier are not fully known. We describe the current knowledge and possible networking of the TLR4-signaling with cellular and molecular mechanisms of TJs, lung epithelial barrier function, and resistance to treatment strategies.

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