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Airway remodeling is a critical factor determining the pathogenesis and treatment sensitivity of severe asthma (SA) or uncontrolled asthma (UA). The activation of epithelial-mesenchymal trophic units (EMTUs) regulated by airway epithelial cells (AECs) has been proven to induce airway remodeling directly. However, the triggers for EMTU activation and the underlying mechanism of airway remodeling are not fully elucidated. Here, we screened the differentially expressed gene cathepsin C (CTSC; also known as dipeptidyl peptidase 1 [DPP-1]) in epithelia of patients with SA and UA using RNA-sequencing data and further verified the increased expression of CTSC in induced sputum of patients with asthma, which was positively correlated with severity and airway remodeling. Moreover, direct instillation of exogenous CTSC induced airway remodeling. Genetic inhibition of CTSC suppressed EMTU activation and airway remodeling in two asthma models with airway remodeling. Mechanistically, increased secretion of CTSC from AECs induced EMTU activation through the p38-mediated pathway, further inducing airway remodeling. Meanwhile, inhibition of CTSC also reduced the infiltration of inflammatory cells and the production of inflammatory factors in the lungs of asthmatic mice. Consequently, targeting CTSC with compound AZD7986 protected against airway inflammation, EMTU activation, and remodeling in the asthma model. Based on the dual effects of CTSC on airway inflammation and remodeling, CTSC is a potential biomarker and therapeutic target for SA or UA.

The mucociliary clearance (MC) system is a vital host defense against infection in the lung. MC system function is dependent on ciliary density, structure, and function and airway surface liquid (ASL) composition and hydration. Animal and human studies indicate that MC rate decreases with age which may contribute to the increased rates of pulmonary infection experienced by older people. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes an anion channel on epithelial surfaces that plays a key role in maintaining ASL hydration. Failure or dysfunction of CFTR could result in the dehydration of airway mucus, depressing MC. Here we use two available databases including bulk (GTEx) and single-cell (CELLxGENE) sequencing data from the lung to determine if CFTR expression decreases with age. Bulk expression data and single-cell expression data from goblet, club, and respiratory basal cells all demonstrated patterns of decreasing CFTR expression with age. Ciliated airway cells did not. Secretory cells (including club and goblet cells) and basal cells are the largest source of CFTR expression in the airway. This indicates that changes in CFTR expression and ASL dehydration may contribute to the decreasing MC associated with aging., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Rhinoviruses (RV) are the major cause of common colds in healthy individuals and are associated with acute exacerbations in patients with chronic lung diseases. Yet, no vaccines or effective treatment against RV are available. This study investigated the effect of Euphorbium compositum SN (ECSN6), a multicomponent, multitarget medication made from natural ingredients, on the mucosal barrier network during RV infection., Methods: Mucociliary-differentiated airway epithelial cell cultures were infected with RV or sham, and treated with 20% ECSN6 or placebo twice daily. Barrier integrity was assessed by measuring transepithelial resistance (TER), permeability to inulin, and expression and localization of intercellular junctions proteins (IJ). Ciliary beat frequency (CBF), expression of pro-inflammatory cytokines, antiviral interferons and mucins, and viral load were also measured. C57BL/6 mice were infected intranasally with RV or sham and treated with 40% ECSN6 or placebo twice daily. Inflammation of sinunasal mucosa, localization of E-cadherin, viral load and mucin gene expression were determined., Results: ECSN6-treated, uninfected cell cultures showed small, but significant increase in TER over placebo, which was associated with enhanced localization of E-cadherin and ZO-1 to IJ. In RV-infected cultures, treatment with ECSN6, but not placebo prevented RV-induced (1) reduction in TER, (2) dissociation of E-cadherin and ZO-1 from the IJ, (3) mucin expression, and (4) CBF attenuation. ECSN6 also decreased RV-stimulated expression of pro-inflammatory cytokines and permeability to inulin. Although ECSN6 significantly increased the expression of some antiviral type I and type III interferons, it did not alter viral load. In vivo, ECSN6 reduced RV-A1-induced moderate inflammation of nasal mucosa, beneficially affected RV-A1-induced cytokine responses and Muc5ac mRNA expression and prevented RV-caused dissociation of E-cadherin from the IJ of nasal mucosa without an effect on viral clearance., Conclusions: ECSN6 prevents RV-induced airway mucosal barrier dysfunction and improves the immunological and mucociliary barrier function. ECSN6 may maintain integrity of barrier function by promoting localization of tight and adherence junction proteins to the IJ. This in turn may lead to the observed decrease in RV-induced pro-inflammatory responses in vitro. By improving the innate defenses of the airway mucosal barrier network, ECSN6 may alleviate respiratory symptoms caused by RV infections., Competing Interests: Declarations Ethics approval and consent to participate The collection of trachea and nasal brushings and use of airway basal cells was approved by Institutional Review Board, Temple University, Philadelphia, PA (4407) and University of Michigan Ann Arbor, MI (HUM00052806). The donors had written consents to use their lungs for research if the lungs were rejected for transplantation. All the experiments with animals were approved by Institutional Animal Care and User Committee, Temple University (Protocol # 4904). Consent for publication The donors provided written consent to publish the results if the donated lungs are used in research. All authors consented to publish the results. Competing interests KW, JD and BS were employed by Heel GmbH. The funders were involved in the design of the study, in the writing of the manuscript, and in the decision to publish the results, but did not have any influence on the outcome of the study., (© 2024. The Author(s).)

SARS-CoV-2 initiates infection in the conducting airways, where mucociliary clearance inhibits pathogen penetration. However, it is unclear how mucociliary clearance impacts SARS-CoV-2 spread after infection is established. To investigate viral spread at this site, we perform live imaging of SARS-CoV-2 infected differentiated primary human bronchial epithelium cultures for up to 12 days. Using a fluorescent reporter virus and markers for cilia and mucus, we longitudinally monitor mucus motion, ciliary motion, and infection. Infected cell numbers peak at 4 days post infection, forming characteristic foci that tracked mucus movement. Inhibition of MCC using physical and genetic perturbations limits foci. Later in infection, mucociliary clearance deteriorates. Increased mucus secretion accompanies ciliary motion defects, but mucociliary clearance and vectorial infection spread resume after mucus removal, suggesting that mucus secretion may mediate MCC deterioration. Our work shows that while MCC can facilitate SARS-CoV-2 spread after initial infection, subsequent MCC decreases inhibit spread, revealing a complex interplay between SARS-CoV-2 and MCC., (© 2024. The Author(s).)

Immature innate and adaptive immunity and vulnerability of narrower airways to obstruction increase the susceptibility of infants to severe respiratory syncytial virus (RSV) disease. In this issue of the JCI, Zhao et al. illustrated greater intrinsic susceptibility of pediatric versus adult airway epithelial cells to RSV-induced cytopathology. Using precision cut lung slices (PCLS) and air-liquid interface (ALI) airway epithelial cell cultures, the authors showed that impaired STAT3 activation in RSV-infected pediatric multiciliated cells increased cell apoptosis and viral shedding, which enhanced the spread of infection. Bolstering STAT3 activation and treatment of neonatal mice with apoptosis inhibitors suppressed virus spread, suggesting that enhancing STAT3 activation may provide therapeutic benefit.

Respiratory syncytial virus (RSV) selectively targets ciliated cells in human bronchial epithelium and can cause bronchiolitis and pneumonia, mostly in infants. To identify molecular targets of intervention during RSV infection in infants, we investigated how age regulates RSV interaction with the bronchial epithelium barrier. Employing precision-cut lung slices and air-liquid interface cultures generated from infant and adult human donors, we found robust RSV virus spread and extensive apoptotic cell death only in infant bronchial epithelium. In contrast, adult bronchial epithelium showed no barrier damage and limited RSV infection. Single nuclear RNA-Seq revealed age-related insufficiency of an antiapoptotic STAT3 activation response to RSV infection in infant ciliated cells, which was exploited to facilitate virus spread via the extruded apoptotic ciliated cells carrying RSV. Activation of STAT3 and blockade of apoptosis rendered protection against severe RSV infection in infant bronchial epithelium. Lastly, apoptotic inhibitor treatment of a neonatal mouse model of RSV infection mitigated infection and inflammation in the lung. Taken together, our findings identify a STAT3-mediated antiapoptosis pathway as a target to battle severe RSV disease in infants.

Airway mucous cell metaplasia is a significant feature of many chronic airway diseases, such as chronic obstructive pulmonary disease, cystic fibrosis and asthma. However, the mechanisms underlying this process remain poorly understood. Here, we employed in vivo mouse genetic models to demonstrate that Hippo and p53 (encoded by Trp53) cooperate to modulate the differentiation of club cells into goblet cells. We revealed that ablation of Mst1 (Stk4) and Mst2 (Stk3), encoding the core components of Hippo signaling, significantly reduces mucous metaplasia in the lung airways in a lipopolysaccharide (LPS)-induced lung inflammation murine model while promoting club cell proliferation in a Yap (Yap1)-dependent manner. Additionally, we showed that deleting Mst1/2 is sufficient to suppress p53 deficiency-mediated goblet cell metaplasia. Finally, single-cell RNA-sequencing analysis revealed downregulation of YAP and p53 signaling in goblet cells in human airways. These findings underscore the important role of Hippo and p53 signaling in regulating airway mucous metaplasia., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions., Methods: The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs., Results: Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells., Conclusion: Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases., Competing Interests: The authors declare no potential conflicts of interest in this work., (© 2024 Luo et al.)

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cystic fibrosis related diabetes (CFRD), the main co-morbidity in cystic fibrosis (CF), is associated with higher rates of lung function decline. We hypothesize that airway epithelial barrier function is impaired in CF and is further exacerbated under hyperglycemia, worsening pulmonary outcomes. Using 16HBE cells, we studied the effects of hyperglycemia in airway epithelial barrier function. Results show increased paracellular dye flux in CF cells in response to insulin under hyperglycemia. Gene expression experiments identified claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells. CLDN4 protein localization by confocal microscopy showed that CLDN4 was tightly localized at tight junctions in WT cells, which did not change under hyperglycemia. ln contrast, CLDN4 was less well-localized in CF cells at normal glucose and localization was worsened under hyperglycemia. Treatment with highly effective modulator compounds (ETI) reversed this trend, and CFTR rescue was not affected by insulin or hyperglycemia. Bulk RNA sequencing showed differences in transcriptional responses in CF compared to WT cells under normal or high glucose, highlighting promising targets for future investigation. One of these targets is protein tyrosine phosphatase receptor type G (PTPRG), which has been previously found to play a role in defective Akt signaling and insulin resistance., (© 2024. The Author(s).)

Background: An increasing amount of evidence supports the relevance of epithelium across the wide spectrum of asthma pathobiology. On a clinical ground tezepelumab, selectively binding TSLP, a major epithelial cytokine, has demonstrated to be effective in asthma patients regardless their specific phenotype. In order to avoid the risk of considering tezepelumab as a not-specific option, the present perspective aims to sketch the tezepelumab best eligible patient profile and to propose some hallmarks of epithelial-driven disease by reviewing the published evidence on the drug mechanism of action and efficacy data., Main Body: Although it cannot rely on standardised or exclusive "markers", the relationship between environment and poor asthma control might suggest a major relevance of the epithelial barrier dysfunction. In that light, allergy and asthma exacerbations concomitant with specific exposures (pathogens, pollutants, chemicals), as well as increased susceptibility to infections can be considered as the hallmark of an impaired epithelial immune response. Tezepelumab is effective in allergic patients, being able to reduce asthma exacerbations precipitated by the exposure to seasonal or perennial aeroallergens, including fungi. In addition, tezepelumab reduced the incidence of co-occurring respiratory illness and asthma exacerbations. In terms of inflammation, epithelial immune response has been related to an impaired mucus hypersecretion and plugging. A placebo-controlled trial demonstrated a significant reduction of mucus plugging in treated patient. Airways hyperreactivity (AHR), airways obstruction and remodelling have been described as an expression of epithelial orchestrated immunological activation. Of note, a significantly higher incidence of mannitol negative test in patients treated with tezepelumab when compared to placebo group has been observed. In addition, A 130 mL improvement in pre-BD FEV1 has been described in patients assuming Tezepelumab. The above-mentioned data suggest that bronchial reversibility and AHR can be considered "functional biomarkers" supporting patients' phenotyping and the identification of tezepelumab best responders., Conclusion: Integrating "functional biomarkers" to the inflammatory ones and a better characterization of asthma exacerbations might pave the way to a different and more transversal phenotyping, which overcomes the "restrictive" labels including T2 high, allergic/atopic or T2 low asthma. Precisely defining the disease characteristics and potential targets for a better control even in tezepelumab eligible subjects is essential to avoid the block buster temptation and optimize the personalized medicine approach according to each patient's individuality., (© 2024. The Author(s).)

Cyclosporin A (CsA), an immunosuppressive drug used in transplant recipients, inhibits graft rejection by binding to cyclophilins and competitively inhibiting calcineurin. While concerns about respiratory infections in immunosuppressed patients exist, contradictory data emerged during the COVID-19 pandemic, prompting investigations into CsA's impact on viral infections. This study explores CsA's antiviral effects on SARS-CoV-2 Omicron BA.1, Delta variants, and human parainfluenza virus 3 (HPIV3) using an ex vivo model of human airway epithelium (HAE). CsA exhibited a dose-dependent antiviral effect against the SARS-CoV-2 Delta variant, reducing viral load over 10 days. However, no significant impact was observed against SARS-CoV-2 Omicron or HPIV3, indicating a virus-specific effect. At high concentrations, CsA was associated with an increase of IL-8 and a decrease of IFNλ expression in infected and noninfected HAE. This study highlights the complexity of CsA's antiviral mechanisms, more likely involving intricate inflammatory pathways and interactions with specific viral proteins. The research provides novel insights into CsA's effects on respiratory viruses, emphasizing the need for understanding drug-virus interactions in optimizing therapeutic approaches for transplant recipients and advancing knowledge on immunosuppressive treatments' implications on respiratory viral infections. Limitations include the model's inability to assess T lymphocyte activation, suggesting the necessity for further comprehensive studies to decipher the intricate dynamics of immunosuppressive treatments on respiratory viral infections., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Titanium dioxide fine particles (TiO 2 -FPs) and nanoparticles (TiO 2 -NPs) are the most widely used whitening pigments worldwide. Inhalation of TiO 2 -FPs and TiO 2 -NPs can be harmful as it triggers toxicity in the airway epithelial cells. The airway epithelium serves as the respiratory system's first line of defense in which airway epithelial cells are significant targets of inhaled pathogens and environmental particles. Our group previously found that TiO 2 -NPs lead to a disrupted barrier in the polarized airway epithelial cells. However, the effect of TiO 2 -FPs on the respiratory epithelial barrier has not been examined closely. In this study, we aimed to compare the effects of TiO 2 -FPs and TiO 2 -NPs on the structure and function of the airway epithelial barrier. Additionally, we hypothesized that 8-Bromo-cAMP, a cyclic adenosine monophosphate (cAMP) derivative, would alleviate the disruptive effects of both TiO 2 -FPs and TiO 2 -NPs. We observed increased epithelial membrane permeability in both TiO 2 -FPs and TiO 2 -NPs after exposure to 16HBE cells. Immunofluorescent labeling showed that both particle sizes disrupted the structural integrity of airway epithelial tight junctions and adherens junctions. TiO 2 -FPs had a slightly more, but insignificant impact on the epithelial barrier disruption than TiO 2 -NPs. Treatment with 8-Bromo-cAMP significantly attenuated the barrier-disrupting impact of both TiO 2 -FPs and TiO 2 -NPs on cell monolayers. Our study demonstrates that both TiO 2 -FPs and TiO 2 -NPs cause comparable barrier disruption and suggests a protective role for cAMP signaling. The observed effects of TiO 2 -FPs and TiO 2 -NPs provide a necessary understanding for characterizing the pathways involved in the defensive role of the cAMP pathway on TiO 2 -induced airway barrier disruption.

Background: In recent years, e-cigarettes have been used as alternatives among adult smokers. However, the impact of e-cigarette use on human bronchial epithelial (HBE) cells remains controversial., Methods: We collected primary HBE cells of healthy nonsmokers and chronic obstructive pulmonary disease (COPD) smokers, and analyzed the impact of e- cigarette vapor extract (ECE) or cigarette smoke extract (CSE) on HBE cell differentiation and injury by single-cell RNA sequencing, immunostaining, HE staining, qPCR and ELISA. We obtained serum and sputum from healthy non- smokers, smokers and e-cigarette users, and analyzed cell injury markers and mucin proteins., Results: ECE treatment led to a distinct differentiation program of ciliated cells and unique patterns of their cell-cell communications compared with CSE. ECE treatment caused increased Notch signaling strength in a ciliated cell subpopulation, and HBE cell remodeling and injury including hypoplasia of ciliated cells and club cells, and shorter cilia. ECE-induced hypoplasia of ciliated cells and shorter cilia were ameliorated by the Notch signaling inhibition., Conclusions: This study reveals distinct characteristics in e-cigarette vapor-induced airway epithelial remodeling, pointing to Notch signaling pathway as a potential targeted intervention for e-cigarette vapor-caused ciliated cell differentiation defects and cilia injury. In addition, a decrease in SCGB1A1 proteins is associated with e- cigarette users, indicating a potential lung injury marker for e-cigarette users., (© 2024. The Author(s).)

Effective treatment and immunoprophylaxis of viral respiratory infections with neutralizing monoclonal antibodies (mAbs) require maintaining inhibitory concentrations of mAbs at the airway surface. While engineered mAbs with increased affinity to the neonatal Fc receptor (FcRn) are increasingly employed, little is known how increased affinity of Fc to FcRn influences basal-to-apical transepithelial transport (transcytosis) of mAbs across the airway epithelium. To investigate this, we utilized a model of well-differentiated human airway epithelium (WD-HAE) that exhibited robust FcRn expression, and measured the transepithelial transport of a mAb against SARS-CoV-2 Spike protein (CR3022) with either wildtype IgG 1 -Fc or Fc modified with YTE or LS mutations known to increase affinity for FcRn. Despite the marked differences in the affinity of these CR3022 variants for FcRn, we did not find substantial differences in basal-to-apical transport reflective of systemic dosing, or apical-to-basal transport reflective of inhaled dosing, compared to the transport of wildtype IgG 1 -Fc. These results suggest increasing FcRn affinity may only have limited influence over transcytosis rates of systemically dosed mAbs across the human airway epithelium over short time scales. Over longer time scales, the elevated circulating levels of mAbs with greater FcRn affinity, due to more effective FcRn-mediated recycling, may better resupply mAb into the respiratory tract, leading to more effective extended immunoprophylaxis., Competing Interests: Authors LZ, MP, and KW were employed by the company ZabBio. Author SL is founder of Mucommune, LLC and currently serves as its interim CEO. Author SL is also founder of Inhalon Biopharma, Inc, and currently serves as its CSO as well as on its Board of Director and Scientific Advisory Board. Author SL has equity interests in both Mucommune and Inhalon Biopharma; Author SL’s relationships with Mucommune and Inhalon are subject to certain restrictions under university policy. The terms of these arrangements are managed by UNC-CH in accordance with its conflict-of-interest policies. Author SL was inventor of intellectual property that has been licensed to Inhalon Biopharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Togami, Wolf, Olson, Card, Shen, Schaefer, Okuda, Zeitlin, Pauly, Whaley, Pickles and Lai.)

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.

The airway surface liquid (ASL) plays a crucial role in lung defense mechanisms, and its composition and volume are regulated by the airway epithelium. The cystic fibrosis transmembrane conductance regulator (CFTR) is abundantly expressed in a rare airway epithelial cell type called an ionocyte. Recently, we demonstrated that ionocytes can increase liquid absorption through apical CFTR and basolateral barttin/chloride channels, while airway secretory cells mediate liquid secretion through apical CFTR channels and basolateral NKCC1 transporters. Th2-driven (IL-4/IL-13) airway diseases, such as asthma, cause goblet cell metaplasia, accompanied by increased mucus production and airway secretions. In this study, we investigate the effect of IL-13 on chloride and liquid transport performed by ionocytes. IL-13 treatment of human airway epithelia was associated with reduced epithelial liquid absorption rates and increased ASL volume. Additionally, IL-13 treatment reduced the abundance of CFTR-positive ionocytes and increased the abundance of CFTR-positive secretory cells. Increasing ionocyte abundance attenuated liquid secretion caused by IL-13. Finally, CFTR-positive ionocytes were less common in asthma and chronic obstructive pulmonary disease and were associated with airflow obstruction. Our findings suggest that loss of CFTR in ionocytes contributes to the liquid secretion observed in IL-13-mediated airway diseases.

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers., Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract., Results: The nano-micelles, with a particle size of <100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (C max ) and AUC 0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h., Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Kang et al.)

Secretory leukocyte protease inhibitor (SLPI) is an important cationic protein involved in innate airway immunity and highly expressed in mucosal secretions, shown to target and inhibit neutrophil elastase (NE), cathepsin G and trypsin activity to limit proteolytic activity. In addition to the potent anti-protease activity, SLPI has been demonstrated to exert a direct anti-inflammatory effect, which is mediated via increased inhibition and competitive binding of NF-κB, regulating immune responses through limiting transcription of pro-inflammatory gene targets. In muco-obstructive lung disorders, such as Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF), there is an observed elevation in airway SLPI protein concentrations as a result of increased lung inflammation and disease progression. However, studies have identified COPD patients presenting with diminished SLPI concentrations. Furthermore, there is a decrease in SLPI concentrations through cleavage and subsequent inactivation by NE degradation in Pseudomonas aeruginosa infected people with CF (pwCF). These observations suggest reduced SLPI protein levels may contribute to the compromising of airway immunity indicating a potential role of decreased SLPI levels in the pathogenesis of muco-obstructive lung disease. The Beta Epithelial Na+ Channel transgenic (ENaC-Tg) mouse model phenotype exhibits characteristics which replicate the pathological features observed in conditions such as COPD and CF, including mucus accumulation, alterations in airway morphology and increased pulmonary inflammation. To evaluate the effect of SLPI in muco-obstructive pulmonary disease, ENaC-Tg mice were crossed with SLPI knock-out (SLPI -/- ) mice, generating a ENaC-Tg/SLPI -/- colony to further investigate the role of SLPI in chronic lung disease and determine the effect of its ablation on disease pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brown, Dougan, Ferris, Delaney, Houston, Rodgers, Downey, Mall, Connolly, Small, Weldon and Taggart.)

Mucosal barrier tissues and their mucosal associated lymphoid tissues (MALT) are attractive targets for vaccines and immunotherapies due to their roles in both priming and regulating adaptive immune responses. The upper and lower respiratory mucosae, in particular, possess unique properties: a vast surface area responsible for frontline protection against inhaled pathogens but also simultaneous tight regulation of homeostasis against a continuous backdrop of non-pathogenic antigen exposure. Within the upper and lower respiratory tract, the nasal and bronchial associated lymphoid tissues (NALT and BALT, respectively) are key sites where antigen-specific immune responses are orchestrated against inhaled antigens, serving as critical training grounds for adaptive immunity. Many infectious diseases are transmitted via respiratory mucosal sites, highlighting the need for vaccines that can activate resident frontline immune protection in these tissues to block infection. While traditional parenteral vaccines that are injected tend to elicit weak immunity in mucosal tissues, mucosal vaccines (i.e., that are administered intranasally) are capable of eliciting both systemic and mucosal immunity in tandem by initiating immune responses in the MALT. In contrast, administering antigen to mucosal tissues in the absence of adjuvant or costimulatory signals can instead induce antigen-specific tolerance by exploiting regulatory mechanisms inherent to MALT, holding potential for mucosal immunotherapies to treat autoimmunity. Yet despite being well motivated by mucosal biology, development of both mucosal subunit vaccines and immunotherapies has historically been plagued by poor drug delivery across mucosal barriers, resulting in weak efficacy, short-lived responses, and to-date a lack of clinical translation. Development of engineering strategies that can overcome barriers to mucosal delivery are thus critical for translation of mucosal subunit vaccines and immunotherapies. This review covers engineering strategies to enhance mucosal uptake via active targeting and passive transport mechanisms, with a parallel focus on mechanisms of immune activation and regulation in the respiratory mucosa. By combining engineering strategies for enhanced mucosal delivery with a better understanding of immune mechanisms in the NALT and BALT, we hope to illustrate the potential of these mucosal sites as targets for immunomodulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Seefeld, Templeton, Lehtinen, Sinclair, Yadav and Hartwell.)

Background/aim: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle., Materials and Methods: Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (I sc ) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10 -5 M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations., Results: The amiloride effect on normal epithelium led to a significant decrease in I sc [ΔI=33±5.92 μA/cm 2 ; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm 2 )]. After incubation with CSE, ENaC I sc was significantly reduced (ΔI=14.80±1.96 μA/cm 2 ; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells., Conclusion: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Although CF affects multiple organs, lung disease is the main cause of morbidity and mortality, and gene therapy is expected to provide a mutation-agnostic option for treatment. SP-101 is a recombinant adeno-associated virus (AAV) gene therapy vector carrying a human CFTR minigene, hCFTRΔR , and is being investigated as an inhalation treatment for people with CF. To further understand SP-101 activity, in vitro studies were performed in human airway epithelia (HAE) derived from multiple CF and non-CF donors. SP-101 restored CFTR-mediated chloride conductance, measured via Ussing chamber assay, at a multiplicity of infection (MOI) as low as 5E2 in the presence of doxorubicin, a small molecule known to augment AAV transduction. Functional correction of CF HAE increased with increasing MOI and doxorubicin concentration and correlated with increasing cell-associated vector genomes and hCFTRΔR mRNA expression. Tropism studies using a fluorescent reporter vector and single-cell mRNA sequencing of SP-101-mediated hCFTRΔR mRNA demonstrated broad expression in all cell types after apical transduction, including secretory, ciliated, and basal cells. In summary, SP-101, particularly in combination with doxorubicin, shows promise for a novel CF treatment strategy and strongly supports continued development.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity. Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits. Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner. Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Interaction between viruses and bacteria during the development of infectious diseases is a complex question that requires continuous study. In this study, we explored the interactions between pseudorabies virus (PRV) and Pasteurella multocida (PM), which are recognized as the primary and secondary agents of porcine respiratory disease complex (PRDC), respectively. In vivo tests using mouse models demonstrated that intranasal inoculation with PRV at a sublethal dose induced disruption of murine respiratory barrier and promoted the invasion and damages caused by PM through respiratory infection. Inoculation with PRV also disrupted the barrier function of murine and porcine respiratory epithelial cells, and accelerated the adherence and invasion of PM to the cells. In mechanism, PRV infection resulted in decreased expression of tight junction proteins (ZO-1, occludin) and adherens junction proteins (β-catenin, E-cadherin) between neighboring respiratory epithelial cells. Additionally, PRV inoculation at an early stage downregulated multiple biological processes contributing to epithelial adhesion and barrier functions while upregulating signals beneficial for respiratory barrier disruption (e.g., the HIF-1α signaling). Furthermore, PRV infection also stimulated the upregulation of cellular receptors (CAM5, ICAM2, ACAN, and DSCAM) that promote bacterial adherence. The data presented in this study provide insights into the understanding of virus-bacteria interactions in PRDC and may also contribute to understanding the mechanisms of secondary infections caused by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine., Importance: Co-infections caused by viral and bacterial agents are common in both medical and veterinary medicine, but the related mechanisms are not fully understood. This study investigated the interactions between the zoonotic pathogens PRV and PM during the development of respiratory infections in both cell and mouse models, and reported the possible mechanisms which included: (i) the primary infection of PRV may induce the disruption and/or damage of mammal respiratory barrier, thereby contributing to the invasion of PM; (ii) PRV infection at early stage accelerates the transcription and/or expression of several cellular receptors that are beneficial for bacterial adherence. This study may shed a light on understanding the mechanisms on the secondary infection of PM promoted by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine., Competing Interests: The authors declare no conflict of interest.

The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H2O2) and hypothiocyanite (OSCN-), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H2O2 exposure that does not recapitulate the complex H2O2/LPO/SCN- system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H2O2/OSCN- concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H2O2/OSCN- were steadily produced from 7h and up to 25h, but OSCN- was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H2O2 of roughly 50% for 105 and 107 colony-forming unit (CFU)/mL inocula, while 102 and 104 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P. aeruginosa, S. aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response., Competing Interests: the authors have declared that no competing interests exist., (Copyright: © 2024 Petithomme-Nanrocki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fine particulate matter (PM 2.5 ) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM 2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (I sc ) and airway surface liquid (ASL) significantly decreased after PM 2.5 exposure. In addition, PM 2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM 2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM 2.5 . Together, our study reveals that PM 2.5 impairs the ATP-induced transepithelial anion I sc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM 2.5 -mediated airway epithelial injury., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The production of nanoparticles has recently surged due to their varied applications in the biomedical, pharmaceutical, textile, and electronic sectors. However, this rapid increase in nanoparticle manufacturing has raised concerns about environmental pollution, particularly its potential adverse effects on human health. Among the various concerns, inhalation exposure to nanoparticles poses significant risks, especially affecting the respiratory system. Airway epithelial cells play a crucial role as the primary defense against inhaled particulate matter and pathogens. Studies have shown that nanoparticles can disrupt the airway epithelial barrier, triggering inflammatory responses, generating reactive oxygen species, and compromising cell viability. However, our understanding of how different types of nanoparticles specifically impact the airway epithelial barrier remains limited. Both in vitro cell culture and in vivo murine models are commonly utilized to investigate nanoparticle-induced cellular responses and barrier dysfunction. This review discusses the methodologies frequently employed to assess nanoparticle toxicity and barrier disruption. Furthermore, we analyze and compare the distinct effects of various nanoparticle types on the airway epithelial barrier. By elucidating the diverse responses elicited by different nanoparticles, we aim to provide insights that can guide future research endeavors in assessing and mitigating the potential risks associated with nanoparticle exposure.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases., (© 2024. The Author(s).)

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens., (© 2024. The Author(s).)

Background: Cystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis., Methods: Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement., Results: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures., Conclusion: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD., (© 2024. The Author(s).)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses.

Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodeling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodeling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to fully replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodeling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed gland-like structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy., 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.)

Mucus provides a protective barrier that is crucial for host defense in the lungs. However, excessive or abnormal mucus can have pathophysiological consequences in many pulmonary diseases, including asthma. Patients with asthma are treated with agents that relax airway smooth muscle and reduce airway inflammation, but responses are often inadequate. In part, this is due to the inability of existing therapeutic agents to directly target mucus. Accordingly, there is a critical need to better understand how mucus hypersecretion and airway plugging are affected by the epithelial cells that synthesize, secrete, and transport mucus components. This review highlights recent advances in the biology of mucin glycoproteins with a specific focus on MUC5AC and MUC5B, the chief macromolecular components of airway mucus. An improved mechanistic understanding of key steps in mucin production and secretion will help reveal novel potential therapeutic strategies., (Published by Elsevier B.V.)

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that IL-13-activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibited viscoelastic liquid behavior and that mucus secreted by IL-13-activated HAECs exhibited solid-like behavior caused by mucin cross-linking. In addition, IL-13-activated HAECs shows increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that was prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), was increased in IL-13-activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings was increased in patients with asthma with high airway mucus plug scores. Together, our results show that IL-13-activated HAECs autonomously generated pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF, and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ΔF508-CF epithelia with TNF-α and IL-17, two inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl- (Na+/K+/2Cl- cotransport) and HCO3- (carbonic anhydrases and Na+/HCO3- cotransport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions but prevented the rise in viscosity in epithelia treated with TNF-α+IL-17. Finally, antiinflammatory drugs limited CFTR modulator responses in TNF-α+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits and limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.

Background: Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of pulmonary diseases, even after accounting for their smoking status. Cytotoxic CD8 T-cells are likely implicated in this phenomenon and may act as a double-edged sword. While being essential in viral infection control, their hyperactivation can also contribute to lung mucosal tissue damage. The effects of HIV and smoking on pulmonary mucosal CD8 T-cell dynamics has been a neglected area of research, which we address herein., Methods: Bronchoalveolar lavage (BAL) fluid were obtained from ART-treated PLWH (median duration of supressed viral load: 9 years; smokers: n = 14; non-smokers: n = 21) and HIV-uninfected controls (smokers: n = 11; non-smokers: n = 20) without any respiratory symptoms or active infection. Lymphocytes were isolated and CD8 T-cell subsets and homing markers were characterized by multiparametric flow cytometry., Results: Both smoking and HIV infection were independently associated with a significant increase in frequencies of total pulmonary mucosal CD8 T-cell. BAL CD8 T-cells were primarily CD69 + expressing CD103 and/or CD49a, at least one of the two granzymes (GzmA/GzmB), and little Perforin. Higher expression levels of CD103, CD69, and GzmB were observed in smokers versus non-smokers. The ex vivo phenotype of GzmA + and GzmB + cells revealed increased expression of CD103 and CXCR6 in smokers, while PLWH displayed elevated levels of CX3CR1 compared to controls., Conclusion: Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. Smoking likely increases recruitment and retention of GzmB + CD8 Trm via CXCR6 and CD103. Heightened CX3CR1 expression could be associated with CD8 non-Trm recruitment from the periphery in PLWH., (© 2024. The Author(s).)

Introduction: Exacerbations of chronic obstructive pulmonary disease (COPD) increase mortality risk and can lead to accelerated loss of lung function. The increased inflammatory response during exacerbations contributes to worsening of airflow limitation, but whether it also impacts epithelial repair is unclear. Therefore, we studied the effect of the soluble factor micro-environment during COPD exacerbations on epithelial repair using an exacerbation cocktail (EC), composed of four factors that are increased in COPD lungs during exacerbations (IL-1β, IL-6, IL-8, TNF-α)., Methods: Mouse organoids (primary CD31-CD45-Epcam+ cells co-cultured with CCL206 fibroblasts) were used to study epithelial progenitor behavior. Mature epithelial cell responses were evaluated using mouse precision cut lung slices (PCLS). The expression of epithelial supportive factors was assessed in CCL206 fibroblasts and primary human lung fibroblasts., Results: EC exposure increased the number and size of organoids formed, and upregulated Lamp3 , Muc5ac and Muc5b expression in day 14 organoids. In PCLS, EC imparted no effect on epithelial marker expression. Pre-treatment of CCL206 fibroblasts with EC was sufficient to increase organoid formation. Additionally, the expression of Il33 , Tgfa and Areg was increased in CCL206 fibroblasts from EC treated organoids, but these factors individually did not affect organoid formation or size. However, TGF-α downregulated Foxj1 expression and upregulated Aqp5 expression in day 14 organoids., Conclusions: EC exposure stimulates organoid formation and growth, but it alters epithelial differentiation. EC changes the epithelial progenitor support function of fibroblasts which contributes to observed effects on epithelial progenitors., Competing Interests: RK, JB, and RG receive unrestricted research funds from Boehringer Ingelheim to the institution. KG-K is an employee of Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kortekaas, Geillinger-Kästle, Fuentes-Mateos, van Orsoy, Al-Alyan, Burgess and Gosens.)

Superficial, systemic microcirculations, distinct from the pulmonary circulation, supply the mucosae of human nasal and conducting airways. Non-injurious, inflammatory challenges of the airway mucosa cause extravasation without overt mucosal oedema. Instead, likely reflecting minimal increases in basolateral hydrostatic pressure, circulating proteins/peptides of all sizes are transmitted paracellularly across the juxtaposed epithelial barrier. Thus, small volumes of extravasated, unfiltered bulk plasma appear on the mucosal surface at nasal and bronchial sites of challenge. Importantly, the plasma-exuding mucosa maintains barrier integrity against penetrability of inhaled molecules. Thus, one-way epithelial penetrability, strict localization, and well-controlled magnitude and duration are basic characteristics of the plasma exudation response in human intact airways. In vivo experiments in human-like airways demonstrate that local plasma exudation is also induced by non-sanguineous removal of epithelium over an intact basement membrane. This humoral response results in a protective, repair-promoting barrier kept together by a fibrin-fibronectin net. Plasma exudation stops once the provisional barrier is substituted by a new cellular cover consisting of speedily migrating repair cells, which may emanate from all types of epithelial cells bordering the denuded patch. Exuded plasma on the surface of human airways reflects physiological microvascular-epithelial cooperation in first line mucosal defense at sites of intact and regenerating epithelium., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Identifying the molecular mechanisms behind SARS-CoV-2 disparities and similarities will help find new treatments. The present study determines networks' shared and non-shared (specific) crucial elements in response to HCoV-229E and SARS-CoV-2 viruses to recommend candidate medications., Methods: We retrieved the omics data on respiratory cells infected with HCoV-229E and SARS-CoV-2, constructed PPIN and GRN, and detected clusters and motifs. Using a drug-gene interaction network, we determined the similarities and disparities of mechanisms behind their host response and drug-repurposed., Results: CXCL1, KLHL21, SMAD3, HIF1A, and STAT1 were the shared DEGs between both viruses' protein-protein interaction network (PPIN) and gene regulatory network (GRN). The NPM1 was a specific critical node for HCoV-229E and was a Hub-Bottleneck shared between PPI and GRN in HCoV-229E. The HLA-F, ADCY5, TRIM14, RPF1, and FGA were the seed proteins in subnetworks of the SARS-CoV-2 PPI network, and HSPA1A and RPL26 proteins were the seed in subnetworks of the PPI network of HCOV-229E. TRIM14, STAT2, and HLA-F played the same role for SARS-CoV-2. Top enriched KEGG pathways included cell cycle and proteasome in HCoV-229E and RIG-I-like receptor, Chemokine, Cytokine-cytokine, NOD-like receptor, and TNF signaling pathways in SARS-CoV-2. We suggest some candidate medications for COVID-19 patient lungs, including Noscapine, Isoetharine mesylate, Cycloserine, Ethamsylate, Cetylpyridinium, Tretinoin, Ixazomib, Vorinostat, Venetoclax, Vorinostat, Ixazomib, Venetoclax, and epoetin alfa for further in-vitro and in-vivo investigations., Conclusion: We suggested CXCL1, KLHL21, SMAD3, HIF1A, and STAT1, ADCY5, TRIM14, RPF1, and FGA, STAT2, and HLA-F as critical genes and Cetylpyridinium, Cycloserine, Noscapine, Ethamsylate, Epoetin alfa, Isoetharine mesylate, Ribavirin, and Tretinoin drugs to study further their importance in treating COVID-19 lung complications., (© 2024. The Author(s), under exclusive licence to Tehran University of Medical Sciences.)

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rapsinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC + mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma., (© 2024. The Author(s).)

Infants are highly susceptible to invasive respiratory and gastrointestinal infections. To elucidate the age-dependent mechanism(s) that drive bacterial spread from the mucosa, we developed an infant mouse model using the prevalent pediatric respiratory pathogen, Streptococcus pneumoniae (Spn). Despite similar upper respiratory tract (URT) colonization levels, the survival rate of Spn-infected infant mice was significantly decreased compared to adults and corresponded with Spn dissemination to the bloodstream. An increased rate of pneumococcal bacteremia in early life beyond the newborn period was attributed to increased bacterial translocation across the URT barrier. Bacterial dissemination in infant mice was independent of URT monocyte or neutrophil infiltration, phagocyte-derived ROS or RNS, inflammation mediated by toll-like receptor 2 or interleukin 1 receptor signaling, or the pore-forming toxin pneumolysin. Using molecular barcoding of Spn, we found that only a minority of bacterial clones in the nasopharynx disseminated to the blood in infant mice, indicating the absence of robust URT barrier breakdown. Rather, transcriptional profiling of the URT epithelium revealed a failure of infant mice to upregulate genes involved in the tight junction pathway. Expression of many such genes was also decreased in early life in humans. Infant mice also showed increased URT barrier permeability and delayed mucociliary clearance during the first two weeks of life, which corresponded with tighter attachment of bacteria to the respiratory epithelium. Together, these results demonstrate a window of vulnerability during postnatal development when altered mucosal barrier function facilitates bacterial dissemination., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lokken-Toyli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: The anti-inflammatory effect of green tea extract (GTE) has been confirmed in asthmatic mice, however, the pharmacological mechanism is not fully elucidated., Methods: To investigate the therapeutic efficacy of GTE in asthma and identify specific pathways, murine model of allergic asthma was established by ovalbumin (OVA) sensitization and the challenge for 4 weeks, with oral treatment using GTE and dexamethasone (DEX). Inflammatory cell counts, cytokines, OVA-specific IgE, airway hyperreactivity, and antioxidant markers in the lung were evaluated. Also, pulmonary histopathological analysis and western blotting were performed. In vitro , we established the model by stimulating the human airway epithelial cell line NCI-H292 using lipopolysaccharide, and treating with GTE and mitogen-activated protein kinases (MAPKs) inhibitors., Results: The GTE100 and GTE400 groups showed a decrease in airway hyperresponsiveness and the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) compared to the OVA group. GTE treatment also reduced interleukin (IL)-13, IL-5, and IL-4 levels in the BALF, and OVA-specific immunoglobulin E levels in the serum compared to those in the OVA group. GTE treatment decreased OVA-induced mucus secretion and airway inflammation. In addition, GTE suppressed the oxidative stress, and phosphorylation of MAPKs, which generally occurs after exposure to OVA. GTE administration also reduced matrix metalloproteinase-9 activity and protein levels., Conclusion: GTE effectively inhibited asthmatic respiratory inflammation and mucus hyperproduction induced by OVA inhalation. These results suggest that GTE has the potential to be used for the treatment of asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Kim, Jeong, Kim, Chung, Kim, Hong, Kwon, Ko and Kim.)

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection., (© 2024. The Author(s).)

Background: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium., Methods: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties., Results: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells., Conclusion: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium., (© 2024. The Author(s).)