Human Airway Epithelial Cells Research Articles

Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.

Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE-engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal 'dead' single-guide RNAs-we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic 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.MethodsSmall 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.ResultsWe 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.ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic 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.MethodsSmall 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.ResultsWe 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.ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

Magnetoactive, Kirigami-Inspired Hammocks to Probe Lung Epithelial Cell Function

Abstract Introduction Mechanical forces provide critical biological signals to cells. Within the distal lung, tensile forces act across the basement membrane and epithelial cells atop. Stretching devices have supported studies of mechanical forces in distal lung epithelium to gain mechanistic insights into pulmonary diseases. However, the integration of curvature into devices applying mechanical forces onto lung epithelial cell monolayers has remained challenging. To address this, we developed a hammock-shaped platform that offers desired curvature and mechanical forces to lung epithelial monolayers. Methods We developed hammocks using polyethylene terephthalate (PET)-based membranes and magnetic-particle modified silicone elastomer films within a 48-well plate that mimic the alveolar curvature and tensile forces during breathing. These hammocks were engineered and characterized for mechanical and cell-adhesive properties to facilitate cell culture. Using human small airway epithelial cells (SAECs), we measured monolayer formation and mechanosensing using F-Actin staining and immunofluorescence for cytokeratin to visualize intermediate filaments. Results We demonstrate a multi-functional design that facilitates a range of curvatures along with the incorporation of magnetic elements for dynamic actuation to induce mechanical forces. Using this system, we then showed that SAECs remain viable, proliferate, and form an epithelial cell monolayer across the entire hammock. By further applying mechanical stimulation via magnetic actuation, we observed an increase in proliferation and strengthening of the cytoskeleton, suggesting an increase in mechanosensing. Conclusion This hammock strategy provides an easily accessible and tunable cell culture platform for mimicking distal lung mechanical forces in vitro. We anticipate the promise of this culture platform for mechanistic studies, multi-modal stimulation, and drug or small molecule testing, extendable to other cell types and organ systems.

Magnetoactive, Kirigami-Inspired Hammocks to Probe Lung Epithelial Cell Function

Abstract Introduction Mechanical forces provide critical biological signals to cells. Within the distal lung, tensile forces act across the basement membrane and epithelial cells atop. Stretching devices have supported studies of mechanical forces in distal lung epithelium to gain mechanistic insights into pulmonary diseases. However, the integration of curvature into devices applying mechanical forces onto lung epithelial cell monolayers has remained challenging. To address this, we developed a hammock-shaped platform that offers desired curvature and mechanical forces to lung epithelial monolayers. Methods We developed hammocks using polyethylene terephthalate (PET)-based membranes and magnetic-particle modified silicone elastomer films within a 48-well plate that mimic the alveolar curvature and tensile forces during breathing. These hammocks were engineered and characterized for mechanical and cell-adhesive properties to facilitate cell culture. Using human small airway epithelial cells (SAECs), we measured monolayer formation and mechanosensing using F-Actin staining and immunofluorescence for cytokeratin to visualize intermediate filaments. Results We demonstrate a multi-functional design that facilitates a range of curvatures along with the incorporation of magnetic elements for dynamic actuation to induce mechanical forces. Using this system, we then showed that SAECs remain viable, proliferate, and form an epithelial cell monolayer across the entire hammock. By further applying mechanical stimulation via magnetic actuation, we observed an increase in proliferation and strengthening of the cytoskeleton, suggesting an increase in mechanosensing. Conclusion This hammock strategy provides an easily accessible and tunable cell culture platform for mimicking distal lung mechanical forces in vitro. We anticipate the promise of this culture platform for mechanistic studies, multi-modal stimulation, and drug or small molecule testing, extendable to other cell types and organ systems.

Peroxidase-mediated mucin cross-linking drives pathologic mucus gel formation in IL-13-stimulated airway epithelial cells.

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 interleukin 13 (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 exhibits viscoelastic liquid behavior and that mucus secreted by IL-13 activated HAECs exhibits solid-like behavior caused by mucin cross-linking. In addition, IL-13 activated HAECs show increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that is prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), is 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 is increased in asthma patients with high airway mucus plug scores. Together, our results show that IL-13 activated HAECs autonomously generate pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

Comprehensive analysis of resilience of human airway epithelial barrier against short-term PM2.5 inorganic dust exposure using invitro microfluidic chip and exvivo human airway models.

The updated World Health Organization (WHO) air quality guideline recommends an annual mean concentration of fine particulate matter (PM2.5) not exceeding 5 or 15 μg/m3 in the short-term (24 h) for no more than 3-4 days annually. However, more than 90% of the global population is currently exposed to daily concentrations surpassing these limits, especially during extreme weather conditions and due to transboundary dust transport influenced by climate change. Herein, the effect of respirable <PM2.5 inorganic silica particle exposures on epithelial barrier integrity was simultaneously evaluated within the biomimetic microfluidic platform-based airway epithelial barrier (AEB)-on-a-chip and human bronchoscopic exvivo airway tissue models, comparatively. Silica particles at an average size of 1 μm, referred to as <PM2.5, dose-dependently tested by MTT and LDH analyses. The elicited dose of 800 μg/mL was applied to human airway epithelial cells (Calu-3) seeded to the membrane at air-liquid interface in the AEB-on-a-chip platform, which is operated under static and dynamic conditions and to exvivo human bronchoscopy bronchial tissue slices for 72 h. For both models, healthy and exposed groups were comparatively investigated. Computational fluid dynamics simulations were performed to assess shear stress profiles under different flow conditions. Qualitative and quantitative analyses were carried out to evaluate the resilience of the epithelial barrier via cell survivability, morphology, barrier integrity, permeability, and inflammation. In the AEB-on-a-chip platform, short-term exposure to 800 μg/mL PM2.5 disrupted AEB integrity via increasing barrier permeability, decreasing cell adhesion-barrier markers such as ZO-1, Vinculin, ACE2, and CD31, impaired cell viability and increased the expression levels of proinflammatory markers; IFNs, IL-6, IL-1s, TNF-α, CD68, CD80, and Inos, mostly under dynamic conditions. Besides, decreased tissue viability, impaired tissue integrity via decreasing of Vinculin, ACE2, β-catenin, and E-cadherin, and also proinflammatory response with elevated CD68, IL-1α, IL-6, IFN-Ɣ, Inos, and CD80 markers, were observed after PM2.5 exposure in exvivo tissue. The duration and concentration of PM2.5 that can be exposed during extreme weather conditions and natural events aligns with our exposure model (0-800 μg/mL 72 h). At this level of exposure, the resilience of the epithelial barrier is demonstrated by both AEB-on-a-chip platform emulating dynamic forces in the body and exvivo bronchial biopsy slices. Lung-on-a-chip models will serve as reliable exposure models in this context.

Cell Culture Differentiation and Proliferation Conditions Influence the In Vitro Regeneration of the Human Airway Epithelium.

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGMTM, PneumaCultTM, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCultTM-ALI and Half&Half, stronger EGF signaling from basal cells in BEGMTM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCultTM-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

Human bronchopulmonary disposition and plasma pharmacokinetics of oral bemnifosbuvir (AT-527), an experimental guanosine nucleotide prodrug for COVID-19.

Bemnifosbuvir (AT-527) is a novel oral guanosine nucleotide antiviral drug for the treatment of persons with COVID-19. Direct assessment of drug disposition in the lungs, via bronchoalveolar lavage, is necessary to ensure antiviral drug levels at the primary site of SARS-CoV-2 infection are achieved. This Phase 1 study in healthy subjects aimed to assess the bronchopulmonary pharmacokinetics, safety and tolerability of repeated doses of bemnifosbuvir. A total of 24 subjects were assigned to receive bemnifosbuvir twice daily at doses of 275, 550 or 825 mg for up to 3.5 days. AT-511, the free base of bemnifosbuvir, was largely eliminated from the plasma within 6 h post dose in all dosing groups. Antiviral drug levels of bemnifosbuvir were consistently achieved in the lungs with bemnifosbuvir 550 mg twice daily. The mean level of the guanosine nucleoside metabolite AT-273, the surrogate of the active triphosphate metabolite of the drug, measured in the epithelial lining fluid of the lungs was 0.62 µM at 4-5 h post dose. This exceeded the target in vitro 90% effective concentration (EC90) of 0.5 µM for antiviral drug exposure against SARS-CoV-2 replication in human airway epithelial cells. Bemnifosbuvir was well tolerated across all doses tested, and most treatment-emergent adverse events reported were mild in severity and resolved. The favourable pharmacokinetics and safety profile of bemnifosbuvir demonstrates its potential as an oral antiviral treatment for COVID-19, with 550 mg bemnifosbuvir twice daily currently under further clinical evaluation in persons with COVID-19.

Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236

Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 invivo, we conducted intranasal infection experiments in hamsters. In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

Blockade of endolysosomal acidification suppresses TLR3-mediated proinflammatory signaling in airway epithelial cells

BackgroundEndolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm. ObjectivesHere we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. MethodsHSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. ResultsWe show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. ConclusionThese findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs. Clinical implicationThese findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.

Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm.

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.

AmiA and AliA peptide ligands, found in Klebsiella pneumoniae, are imported into pneumococci and alter the transcriptome

Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.

Effect of Nonpharmaceutical Interventions in Preventing COVID-19 on the Circulation of Avian Influenza Virus in Wuhan, Hubei Province, China

Background. In late 2019, several medical institutions in Wuhan, Hubei Province, China, reported cases of unexplained pneumonia. A novel coronavirus was isolated from human airway epithelial cells causing coronavirus disease-2019 (COVID-19). In recent years, many nonpharmaceutical interventions (NPIs) have been implemented to stop COVID-19 epidemic. This study aimed to explore the effect of NPIs on the circulation of avian influenza virus (AIV) in Wuhan. Materials and Methods. External environmental samples were collected and subjected to viral RNA extraction. Real-time quantitative polymerase chain reaction was used to detect the H5, H7, and H9 subtypes of AIV. Statistical analyses were performed using the chi-square test and binary logistic regression in SPSS 20.0 software. Results. A total of 2,451 external environmental samples were collected from seven districts from 2018 to 2022 in Wuhan, comprising 1,041 samples collected before COVID-19 and 1,410 samples after COVID-19. After COVID-19, the positive rate of AIV decreased significantly with the implementation of NPIs. The dominant subtype was the H9 subtype, followed by the H5 subtype. The positive rates of AIV in live poultry markets and poultry free-range sites were reduced significantly through the implementation of NPIs. Among the different sample types, higher positive rates of AIV were found in chopping boards, sewage, and cages. The positive rate of AIV was higher in trafficked source samples than that in autotrophic source samples. Conclusions. This study identified the characteristics of AIV in terms of different districts, surveillance sites, sample types, and bird sources in Wuhan. This study conducted a multifactorial analysis of the factors affecting AIV infection and provided a theoretical basis and guidance for the future prevention and control of AIV in Wuhan.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

House dust mite allergen Der f 2 drives IL-6 and GM–CSF expression in airway epithelial cells via p38 MAPK/NF-κB signaling

Objective Der f 2, a major allergen derived from Dermatophagoides farinae, is a leading cause of allergic asthma. IL-6 and GM–CSF play essential roles in the exacerbation of asthma. However, the mechanical act by which Der f 2 mediates the expression of IL-6, IL-8, and GM–CSF in airway epithelial cells remains incompletely elucidated. Herein, we aimed to explore the effect of Der f 2 on IL-6 and GM–CSF expression in the human airway epithelial cell BEAS-2B and A549. Methods Recombinant Der f 2 (rDf2) was acquired using Pichia pastoris. BEAS-2B and A549 cells were used as cell model. The expression of genes and proteins and the involvement of the signaling cascade were assessed using RT–PCR, quantitative real-time PCR (qPCR), Western blotting, and ELISA, respectively. Results Our findings showed that rDf2 significantly induced mRNA expression and protein production of IL-6 and GM-CSF in BEAS-2B and A549 cells. In contrast, rDf2 did not influence IL-8 expression or production in both cells. Mechanistic studies revealed that rDf2 triggered activation of the p38 MAPK and JNK. Inhibition of p38, but not JNK, significantly attenuated rDf2-induced IL-6 and GM–CSF expression and production. Conclusion This study demonstrates that Der f 2 promotes the expression and production of the pro-inflammatory cytokines IL-6 and GM–CSF in airway epithelial cells via activation of the p38 signaling pathway. These findings provide insights into the molecular mechanisms that Der f 2 may exacerbate airway inflammation.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma

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.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity

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. HIGHLIGHTS Lung 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.