ABSTRACT Chronic antibiotic‐resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a ‘pulmotype’ detected in ∼34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by reducing Elexacaftor/Tezacaftor/Ivacaftor (ETI) stimulated CF pHBEC CFTR Cl− secretion, enhancing virulence and biofilm formation, and upregulating the stress response and pro‐inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. ETI, a highly effective modulator therapy, did not ameliorate the response of CF pHBEC or return it to WT levels. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.

The World Health Organization and the U.S. Centre for Disease Control and Prevention have reported that antibiotic-resistant infections with Pseudomonas aeruginosa present a significant health risk worldwide. In the genetic disease cystic fibrosis (CF), chronic antibiotic-resistant Pseudomonas lung infections and persistent inflammation remain the leading causes of mortality. While highly effective modulator therapy (HEMT) dramatically improves lung function in CF, they fail to eradicate chronic infections or eliminate the associated hyperinflammatory state. Thus, there is an urgent need for innovative therapies that can simultaneously eliminate antibiotic-resistant P. aeruginosa lung infection and the attendant hyperinflammatory lung environment. Mesenchymal stromal cell-derived extracellular particles (MSC EPs) represent a promising solution, offering potent anti-inflammatory and antimicrobial properties while being safe and non-toxic. This study demonstrates, using a CF mouse model of infection, that MSC EPs reduce acute P. aeruginosa lung infection and inflammation. As the first investigation of MSC EPs in CF mice, this research underscores the dual effects of MSC EPs; reducing inflammation and bacterial burden. These findings mark an important advancement in antimicrobial therapy, addressing the unmet need for reducing antibiotic-resistant infections and hyperinflammation in CF as well as other diseases with chronic, antibiotic-resistant P. aeruginosa infections.

ABSTRACT The goals of this study were to develop a model to study host-pathogen interactions in primary human colon organoids and to test the hypothesis that Bacteroides fragilis toxin (BFT-2) secreted in outer membrane vesicles (OMVs) modulates mucosal immunity and CFTR Cl⁻ secretion. Since Bacteroides species reside in mucus, OMVs are likely to represent a mechanism of communication between Bacteroides and the host. Two strains of Bacteroides were studied: enterotoxigenic Bacteroides fragilis (ETBF), which produces BFT-2, and the non-toxigenic Bacteroides fragilis strain NCTC 9343 (NTBF), which does not produce BFT-2. We also utilized two additional strains of Bacteroides fragilis: one in which bft-2 was knocked out (ETBF Δbft) and one that was engineered to contain bft-2 (NTBF+bft). We report that Bacteroides fragilis OMVs reduced CFTR Cl⁻ secretion, but had no effect on tight junction or cell adhesion proteins, transepithelial resistance (TER), or cytokine secretion by primary human colon organoids. NTBF OMVs containing BFT-2 were more effective in reducing CFTR Cl- secretion than NTBF lacking BFT-2. By contrast, deletion of bft in ETBF did not have a significant effect on CFTR Cl⁻ secretion compared to ETBF. We conclude that OMVs secreted by Bacteroides can be an important mechanism of host-pathogen interactions in the colon by reducing CFTR Cl⁻ secretion, and that the effect of BFT-2 on CFTR Cl⁻ secretion is dependent on the strain of Bacteroides fragilis.

This review examines the role of bacterial extracellular vesicles (BEVs) in shaping interactions between bacteria and their human hosts. Produced by both Gram-positive and Gram-negative bacteria during infections, BEVs play a pivotal role in host–pathogen dynamics without necessitating direct cell-to-cell contact. The article explores how BEVs engage with host cells, transporting short interfering RNAs (sRNAs) and transfer RNA-derived fragments (tRFs) to host cells and modulate the immune response by influencing key signaling pathways in diseases such as cystic fibrosis. The article particularly focuses on how BEVs contribute to biofilms and chronic infections through epigenetic modifications that alter immune responses in lung epithelial and immune cells. Additionally, the review identifies gaps in current knowledge and suggests directions for future research on BEVs.

Background and aims Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and their extracellular vesicles (EVs) reduce lung inflammation and fibrosis in a variety of model systems, including in a Cystic Fibrosis (CF) mouse model. Many components of MSC-derived EVs, including cytokines, antimicrobial peptides, and miRNAs have been implicated in their anti-inflammatory effects. However, a major gap in our knowledge of using MSC as a therapeutic intervention for people with CF (pwCF) is whether the CF airway environment compromises miRNA cargo in hBM-MSC-derived EVs. Methods To assess this, hBM-MSCs were exposed to cell culture media (control) or to bronchoalveolar lavage fluid (BALF) obtained from pwCF or healthy controls (HC) and compositional analysis of EV miRNA content was conducted. Results Thirteen miRNAs (each ≥1% of the total miRNA content) were identified that collectively account for ∼70% of the miRNA content of EVs. These miRNAs were remarkably stable across treatments. To infer potential therapeutic effects, we identified predicted gene targets of these miRNAs and performed pathway enrichment analysis. Gene pathway analysis revealed that many of the 13 miRNAs are predicted to inhibit TLR signaling, NF-κB activation, TGF-β-mediated fibrosis, and cytokine production. Conclusions These results indicate that miRNAs secreted by hBM-MSCs in EVs may contribute to the observed anti-inflammatory and anti-fibrotic effects in experimental models and that exposure to CF BALF does not significantly diminish the abundance of the 13 miRNAs.

Cystic Fibrosis (CF) is a multiorgan disease caused by mutations in the CFTR gene, leading to chronic pulmonary infections and hyperinflammation. Among pathogens colonizing the CF lung, Pseudomonas aeruginosa is predominant, infecting over 50% of adults with CF, and becoming antibiotic-resistant over time. Current therapies for CF, while providing tremendous benefits, fail to eliminate persistent bacterial infections, chronic inflammation, and irreversible lung damage, necessitating novel therapeutic strategies. Our group engineered mesenchymal stromal cell derived extracellular vesicles (MSC EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. MSC EVs are low-immunogenicity platforms with innate antimicrobial and immunomodulatory properties, while let-7b-5p reduces biofilm formation and inflammation. In a preclinical CF mice model, we reported that let-7b-5p-loaded MSC EVs reduced P. aeruginosa burden, immune cells, and proinflammatory cytokines in the lungs. We hypothesize four complementary mechanisms for the observed in-vivo effects of the let-7b-5p loaded MSC EVs: antimicrobial activity, anti-inflammatory properties, inhibition of antibiotic-resistant P. aeruginosa biofilm formation in CF airways, and stimulation of anti-inflammatory macrophage behaviors. This study focused on the second and third mechanisms and demonstrates that MSC EVs engineered to contain let-7b-5p effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs) while also reducing P. aeruginosa-induced inflammation. This approach holds promise for improving outcomes for people with CF. Future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, leading to chronic pulmonary infections and hyperinflammation. Among pathogens colonizing the CF lung, Pseudomonas aeruginosa is predominant, infecting over 50% of adults with CF, and becoming antibiotic-resistant over time. Current therapies for CF, while providing tremendous benefits, fail to eliminate persistent bacterial infections, chronic inflammation, and irreversible lung damage, necessitating novel therapeutic strategies. Our group engineered mesenchymal stromal cell-derived extracellular vesicles (MSC EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. MSC EVs are low-immunogenicity platforms with innate antimicrobial and immunomodulatory properties, whereas let-7b-5p reduces inflammation. This study demonstrates that MSC EVs effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs), and let-7b-5p loading into MSC EVs conferred additional anti-inflammatory effects by reducing P. aeruginosa-induced IL-8 secretion by pHBECs. This approach holds promise for improving outcomes for people with CF, and future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.NEW & NOTEWORTHY This is the first study demonstrating that mesenchymal stromal cell extracellular vesicles (MSC EVs) block antibiotic-resistant P. aeruginosa biofilm formation and that let-7b-5p-loaded MSC EVs reduce inflammation in CF primary human bronchial epithelial cells.

The acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression but effects on microRNA (miRNA) content of MSC-extracellular vesicle (EVs) remain unknown. To assess this, sequencing analysis of EV-miRNAs prepared from human bone marrow-derived MSCs (hMSCs) exposed ex vivo to bronchoalveolar lavage fluid (BALF) from ARDS patients or healthy volunteers (HV) identified a number of differentially expressed miRNAs. Discriminant, differential expression, and functional enrichment analyses identified 14 miRNAs significantly changed following ARDS versus HV BALF exposure. Network analysis showed 4 (miR-760, miR-3175, miR-885-3p, and miR-766-3p) of the 14 EV-miRNAs formed a regulatory “hub”, suggesting co-targeting of specific gene pathways. In silico prediction identified a number of pathways important in lung injury. Two miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential effects on CFTR Cl- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. The potential clinical impact of these finding highlights the need for further studies assessing the role of hMSC-EV miRNAs in regulating lung inflammation and mucociliary clearance.

Quantitative PCR (qPCR) remains a widely used, cost-effective method for RNA quantitation, yet many published studies inadequately comply with MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) and FAIR (Findable, Accessible, Interoperable, Reproducible) data principles. Widespread reliance on the 2-ΔΔCT method often overlooks critical factors such as amplification efficiency variability and reference gene stability. Furthermore, the absence of raw data and analysis code limits the community's ability to evaluate potential biases and reproduce findings. Here, we primarily aim to encourage researchers to share raw qPCR fluorescence data along with detailed analysis scripts that start from raw input and produce final figures and statistical tests. Using our recently published dataset, we model the complete qPCR analytical workflow-from raw fluorescence curves through to differential expression-highlighting key decision points that can influence results. We provide fully documented R scripts illustrating how ANCOVA (Analysis of Covariance), a flexible multivariable linear modeling approach, generally offers greater statistical power and robustness compared to 2-ΔΔCT. Additionally, simulations support ANCOVA's applicability across diverse experimental conditions. We also demonstrate how general-purpose data repositories (e.g., figshare) and code repositories (e.g., GitHub) facilitate adherence to FAIR principles and promote transparency in qPCR research. Finally, we offer graphical examples that transparently depict both target and reference gene behavior within the same figure, enhancing interpretability. This work establishes practical resources and conceptual foundations to improve rigor, reproducibility, and openness in qPCR data analysis.Image 1.

Background Adult people with cystic fibrosis (PwCF) have a higher risk of end-stage kidney disease than the general population. The nature and mechanism of kidney disease in CF are unknown. This study quantifies urinary kidney injury markers and examines the hypothesis that neutrophil activation and lung infection are associated with early kidney injury in CF. Methods Urinary total protein, albumin, and markers of kidney injury and neutrophil activation, normalized to creatinine, as well as urinary immune cells, were quantified in adult CF and healthy cohorts. Infection burden and chronicity were defined by sputum culture and serum titers of anti-bacterial antibodies. Results PwCF had increased urinary protein levels, consisting of low-molecular-weight tubular injury markers, independent of glomerular filtration rate (eGFR). This finding suggests subclinical renal injury processes. Urinary analysis of the CF cohort identified different associations of urinary injury markers with aminoglycoside exposure, lung function, and neutrophil activation. High urinary KIM-1 levels and increased prevalence of neutrophils among urine immune cells correlated with decreased lung function in PwCF. The relationship between tubular injury and reduced lung function was most prominent in patients harboring chronic Pseudomonas aeruginosa infection. Conclusions Increased urinary tubular injury markers in PwCF suggest early subclinical renal injury not readily detected by eGFR. The strong association of high urinary KIM-1 and neutrophils with diminished lung function and high Pseudomonas aeruginosa burden suggests that pulmonary disease may contribute to renal injury in CF.

E.PathDash facilitates re-analysis of gene expression data from pathogens clinically relevant to chronic respiratory diseases, including a total of 48 studies, 548 samples, and 404 unique treatment comparisons. The application enables users to assess broad biological stress responses at the KEGG pathway or gene ontology level and also provides data for individual genes. E.PathDash reduces the time required to gain access to data from multiple hours per data set to seconds. Users can download high-quality images such as volcano plots and boxplots, differential gene expression results, and raw count data, making it fully interoperable with other tools. Importantly, users can rapidly toggle between experimental comparisons and different studies of the same phenomenon, enabling them to judge the extent to which observed responses are reproducible. As a proof of principle, we invited two cystic fibrosis scientists to use the application to explore scientific questions relevant to their specific research areas. Reassuringly, pathway activation analysis recapitulated results reported in original publications, but it also yielded new insights into pathogen responses to changes in their environments, validating the utility of the application. All software and data are freely accessible, and the application is available at scangeo.dartmouth.edu/EPathDash. IMPORTANCE:Chronic respiratory illnesses impose a high disease burden on our communities and people with respiratory diseases are susceptible to robust bacterial infections from pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, that contribute to morbidity and mortality. Public gene expression datasets generated from these and other pathogens are abundantly available and an important resource for synthesizing existing pathogenic research, leading to interventions that improve patient outcomes. However, it can take many hours or weeks to render publicly available datasets usable; significant time and skills are needed to clean, standardize, and apply reproducible and robust bioinformatic pipelines to the data. Through collaboration with two microbiologists, we have shown that E.PathDash addresses this problem, enabling them to elucidate pathogen responses to a variety of over 400 experimental conditions and generate mechanistic hypotheses for cell-level behavior in response to disease-relevant exposures, all in a fraction of the time.

The availability of multiple publicly-available datasets studying the same phenomenon has the promise of accelerating scientific discovery. Meta-analysis can address issues of reproducibility and often increase power. The promise of meta-analysis is especially germane to rarer diseases like cystic fibrosis (CF), which affects roughly 100,000 people worldwide. A recent search of the National Institute of Health's Gene Expression Omnibus revealed 1.3 million data sets related to cancer compared to about 2,000 related to CF. These studies are highly diverse, involving different tissues, animal models, treatments, and clinical covariates. In our search for gene expression studies of primary human airway epithelial cells, we identified three studies with compatible methodologies and sufficient metadata: GSE139078, Sala Study, and PRJEB9292. Even so, experimental designs were not identical, and we identified significant batch effects that would have complicated functional analysis. Here we present quantile discretization and Bayesian network construction using the Hill climb method as a powerful tool to overcome experimental differences and reveal biologically relevant responses to the CF genotype itself, exposure to virus, bacteria, and drugs used to treat CF. Functional patterns revealed by cluster Profiler included interferon signaling, interferon gamma signaling, interleukins 4 and 13 signaling, interleukin 6 signaling, interleukin 21 signaling, and inactivation of CSF3/G-CSF signaling pathways showing significant alterations. These pathways were consistently associated with higher gene expression in CF epithelial cells compared to non-CF cells, suggesting that targeting these pathways could improve clinical outcomes. The success of quantile discretization and Bayesian network analysis in the context of CF suggests that these approaches might be applicable to other contexts where exactly comparable data sets are hard to find.

BACKGROUND:Exposure to arsenic (As) in well water is a well-documented public health issue for Maine and New Hampshire, as well as for other states in the United States and abroad. Arsenic contamination of well water in these locations is primarily attributed to metasedimentary bedrock that leaches As into groundwater. However, As can also enter groundwater reserves from soils contaminated by the historical use of arsenical pesticides. Approximately half of the households in Maine and New Hampshire rely on private wells, many of which have elevated As. Arsenic exposure has been associated with an increased risk of cancer, cardiovascular disease, reduced infection resistance, and lower intelligence quotient in children. Despite these known health impacts, well water testing and treatment are not universal. OBJECTIVES:We have approached the problem of low well water testing rates in Maine and New Hampshire communities by developing the All About Arsenic (AAA) project, which engages secondary school teachers and students as citizen scientists in collecting well water samples for analysis of As and other toxic metals and supports their outreach efforts to their communities. METHODS:We assessed this project's public health impact by analyzing student data relative to existing well water quality datasets in both states. In addition, we surveyed private well owners who contributed well water samples to the project to determine the actions taken to mitigate As in well water. RESULTS:Students collected 3,070 drinking water samples for metals testing, and 752 exceeded New Hampshire's As standard of 5 ppb. The AAA data has more than doubled the amount of information available to public health agencies about well water quality in multiple municipalities across both states. Students also collected information about well types and treatment systems. Their data reveal that some homeowners did not know what type of wells they had or whether they had filtration systems. Those with filtration systems were often unaware of the type of system, what the system was filtering for, or whether the system was designed to remove As. Through interviews with pilot survey participants, we learned that some had begun mitigating their exposure to As and other toxic metals in response to test results from the AAA project. DISCUSSION:A school-based approach to collecting and analyzing private well water samples can successfully reach communities with low testing rates for toxic elements, such as As and other metals. Importantly, information generated through the program can impact household decision-making, and students can influence local and state policymaking by sharing information in their communities. https://doi.org/10.1289/EHP13421.

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in cystic fibrosis (CF) by improving CFTR-mediated Cl- and HCO3- secretion by airway epithelial cells (AECs), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, have numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression, we exposed primary human AEC to ETI for 48 h and interrogated the transcriptome by RNA-seq and qPCR. ETI increased CFTR Cl- secretion, and defensin gene expression (DEFB1), an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI decreased MMP10 and MMP12 gene expression, suggesting that ETI may reduce proteolytic-induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmed DEFB1, HMOX1, MMP10, and MMP12 gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation, and MHC class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug-induced increases in DEFB1 and that ETI may reduce lung damage by reducing MMP10 and MMP12 gene expression. Moreover, pathway analysis also identified several other genes responsible for the ETI-induced reduction in inflammation observed in pwCF. NEW & NOTEWORTHY Gene expression responses by CF AECs exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to enhance resistance to bacterial infection by increasing levels of beta-defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10 and MMP12 and reduce airway inflammation by repressing proinflammatory cytokine secretion by CF AECs.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Our findings show that extracellular vesicles secreted by primary human bronchial epithelial cells significantly reduce Pseudomonas aeruginosa burden, inflammation, and weight loss in a cystic fibrosis mouse model of infection.

This article reviews the role of outer membrane vesicles (OMVs) in mediating the interaction between Gram-negative bacteria and their human hosts. OMVs are produced by a diverse range of Gram-negative bacteria during infection and play a critical role in facilitating host–pathogen interactions without requiring direct cell-to-cell contact. This article describes the mechanisms by which OMVs are formed and subsequently interact with host cells, leading to the transport of microbial protein virulence factors and short interfering RNAs (sRNA) to their host targets, exerting their immunomodulatory effects by targeting specific host signaling pathways. Specifically, this review highlights mechanisms by which OMVs facilitate chronic infection through epigenetic modification of the host immune response. Finally, this review identifies critical knowledge gaps in the field and offers potential avenues for future OMV research, specifically regarding rigor and reproducibility in OMV isolation and characterization methods.

more than 90% of CFTR transcripts.The functional relevance of pulmonary ionocytes in modulating airway ion and fluid transport and CF pathogenesis remains unknown, and new human model systems are needed to examine their biological roles.We have shown the successful directed differentiation of human induced pluripotent stem cells (iPSCs) to airway epithelium (iPSC-airway) with generation of major airway cell types and pulmonary ionocytes that are transcriptomically similar to primary ionocytes.We have also shown that lentiviral over-expression of FOXI1 in iPSC-airways led to increased ASCL3 and CFTR transcripts.To address the role of the pulmonary ionocyte in airway epithelial function, we report the generation of FOXI1-deficient iPSCs and characterization of their progeny after differentiation into airway epithelia.Methods: FOXI1-mutant iPSCs were generated using a Cas9/CRISPR system.After Neon transfection of TrueCut Cas9:sgRNA ribonucleoproteins into iPSCs (clone 1157.2),subclones were screened for FOXI1 disruption, and biallelic frameshift indel formation was confirmed by Sanger and nanopore sequencing.FOXI1-mutant and syngeneic FOXI1-unedited iPSCs were differentiated to airway epithelium and cultured at air-liquid interface (ALI) using our previously published directed differentiation protocol.ALI cultures were harvested for transcriptional analysis and immunofluorescence microscopy.Results: We generated an euploid FOXI1-mutant clone containing a frameshift indel at AA45, upstream from the Forkhead and DNA binding domain of FOXI1, and performed stepwise, directed differentiation of the FOXI1-mutant and syngeneic unedited iPSCs to iPSC-airway.Both lines had similar efficiencies of forming endoderm (96%, 91%), lung progenitors (9.86%, 11.5%), and airway basal cells (90%, 91%).Basal cells derived from both iPSC clones could be expanded at ALI to generate pseudostratified epithelium.FOXI1-mutant iPSC-airway showed minimal or undetectable transcript levels of FOXI1, ASLC3, and the mature ionocyte marker BSND and low transcript levels of CFTR (Figure 1A).There were no significant differences in the airway canonical markers of TP63 (basal), FOXJ1). Immunofluorescence staining demonstrated complete absence of FOXIand BSND-positive cells in the FOXI1-mutant line.The syngeneic unedited iPSC-airway showed FOXI1+ BSND+ cells with morphology similar to primary ionocytes from human bronchial epithelial cell culture (Figure 1B).Conclusions: We generated a FOXI1-mutant iPSC line with absence of pulmonary ionocytes when the line is differentiated to iPSC-airway epithelium.Disruption of FOXI1 did not seem to affect the efficiency of differentiation or generation of major airway cell types.This physiologically relevant, renewable, in vitro system can be used to interrogate the effect of FOXI1 on human airway epithelial functional parameters of ion and fluid transport, rheology, and ciliary motion.

Pseudomonas aeruginosa is a causative agent of a wide range of infections, including chronic infections associated with cystic fibrosis. These P. aeruginosa infections are difficult to treat and often have negative outcomes.