Long-COVID improves in 50% of patients after a year in a Midwestern cohort.

Background: Many of those infected with COVID-19 experience long-term disability due to persistent symptoms known as Long-COVID, which include ongoing respiratory issues, loss of taste and smell, and impaired daily functioning. Research Question: This study aims to better understand the chronology of long-COVID symptoms. Study Design and Methods: We prospectively enrolled 403 adults from the University of Iowa long-COVID clinic (June 2020 to February 2022). Participants provided symptom data during acute illness, symptom progression, and other clinical characteristics. Patients in this registry received a survey containing questions including current symptoms and status since long-COVID diagnosis (sliding status scale, PHQ2, GAD2, MMRC). Those >12 months since acute-COVID diagnosis had chart review done to track their symptomology. Results: Of 403 participants contacted, 129 (32%) responded. The mean age (in years) was 50.17 +/-14.28, with 31.8% male and 68.2% female. Severity of acute covid treatment was stratified by treatment in the outpatient (70.5%), inpatient (16.3%), or ICU (13.2%) settings. 51.2% reported subjective improvement (sliding scale scores of 67-100) since long-COVID onset. Ages 18-29 reported significantly higher subjective status scores. Subjective status scores were unaffected by severity. 102 respondents were >12 months from their initial COVID-19 diagnosis and were tracked for longitudinal symptom persistence. All symptoms tracked had variance (mean fraction 0.58, range 0.34-0.75) in the reported symptoms at the time of long-COVID presentation when compared with patient survey report. 48 reported persistent dyspnea, 23 (48%) had resolved it at time of survey. For fatigue, 44 had persistence, 12 (27%) resolved. Interpretation: Overall, 51.2% respondents improved since their long-COVID began. Pulmonary symptoms were more persistent than neuromuscular symptoms (anosmia, dysgeusia, myalgias). Gender, time since acute COVID infection, and its severity didn't affect subjective status or symptoms. This study highlights recall bias that may be prevalent in other long-COVID research reliant on participant memory.

Eosinophil expression of Triggering receptor expressed on myeloid cells-1 (TREM-1) restricts type 2 lung inflammation.

Asthma affects 25 million Americans and recent advances in treatment are effective for only a portion of severe asthma patients. Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an innate receptor that canonically amplifies inflammatory signaling in neutrophils and monocytes, plays a central role in regulating lung inflammation. It is unknown how TREM-1 contributes to allergic asthma pathology. Utilizing a murine model of asthma, flow cytometry revealed TREM-1+ eosinophils in the lung tissue and airway during allergic airway inflammation. TREM-1 expression was restricted to recruited, inflammatory eosinophils. Expression was induced on bone marrow derived eosinophils by incubation with IL-33, LPS, or GM-CSF. Compared to TREM-1- airway eosinophils, TREM-1+ eosinophils were enriched for pro-inflammatory gene sets including migration, respiratory burst, and cytokine production. Unexpectedly, eosinophil-specific ablation of TREM-1 exacerbated airway IL-5 production, airway MUC5AC production, and lung tissue eosinophil accumulation. Further investigation of transcriptional data revealed apoptosis and superoxide generation related gene sets were enriched in TREM-1+ eosinophils. Consistent with these findings, Annexin V and Caspase 3/7 staining demonstrated higher rates of apoptosis among TREM-1+ eosinophils compared to TREM-1- eosinophils in the inflammatory airway. In vitro, Trem1/3-/- bone marrow derived eosinophils consumed less oxygen than WT in response to PMA, suggesting that TREM-1 promotes superoxide generation in eosinophils. These data reveal protein level expression of TREM-1 by eosinophils, define a population of TREM-1+ inflammatory eosinophils, and demonstrate that eosinophil TREM-1 restricts key features of type 2 lung inflammation.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

IL-13 induced inflammation increases DPP4 abundance but does not enhance MERS-CoV replication in airway epithelia.

BACKGROUND: Chronic pulmonary conditions such as asthma and COPD increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). METHODS: We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine IL-13, examining how this impacted DPP4 protein levels along with MERS-CoV entry and replication. RESULTS: IL-13 exposure for 3 days led to increased DPP4 protein abundance, while a 21-day treatment increased DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly impacted by IL-13 treatment. CONCLUSIONS: Our results suggest that increased DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13 induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis.

Background: Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. Methods: We identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main Results: Between 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus . We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS - mutL - hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. Conclusions: Linezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key Point: Linezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

FXYD3 facilitates Na+ and liquid absorption across human airway epithelia by increasing the transport capacity of the Na/K ATPase.

Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced FXYD3 expression using siRNA. After permeabilizing the apical membrane with nystatin, epithelia pretreated with FXYD3-targeting siRNA had lower ouabain-sensitive short-circuit currents than control epithelia. FXYD3-targeting siRNA also reduced amiloride-sensitive short-circuit currents and liquid absorption across intact epithelia. These data are consistent with FXYD3 facilitating Na+ and liquid absorption. FXYD3 may be needed to maintain the high rates of Na+ and fluid absorption observed for airway and other FXYD3-expressing epithelia.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Vitamin D-mediated effects on airway innate immunity in vitro.

INTRODUCTION: Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune mechanisms in vitro. METHODS: Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs. RESULTS: Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1. CONCLUSION: While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.

Quantitative Chest CT Assessment of Small Airways Disease in Post-Acute SARS-CoV-2 Infection.

Background The long-term effects of SARS-CoV-2 infection on pulmonary structure and function remain incompletely characterized. Purpose To test whether SARS-CoV-2 infection leads to small airways disease in patients with persistent symptoms. Materials and Methods In this single-center study at a university teaching hospital, adults with confirmed COVID-19 who remained symptomatic more than 30 days following diagnosis were prospectively enrolled from June to December 2020 and compared with healthy participants (controls) prospectively enrolled from March to August 2018. Participants with post-acute sequelae of COVID-19 (PASC) were classified as ambulatory, hospitalized, or having required the intensive care unit (ICU) based on the highest level of care received during acute infection. Symptoms, pulmonary function tests, and chest CT images were collected. Quantitative CT analysis was performed using supervised machine learning to measure regional ground-glass opacity (GGO) and using inspiratory and expiratory image-matching to measure regional air trapping. Univariable analyses and multivariable linear regression were used to compare groups. Results Overall, 100 participants with PASC (median age, 48 years; 66 women) were evaluated and compared with 106 matched healthy controls; 67% (67 of 100) of the participants with PASC were classified as ambulatory, 17% (17 of 100) were hospitalized, and 16% (16 of 100) required the ICU. In the hospitalized and ICU groups, the mean percentage of total lung classified as GGO was 13.2% and 28.7%, respectively, and was higher than that in the ambulatory group (3.7%, P < .001 for both comparisons). The mean percentage of total lung affected by air trapping was 25.4%, 34.6%, and 27.3% in the ambulatory, hospitalized, and ICU groups, respectively, and 7.2% in healthy controls (P < .001). Air trapping correlated with the residual volume-to-total lung capacity ratio (ρ = 0.6, P < .001). Conclusion In survivors of COVID-19, small airways disease occurred independently of initial infection severity. The long-term consequences are unknown. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Elicker in this issue.

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells.

RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.

Lentiviral vectors transduce lung stem cells without disrupting plasticity.

Life-long expression of a gene therapy agent likely requires targeting stem cells. Here we ask the question: does viral vector transduction or ectopic expression of a therapeutic transgene preclude airway stem cell function? We used a lentiviral vector containing a GFP or cystic fibrosis transmembrane conductance regulator (CFTR) transgene to transduce primary airway basal cells from human cystic fibrosis (CF) or non-CF lung donors and monitored expression and function after differentiation. Ussing chamber measurements confirmed CFTR-dependent chloride channel activity in CF donor cells. Immunostaining, quantitative real-time PCR, and single-cell sequencing analysis of cell-type markers indicated that vector transduction or CFTR expression does not alter the formation of pseudostratified, fully differentiated epithelial cell cultures or cell type distribution. These results have important implications for use of gene addition or gene editing strategies as life-long curative approaches for lung genetic diseases.

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers.

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2'-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV's strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

MRSA strains with distinct accessory genes predominate at different ages in cystic fibrosis.

RATIONALE: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. OBJECTIVES: To identify MRSA genes associated with low lung function and potential MRSA transmission in CF. METHODS: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies, classified isolates by accessory gene content, and correlated the accessory genome to clinical outcomes. RESULTS: The most prevalent ST were ST5 (N = 55), ST8 (N = 15), and ST105 (N = 14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three clusters of MRSA were identified by accessory genome content. Cluster A, including ST5 and ST105, was highly prevalent at all ages. Cluster B, including ST8, was more limited to younger patients. Cluster C included 6 distantly related strains. Patients 20 years old and younger infected with Cluster A had lower forced expiratory volume in the first second (FEV1 ) and higher sputum biomass compared to similar-aged patients with Cluster B. CONCLUSIONS: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. The most prevalent cluster of MRSA, including ST5 and ST105, was associated with lower FEV1 . ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators.

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HCO3- secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl-/HCO3- exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3- secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.

Differential gene expression analysis for multi-subject single-cell RNA-sequencing studies with aggregateBioVar.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful studies. As scRNA-seq costs have decreased, collecting data from more than one biological replicate has become more feasible, but careful modeling of different layers of biological variation remains challenging for many users. Here, we propose a statistical model for scRNA-seq gene counts, describe a simple method for estimating model parameters and show that failing to account for additional biological variation in scRNA-seq studies can inflate false discovery rates (FDRs) of statistical tests. RESULTS: First, in a simulation study, we show that when the gene expression distribution of a population of cells varies between subjects, a naïve approach to differential expression analysis will inflate the FDR. We then compare multiple differential expression testing methods on scRNA-seq datasets from human samples and from animal models. These analyses suggest that a naïve approach to differential expression testing could lead to many false discoveries; in contrast, an approach based on pseudobulk counts has better FDR control. AVAILABILITY AND IMPLEMENTATION: A software package, aggregateBioVar, is freely available on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/aggregateBioVar.html) to accommodate compatibility with upstream and downstream methods in scRNA-seq data analysis pipelines. SUPPLEMENTARY INFORMATION: Raw gene-by-cell count matrices for pig scRNA-seq data are available as GEO accession GSE150211. Supplementary data are available at Bioinformatics online.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

TNFα and IL-17 alkalinize airway surface liquid through CFTR and pendrin.

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.