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.

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.

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.

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.

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.

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.

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.

Single-molecule long-read sequencing reveals the chromatin basis of gene expression.

Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remain poorly studied at the single-cell level. Here, we present a new experimental approach, methyltransferase treatment followed by single-molecule long-read sequencing (MeSMLR-seq), for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. MeSMLR-seq offers direct measurements of both nucleosome-occupied and nucleosome-evicted regions on a single DNA molecule, which is challenging for many existing methods. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding the transcription start site for silent and actively transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin status spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming. In addition, we revealed the coupled changes of chromatin accessibility for two neighboring glucose transporter genes in response to changes in glucose concentration.

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.

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.

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.

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.

Detection of smoothly distributed spatial outliers, with applications to identifying the distribution of parenchymal hyperinflation following an airway challenge in asthmatics.

Methacholine challenge tests are used to measure changes in pulmonary function that indicate symptoms of asthma. In addition to pulmonary function tests, which measure global changes in pulmonary function, computed tomography images taken at full inspiration before and after administration of methacholine provide local air volume changes (hyper-inflation post methacholine) at individual acinar units, indicating local airway hyperresponsiveness. Some of the acini may have extreme air volume changes relative to the global average, indicating hyperresponsiveness, and those extreme values may occur in clusters. We propose a Gaussian mixture model with a spatial smoothness penalty to improve prediction of hyperresponsive locations that occur in spatial clusters. A simulation study provides evidence that the spatial smoothness penalty improves prediction under different data-generating mechanisms. We apply this method to computed tomography data from Seoul National University Hospital on five healthy and ten asthmatic subjects. Copyright © 2017 John Wiley & Sons, Ltd.

Epithelial responses to CFTR modulators are improved by inflammatory cytokines and impaired by antiinflammatory drugs.

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

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. 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 interleukin 33, lipopolysaccharide, or granulocyte-macrophage colony-stimulating factor. Compared to TREM-1- airway eosinophils, TREM-1+ eosinophils were enriched for proinflammatory gene sets, including migration, respiratory burst, and cytokine production. Unexpectedly, eosinophil-specific ablation of TREM-1 exacerbated airway interleukin (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 wild-type in response to phorbol myristate acetate, 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.

IL-13 decreases susceptibility to airway epithelial SARS-CoV-2 infection but increases disease severity in vivo.

Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signaling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. We investigated the role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2-expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe respiratory virus-induced lung disease.

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.

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.

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.

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.