Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

BAL Cell Gene Expression in Severe Asthma Reveals Mechanisms of Severe Disease and Influences of Medications.

Rationale: Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.Objectives: To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression.Methods: BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.Measurements and Main Results: Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an in vitro cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and ß-agonist-naive subjects given a ß-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.Conclusions: Gene expression in BAL cells is influenced by factors seldomly considered. Notably, ß-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.

Extreme Trait Whole-Genome Sequencing Identifies PTPRO as a Novel Candidate Gene in Emphysema with Severe Airflow Obstruction.

RATIONALE: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole-genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility. OBJECTIVES: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction. METHODS: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole-genome sequencing results to include only rare variants and conducted single variant tests, region-based tests across the genome, gene-based tests, and exome-wide tests. MEASUREMENTS AND MAIN RESULTS: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10-6), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10-5). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non-Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation. CONCLUSIONS: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

RATIONALE: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). OBJECTIVES: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. METHODS: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. MEASUREMENTS AND MAIN RESULTS: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. CONCLUSIONS: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Genome-Wide Association Mapping of the Antibody Response to Diphtheria, Tetanus and Acellular Pertussis Vaccine in Mice.

The objective of the current study was to investigate the genetics of antibody responses to an acellular pertussis vaccine by a genome-wide association study in mice. Female mice of 28 inbred strains received this vaccine at 6, 8, and 12 weeks of age. The antibody titer and avidity of immunoglobulin (Ig) G specific for diphtheria toxin, pertussis toxin, filamentous hemagglutinin and pertactin were measured at 14 and 24 weeks of age. The magnitude, longevity and avidity of IgG differed significantly among mouse strains. There was significant correlation between antigen-specific IgGs for longevity but not for magnitude and avidity. Association mapping and analysis with PolyPhen software identified 6 genetic markers associated with longevity for all 4 antigens, although the expression levels of these genes did not correlate with longevity phenotype. This study provides novel insights into the genetic basis and potential candidate genes for differences in the IgG responses to vaccination.

Variable Susceptibility to Cigarette Smoke-Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 µm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 µm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction.

Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH-HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH-HFpEF. To develop a mouse model of PH-HFpEF, we exposed 36 mouse strains to 20 weeks of high-fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure-volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH-HFpEF. Of the strains that develop PH-HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD-induced metabolic syndrome and had low variability in hemodynamics. HFD-treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end-diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end-diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD-induced PH-HFpEF. These studies validate an HFD/AKR/J mouse model of PH-HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

Longitudinal multicompartment characterization of host-microbiota interactions in patients with acute respiratory failure.

Critical illness can significantly alter the composition and function of the human microbiome, but few studies have examined these changes over time. Here, we conduct a comprehensive analysis of the oral, lung, and gut microbiota in 479 mechanically ventilated patients (223 females, 256 males) with acute respiratory failure. We use advanced DNA sequencing technologies, including Illumina amplicon sequencing (utilizing 16S and ITS rRNA genes for bacteria and fungi, respectively, in all sample types) and Nanopore metagenomics for lung microbiota. Our results reveal a progressive dysbiosis in all three body compartments, characterized by a reduction in microbial diversity, a decrease in beneficial anaerobes, and an increase in pathogens. We find that clinical factors, such as chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, are associated with specific patterns of dysbiosis. Interestingly, unsupervised clustering of lung microbiota diversity and composition by 16S independently predicted survival and performed better than traditional clinical and host-response predictors. These observations are validated in two separate cohorts of COVID-19 patients, highlighting the potential of lung microbiota as valuable prognostic biomarkers in critical care. Understanding these microbiome changes during critical illness points to new opportunities for microbiota-targeted precision medicine interventions.

Robust airway microbiome signatures in acute respiratory failure and hospital-acquired pneumonia.

Respiratory microbial dysbiosis is associated with acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) in critically ill patients. However, we lack reproducible respiratory microbiome signatures that can increase our understanding of these conditions and potential treatments. Here, we analyze 16S rRNA sequencing data from 2,177 respiratory samples collected from 1,029 critically ill patients (21.7% with ARDS and 26.3% with HAP) and 327 healthy controls, sourced from 17 published studies. After data harmonization and pooling of individual patient data, we identified microbiota signatures associated with ARDS, HAP and prolonged mechanical ventilation. Microbiota signatures for HAP and prolonged mechanical ventilation were characterized by depletion of a core group of microbes typical of healthy respiratory samples, and the ARDS microbiota signature was distinguished by enrichment of potentially pathogenic respiratory microbes, including Pseudomonas and Staphylococcus. Using machine learning models, we identified clinically informative, three- and four-factor signatures that predicted ARDS, HAP and prolonged mechanical ventilation with relatively high accuracy (area under the curve of 0.751, 0.72 and 0.727, respectively). We validated the signatures in an independent prospective cohort of 136 patients on mechanical ventillation and found that patients with microbiome signatures associated with ARDS, HAP or prolonged mechanical ventilation had longer times to successful extubation than patients lacking these signatures (hazard ratios of 1.56 (95% confidence interval (CI) 1.07-2.27), 1.51 (95% CI 1.02-2.23) and 1.50 (95% CI 1.03-2.18), respectively). Thus, we defined and validated robust respiratory microbiome signatures associated with ARDS and HAP that may help to identify promising targets for microbiome therapeutic modulation in critically ill patients.

A common single nucleotide polymorphism is associated with inflammation and critical illness outcomes.

Acute inflammation is heterogeneous in critical illness and predictive of outcome. We hypothesized that genetic variability in novel, yet common, gene variants contributes to this heterogeneity and could stratify patient outcomes. We searched algorithmically for significant differences in systemic inflammatory mediators associated with any of 551,839 SNPs in one derivation (n = 380 patients with blunt trauma) and two validation (n = 75 trauma and n = 537 non-trauma patients) cohorts. This analysis identified rs10404939 in the LYPD4 gene. Trauma patients homozygous for the A allele (rs10404939AA; 27%) had different trajectories of systemic inflammation along with persistently elevated multiple organ dysfunction (MOD) indices vs. patients homozygous for the G allele (rs10404939GG; 26%). rs10404939AA homozygotes in the trauma validation cohort had elevated MOD indices, and non-trauma patients displayed more complex inflammatory networks and worse 90-day survival compared to rs10404939GG homozygotes. Thus, rs10404939 emerged as a common, broadly prognostic SNP in critical illness.

Prognostic Insights from Longitudinal Multicompartment Study of Host-Microbiota Interactions in Critically Ill Patients.

Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.

Prognostic Insights from Longitudinal Multicompartment Study of Host-Microbiota Interactions in Critically Ill Patients.

Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins--towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation.

BACKGROUND: Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. RESULTS: The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin ß-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. CONCLUSION: Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

Genetic Variation in the Magnitude and Longevity of the IgG Subclass Response to a Diphtheria-Tetanus-Acellular Pertussis (DTaP) Vaccine in Mice.

The type of IgG subclasses induced by vaccination is an important determinant of vaccine efficacy because the IgG subclasses vary in their biological function. The goal of this study was to determine the influence of the genetic background on the production and duration of vaccine-induced IgG subclasses. IgG1, IgG2b, and IgG3 titers against diphtheria toxoid (DT), pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (Prn) were measured in mice from 28 different inbred and wild-derived strains vaccinated with an aluminum hydroxide-adjuvanted DTaP vaccine. The titers and duration of vaccine-specific IgG subclass responses were different among mouse strains, indicating that genetic factors contribute to this variation. Statistical associations were used to identify potential mechanisms that contribute to antibody production and longevity. This analysis showed that the mechanisms guiding the magnitude of antibody production were antigen-dependent for IgG1 but antigen-independent for IgG2b and IgG3. However, the mechanisms driving the longevity of antibody titers were antigen-independent for IgG1, IgG2b, and IgG3. The ratio of IgG1 and IgG3 titers identified Th1 and Th2-prone mouse strains. TLR4-deficient C3H/HeJ mice had an enhanced IgG1 response compared with C3H/HeOuJ mice with intact TLR4. This work demonstrates that the genetic background contributes significantly to the magnitude and longevity of vaccine-induced IgG1, IgG2b, and IgG3 titers in mice.

Correction: Synthesis of IL-6 by Hepatocytes Is a Normal Response to Common Hepatic Stimuli.

[This corrects the article DOI: 10.1371/journal.pone.0096053.].

Ex vivo lung perfusion as a human platform for preclinical small molecule testing.

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.