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Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network. NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. [ABSTRACT FROM AUTHOR]

Among asthmatics, there is significant heterogeneity in the clinical presentation and underlying pathophysiological mechanisms, leading to the recognition of multiple disease endotypes (e.g., T2-high vs. T2-low). This heterogeneity extends to severe asthmatics, who may struggle to control symptoms even with high-dose corticosteroid treatment and other therapies. However, there are limited mouse models available to model the spectrum of severe asthma endotypes. We sought to identify a new mouse model of severe asthma by first examining responses to chronic allergen exposure among strains from the Collaborative Cross (CC) mouse genetics reference population, which contains greater genetic diversity than other inbred strain panels previously used for models of asthma. Mice from five CC strains and the often-used classical inbred strain BALB/cJ were chronically exposed to house dust mite (HDM) allergen for five weeks followed by measurements of airway inflammation. CC strain CC011/UncJ (CC011) exhibited extreme responses to HDM including high levels of airway eosinophilia, elevated lung resistance, and extensive airway wall remodeling, and even fatalities among ~ 50% of mice prior to study completion. Compared to BALB/cJ mice, CC011 mice had stronger Th2-mediated airway responses demonstrated by significantly elevated total and HDM-specific IgE and increased Th2 cytokines during tests of antigen recall, but not enhanced ILC2 activation. Airway eosinophilia in CC011 mice was completely dependent upon CD4+ T-cells. Notably, we also found that airway eosinophilia in CC011 mice was resistant to dexamethasone steroid treatment. Thus, the CC011 strain provides a new mouse model of T2-high, severe asthma driven by natural genetic variation likely acting through CD4+ T-cells. Future studies aimed at determining the genetic basis of this phenotype will provide new insights into mechanisms underlying severe asthma. [ABSTRACT FROM AUTHOR]

Ambient ozone (O3) exposure has serious consequences on respiratory health, including airway inflammation and injury. Decades of research have yielded thorough descriptions of these outcomes; however, less is known about the molecular processes that drive them. The aim of this study was to further describe the cellular and molecular responses to O3 exposure in murine airways, with a particular focus on transcriptional responses in 2 critical pulmonary tissue compartments: conducting airways (CA) and airway macrophages (AM). After exposing adult, female C57BL/6J mice to filtered air, 1 or 2 ppm O3, we assessed hallmark responses including airway inflammation (cell counts and cytokine secretion) and injury (epithelial permeability), followed by gene expression profiling of CA and AM by RNA-seq. As expected, we observed concentration-dependent increases in airway inflammation and injury. Conducting airways and AM both exhibited changes in gene expression to both 1 and 2 ppm O3 that were largely compartment-specific. In CA, genes associated with epithelial barrier function, detoxification processes, and cellular proliferation were altered, while O3 affected genes involved in innate immune signaling, cytokine production, and extracellular matrix remodeling in AM. Further, CA and AM also exhibited notable differences in concentration–response expression patterns for large numbers of genes. Overall, our study has described transcriptional responses to acute O3 exposure, revealing both shared and unique gene expression patterns across multiple concentrations of O3 and in 2 important O3-responsive tissues. These profiles provide broad mechanistic insight into pulmonary O3 toxicity, and reveal a variety of targets for focused follow-up studies. [ABSTRACT FROM AUTHOR]

The Collaborative Cross (CC) is a mouse recombinant inbred strain panel that is being developed as a resource for mammalian systems genetics. Here we describe an experiment that uses partially inbred CC lines to evaluate the genetic properties and utility of this emerging resource. Genome-wide analysis of the incipient strains reveals high genetic diversity, balanced allele frequencies, and dense, evenly distributed recombination sites--all ideal qualities for a systems genetics resource. We map discrete, complex, and biomolecular traits and contrast two quantitative trait locus (QTL) mapping approaches. Analysis based on inferred haplotypes improves power, reduces false discovery, and provides information to identify and prioritize candidate genes that is unique to multifounder crosses like the CC. The number of expression QTLs discovered here exceeds all previous efforts at eQTL mapping in mice, and we map local eQTL at 1-Mb resolution. We demonstrate that the genetic diversity of the CC, which derives from random mixing of eight founder strains, results in high phenotypic diversity and enhances our ability to map causative loci underlying complex disease-related traits. [ABSTRACT FROM AUTHOR]

Zoonotic arenavirus infections can result in viral hemorrhagic disease, characterized by platelet loss, petechia, and multi-organ injury. The mechanisms governing these outcomes are likely impacted by virus strain and infection dose, as well as an individual's genetic background and immune constitution. To better understand the processes leading to severe pathogenesis, we compared two strains of inbred mice, C57BL/6J (B6) and FVB/NJ (FVB), that have diametrically opposed outcomes during disseminated lymphocytic choriomeningitis virus (LCMV) infection. Infection caused minimal pathogenesis in B6 mice, whereas FVB mice developed acute hepatitis and perished due, in part, to aberrant NK cell and T cell responses. Susceptible mice showed an outgrowth of cytolytic CD4 + T cells and loss of Treg cells. B6 congenic mice with the FVB allele at a 25Mb locus on chromosome 17 recapitulated FVB pathogenesis upon infection. A locus containing a limited number of variants in immune-related genes greatly impacts survival during infection., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Obesity exacerbates inflammation upon lung injury; however, the mechanisms by which obesity primes pulmonary dysregulation prior to external injury are not well studied. Herein, we tested the hypothesis that obesity dysregulates pulmonary PUFA metabolism that is central to inflammation initiation and resolution. We first show that a high-fat diet (HFD) administered to C57BL/6J mice increased the relative abundance of pulmonary PUFA-containing triglycerides and the concentration of PUFA-derived oxylipins (particularly prostaglandins and hydroxyeicosatetraenoic acids), independent of an increase in total pulmonary PUFAs, prior to onset of pulmonary inflammation. Experiments with a genetic model of obesity (ob/ob) generally recapitulated the effects of the HFD on the pulmonary oxylipin signature. Subsequent pulmonary next-generation RNA sequencing identified complex and unique transcriptional regulation with the HFD. We found the HFD increased pathways related to glycerophospholipid metabolism and immunity, including a unique elevation in B cell differentiation and signaling. Furthermore, we conducted computational integration of lipidomic with transcriptomic data. These analyses identified novel HFD-driven networks between glycerophospholipid metabolism and B cell receptor signaling with specific PUFA-derived pulmonary oxylipins. Finally, we confirmed the hypothesis by demonstrating that the concentration of pulmonary oxylipins, in addition to inflammatory markers, were generally increased in mice consuming a HFD upon ozone-induced acute lung injury. Collectively, these data show that a HFD dysregulates pulmonary PUFA metabolism prior to external lung injury, which may be a mechanism by which obesity primes the lungs to respond poorly to infectious and/or inflammatory challenges., Competing Interests: Conflicts of interest S. R. S. has received industry support on projects and conferences related to n-3 fatty acids from Metagenics Inc., and the Wiley Companies., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute ozone (O 3 ) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O 3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O 3 exposure interactions on AM gene expression and identify transcriptional correlates of O 3 -induced inflammation and injury across six mouse strains, including five Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O 3 for 3 h and measured inflammatory and injury parameters 21 h later. Mice exposed to O 3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O 3 transcriptional response signature across all strains, as well as a set of genes exhibiting strain-by-O 3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contribute to their divergent post-O 3 exposure transcriptional responses. Together, these results suggest that aspects of O 3 -induced respiratory responses are mediated through altered AM transcriptional signatures and further confirm the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.

The Collaborative Cross (CC) is a mouse genetic reference population whose range of applications includes quantitative trait loci (QTL) mapping. The design of a CC QTL mapping study involves multiple decisions, including which and how many strains to use, and how many replicates per strain to phenotype, all viewed within the context of hypothesized QTL architecture. Until now, these decisions have been informed largely by early power analyses that were based on simulated, hypothetical CC genomes. Now that more than 50 CC strains are available and more than 70 CC genomes have been observed, it is possible to characterize power based on realized CC genomes. We report power analyses from extensive simulations and examine several key considerations: 1) the number of strains and biological replicates, 2) the QTL effect size, 3) the presence of population structure, and 4) the distribution of functionally distinct alleles among the founder strains at the QTL. We also provide general power estimates to aide in the design of future experiments. All analyses were conducted with our R package, SPARCC (Simulated Power Analysis in the Realized Collaborative Cross), developed for performing either large scale power analyses or those tailored to particular CC experiments., (Copyright © 2019 Keele et al.)

Differential gene expression in the airway epithelium of patients with asthma versus controls has been reported in several studies. However, there is no consensus on which genes are reproducibly affected in asthma. We sought to identify a consensus list of differentially expressed genes (DEGs) using a meta-analysis approach.We identified eight studies with data that met defined inclusion criteria. These studies comprised 355 cases and 193 controls and involved sampling either bronchial or nasal epithelium. We conducted study-level analyses, followed by a meta-analysis. Likewise, we applied a meta-analysis framework to the results of study-level pathway enrichment.We identified 1273 DEGs, 431 of which had not been identified in previous studies. 450 DEGs exhibited large effect sizes and were robust to study population differences in age, sex, race/ethnicity, medication use, smoking status and exacerbations. The magnitude of differential expression of these 450 genes was highly similar in bronchial and nasal airway epithelia. Meta-analysis of pathway enrichment revealed a number of consistently dysregulated biological pathways, including putative transcriptional and post-transcriptional regulators.In total, we identified a set of genes that is consistently dysregulated in asthma, that links to known and novel biological pathways, and that will inform asthma subtype identification., Competing Interests: Conflict of interest: None declared., (Copyright ©ERS 2018.)

Allergic asthma is common airway disease that is characterized in part by enhanced airway constriction in response to nonspecific stimuli. Genome-wide association studies have identified multiple loci associated with asthma risk in humans, but these studies have not accounted for gene-environment interactions, which are thought to be important factors in asthma. To identify quantitative trait loci (QTL) that regulate responses to a common human allergen, we applied a house dust mite mouse (HDM) model of allergic airway disease (AAD) to 146 incipient lines of the Collaborative Cross (CC) and the CC founder strains. We employed a longitudinal study design in which mice were phenotyped for response to the bronchoconstrictor methacholine both before and after HDM sensitization and challenge using whole body plethysmography (WBP). There was significant variation in methacholine responsiveness due to both strain and HDM treatment, as reflected by changes in the WBP parameter enhanced pause. We also found that distinct QTL regulate baseline [chromosome (Chr) 18] and post-HDM (Chr 19) methacholine responsiveness and that post-HDM airway responsiveness was correlated with other features of AAD. Finally, using invasive measurements of airway mechanics, we tested whether the Chr 19 QTL affects lung resistance per se using C57BL/6J mice and a consomic strain but found that QTL haplotype did not affect lung resistance. We conclude that aspects of baseline and allergen-induced methacholine responsiveness are associated with genetic variation, and that robust detection of airway resistance QTL in genetically diverse mice will be facilitated by direct measurement of airway mechanics., (Copyright © 2016 Kelada.)

Hematological parameters, including red and white blood cell counts and hemoglobin concentration, are widely used clinical indicators of health and disease. These traits are tightly regulated in healthy individuals and are under genetic control. Mutations in key genes that affect hematological parameters have important phenotypic consequences, including multiple variants that affect susceptibility to malarial disease. However, most variation in hematological traits is continuous and is presumably influenced by multiple loci and variants with small phenotypic effects. We used a newly developed mouse resource population, the Collaborative Cross (CC), to identify genetic determinants of hematological parameters. We surveyed the eight founder strains of the CC and performed a mapping study using 131 incipient lines of the CC. Genome scans identified quantitative trait loci for several hematological parameters, including mean red cell volume (Chr 7 and Chr 14), white blood cell count (Chr 18), percent neutrophils/lymphocytes (Chr 11), and monocyte number (Chr 1). We used evolutionary principles and unique bioinformatics resources to reduce the size of candidate intervals and to view functional variation in the context of phylogeny. Many quantitative trait loci regions could be narrowed sufficiently to identify a small number of promising candidate genes. This approach not only expands our knowledge about hematological traits but also demonstrates the unique ability of the CC to elucidate the genetic architecture of complex traits.

The dopamine transporter gene (SLC6A3) is a candidate gene for Parkinson's disease (PD) on the basis of its critical role in dopaminergic neurotransmission. Previously, we identified 22 SNPs in the 5' region of SLC6A3, which segregate as eight haplotypes that differ in transcriptional activity when transfected in rat dopamine-producing cells. In the present work from a case-control study size of 293 cases and 395 controls, we employed a cladistic approach to examine gene-disease association. First, we found strong evidence of balancing selection in this region, as determined by a Tajima's D statistic of 2.97 (P<0.001). Second, we found that the eight haplotypes fit into two main clades and that diplotypes of these clades were marginally associated with PD. Then, after we classified cases and controls by the number of risk alleles, accounting for the well-known 3' region VNTR polymorphism, we found that having two or more risk alleles resulted in a modest but significant increase in PD risk [odds ratio=1.58; 95% confidence interval (CI): 1.03-2.40]. Finally, we detected a significant interaction between occupational pesticide exposure in men and the number of risk alleles. Among pesticide-exposed subjects, the odds ratio for having two or more risk alleles was 5.66 (95% CI: 1.73-18.53). Thus, allelic variants in SLC6A3, which affect gene expression, are associated with PD in this population and may interact with occupational pesticide exposure to increase PD risk.