Transcriptional changes in alveolar macrophages from adults with asthma after intrabronchial allergen challenge.
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ABSTRACT: In recent years, alveolar macrophages (AM) have been shown to play an important role in environmental allergen-induced airway inflammation in asthma. In this study, we investigated the effect of house dust mite (HDM) and lipopolysaccharide (LPS) challenge on the transcriptome of AM from patients with mild asthma. We have shown previously that intrabronchial HDM/LPS challenge induces a mixed eosinophilic and neutrophil airways inflammation in asthma patients(5). Therefore, we hypothesize that exposure of AM to HDM/LPS would upregulate genes associated with eosinophil and neutrophil signaling. AM were harvested from the bronchoalveolar fluid of 8 asthma patients who were challenged with either saline in one lung segment or HDM/LPS in the contralateral lung segment. We performed RNA sequencing and found over 1000 significantly different expressed genes following HDM/LPS challenge. To the best of our knowledge, this is the first investigation to examine gene expression profiles in AM from asthma patients challenged with HDM and LPS in vivo. Using a well-controlled study design allowing paired analysis of saline and HDM/LPS effects in the same patient, we identified different transcriptional profiles in AM as consequence of local exposure to HDM and LPS. These findings may provide a better understanding of AM functions in (exacerbations of) allergic asthma.
Project description:Segmental instillation of lipopolysaccharide (LPS) by bronchoscopy can be used as a model to safely induce transient airway inflammation in the human lung. The LPS challenge model enables investigation of cellular mechanisms involved in pulmonary inflammatory processes as well as pharmacodynamic analysis of investigational drugs for the treatment of respiratory diseases. Aim of this work was to describe the transcriptomic profile of the human segmental LPS challenge model with contextualization to major respiratory diseases. Pre-challenge bronchoalveolar lavage fluid (BAL) and biopsies were sampled from twenty-eight smoking, healthy subjects, followed by segmental LPS challenge and saline control challenge. Twenty-four hours post instillation, BAL and biopsies were collected from the challenged lung segments. Total RNA of cells from BAL and biopsy samples were sequenced for subsequent data analysis. Differential gene expression analysis resulted in 6316 upregulated differentially expressed genes (DEGs) and 241 downregulated DEG in BAL, but only one downregulated DEG in biopsy samples after LPS challenge compared to saline challenge. Upregulated DEG in BAL were related to molecular functions such as “Inflammatory response” or “antimicrobial humoral immune response mediated by antimicrobial peptide”, enriched biological processes such as “chemokine receptor activity”, and upregulated pro-inflammatory pathways such as “Wnt signaling pathway”, “Ras signaling pathway” or “JAK-STAT signaling pathway”. Furthermore, the segmental LPS challenge model resembled aspects of the five most prevalent respiratory diseases chronic obstructive pulmonary disease (COPD), asthma, pneumonia, tuberculosis and lung cancer and featured similarities with acute exacerbations in COPD (AECOPD) and community-acquired pneumonia (CAP). Overall, our study provides extensive information about the transcriptomic profile from BAL cells and mucosal biopsies following LPS challenge in healthy smokers. It expands the knowledge about the LPS challenge model providing potential overlap with respiratory diseases in general and infection-triggered respiratory insults such as AECOPD in particular.
Project description:Neutrophils play important roles in inflammatory airway diseases. Here, we assessed whether apolipoprotein A-I (apoA-I) modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 days. Single cell RNA sequencing was performed on cells recovered in bronchoalveolar lavage fluid (BALF) on day 4. Unsupervised profiling identified 10 clusters of neutrophils in BALF from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8, and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing interferon-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of pro-inflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of pro-inflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apoA-I attenuates acute airway inflammation.
Project description:Background: Inhalation exposure to biological particulate matter (BioPM) from livestock farms may provoke exacerbations in subjects suffering from allergy and asthma. The aim of this study was to use a murine model of allergic asthma to determine the effect of BioPM derived from goat farm on airway allergic responses Methods: Fine (< 2.5 μm) BioPM was collected from an indoor goat stable. Female BALB/c mice were ovalbumin (OVA) sensitized and challenged with OVA or saline as control. The OVA and saline groups were divided in sub-groups and exposed intranasally to different concentrations (0, 0.9, 3, or 9 μg) of goat farm BioPM. Bronchoalveolar lavage fluid (BALF), blood and lung tissues were collected. Results: In saline-challenged mice, goat farm BioPM alone induced a dose-dependent increase in neutrophils in BALF and induced production of macrophage inflammatory protein-3a). In OVA-challenged mice, BioPM significantly enhanced 1) inflammatory cells in BALF, 2) OVA-specific Immunoglobulin (Ig)G1, 3) interleukin-23 production, 4) airway mucus secretion-specific gene expression. RNAseq analysis of lungs indicates that neutrophil chemotaxis and oxidation-reduction processes were the representative genomic pathways in saline and OVA-challenged mice, respectively. Conclusions: A single exposure to goat farm BioPM enhanced airway inflammation in both saline and OVA-challenged allergic mice, with neutrophilic response as Th17 disorder and eosinophilic response as Th2 disorder indicative of the severity of allergic responses. Identification of the mode of action by which farm PM interacts with airway allergic pathways will be useful to design potential therapeutic approaches.
Project description:To profile airway and parenchyma transcriptomes in our mouse model of asthma chronic obstructive pulmonary disease overlap, we employed RNA-Seq (TruSeq Stranded mRNA sample preparation, NovaSeq 6000, 20 million 100 bp single reads) as a discovery tool to identify mRNAs of interest in the development of experimental asthma chronic obstructive pulmonary disease overlap. Experimental asthma: Some mice were sensitized and challenged intranasally with house dust mite (HDM) extract 5 days per week for 3 weeks. In other mice, this was HDM treatment 3 times/week from weeks 4-11. Controls received phosphate buffered saline (PBS). Experimental chronic obstructive pulmonary disease (COPD): Mice were exposed to cigarette smoke (CS) twice per day, 5 days per week for 8 weeks. Control mice were exposed to normal air (AIR). Experimental asthma-COPD overlap (ACO): Mice were sensitized and challenged intranasally with HDM or PBS. From weeks 4-11, mice were also exposed to CS or AIR. Treatment with dexamethasone (DEX): Separate groups of mice were treated with DEX during week 11. Lungs were excised, airway and parenchyma tissue were isolated through blunt dissection, and total RNA isolated.
Project description:Purpose: To test the in vivo effectiveness of a novel function-selective inhibitor of ERK1/2 in a murine model of house dust mite (HDM)-induced allergic asthma. Methods: Mice challenged with HDM for 3 weeks were treated with SF-3-030 or vehicle and asthma phenotype was assessed by histology and lung function measurements. Total RNA from lung samples were subjected to RNAseq analysis to determine the molecular signature perturbed by SF-3-030 treatment in mice. Results: RNA-seq analysis revealed modulation (up- or down-regulation) of expression of a number of genes by HDM challenge. SF-3-030 treatment significantly modulated the expression of genes involved in HDM-induced airway inflammation, cell proliferation, and extracellular matrix production. Conclusion: Function-selective inhibitor of ERK1/2 SF-3-030 mitigates multiple features of asthma in a murine model by modulating expression of genes involved in the regulation of key pathological functions in airway cells.
Project description:Allergic asthmatic, allergy only, asthma only (no allergy), and non-allergic non-asthmatic (control) subjects underwent bronchoscopy with instillation of saline, lipopolysaccharide (LPS), and house dust mite antigen in separate subsegmental bronchi. Bronchoalveolar lavage (BAL) fluid was collected four hours later (three samples per subject). Inflammatory cells from each specimen were isolated and RNA was extracted for microarray analysis. Experiment Overall Design: There are four main phenotypic groups: Experiment Overall Design: 1. control (no allergy or asthma) Experiment Overall Design: 2. allergy only (no asthma) Experiment Overall Design: 3. asthma only (no allergy) Experiment Overall Design: 4. allergy and asthma Experiment Overall Design: and three exposures: saline, house dust mite antigen (HDM), and LPS. Experiment Overall Design: Samples from the different exposures were all collected at the same time: four hours after instillation. The hybridizations were carried out in two main 'batches': samples in batch 1 were processed in mid 2004, samples in batch 2 about a year later in 2005. There is a clear 'batch effect': differences between expression profiles from the two batches (likely caused by technical differences between hybridization and scanning methods). This should be considered when analyzing the data.
Project description:Allergic asthmatic, allergy only, asthma only (no allergy), and non-allergic non-asthmatic (control) subjects underwent bronchoscopy with instillation of saline, lipopolysaccharide (LPS), and house dust mite antigen in separate subsegmental bronchi. Airway epithelial cells were collected four hours later (three samples per subject). RNA was extracted from these cells for microarray analysis. Experiment Overall Design: There are four main phenotypic groups: Experiment Overall Design: 1. control (no allergy or asthma) Experiment Overall Design: 2. allergy only (no asthma) Experiment Overall Design: 3. asthma only (no allergy) Experiment Overall Design: 4. allergy and asthma Experiment Overall Design: and three exposures: saline, house dust mite antigen (HDM), and LPS. Samples from the different exposures were all collected at the same time: four hours after instillation. The hybridizations were carried out in two main "batches": samples in batch 1 were processed in mid 2004, samples in batch 2 about a year later in 2005. There is a clear "batch effect": differences between expression profiles from the two batches (likely caused by technical differences between hybridization and scanning methods). This should be considered when analyzing the data.
Project description:Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in mouse models of asthma. Wild type and DDAH1-transgenic mice were challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. Gene expression in lungs was determined by RNA-Seq and RT-quantitative PCR (qPCR). The expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in serum and BAL fluid were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1 transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1 transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 in airway epithelial cells may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses. mRNA profiles of WT and DDAH1-transgenic mice treated with PBS or house dust mite (HDM).
Project description:Rationale: Lipopolysaccharide (LPS) is ubiquitous in the environment. Inhalation of LPS has been implicated in the pathogenesis and/or severity of several lung diseases, including pneumonia, chronic obstructive pulmonary disease and asthma. Alveolar macrophages are the main resident leukocytes exposed to inhaled antigens. Objectives: To obtain insight into which innate immune pathways become activated within human alveolar macrophages upon exposure to LPS in vivo. In seven healthy humans sterile saline was instilled into a lung segment by bronchoscope, followed by instillation of LPS into the contralateral lung. Six hours later a bilateral bronchoalveolar lavage was performed and whole-genome transcriptional profiling was done (Affymetrix HG-U133 Plus 2.0) on purified alveolar macrophages, comparing cells exposed to saline or LPS from the same individuals.
Project description:Objective and design: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. Methods: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. Results: Hz treatment improved survival outcomes after lethal challenge with LPS or CLPinduced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1β (IL-1β) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. Conclusion: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.