Shot Down Inflamed: Airway Smooth Muscle Bronchodilator Insensitivity in Cystic Fibrosis.
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ABSTRACT:
SUBMITTER: Pascoe CD
PROVIDER: S-EPMC6444628 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
Publications
Shot Down Inflamed: Airway Smooth Muscle Bronchodilator Insensitivity in Cystic Fibrosis.
Pascoe Christopher D CD Halayko Andrew J AJ
American journal of respiratory cell and molecular biology 20190401 4
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Project description:BackgroundPatients with severe asthma are less responsive to the beneficial effects of corticosteroid therapy.ObjectiveWe investigated whether corticosteroid insensitivity was present in airway smooth muscle cells (ASMCs) of patients with severe asthma.MethodsASMCs cultured from bronchial biopsy specimens of nonasthmatic control subjects (n = 12) and patients with nonsevere (n = 10) or severe (n = 10) asthma were compared for the effect of dexamethasone on suppression of TNF-α- and IFN-γ-induced CCL11 (eotaxin), CXCL8 (IL-8), and CX3CL1 (fractalkine) expression. The mechanisms of corticosteroid insensitivity are also determined.ResultsCCL11 release was higher in ASMCs of patients with nonsevere but not severe asthma and nonasthmatic control subjects; CXCL8 and CX3CL1 release were similar in all groups. In patients with severe asthma, dexamethasone caused less suppression of CCL11 and CXCL8 release induced by TNF-α. Dexamethasone potentiated TNF-α- and IFN-γ-induced CX3CL1 release equally in all 3 groups. TNF-α-induced phosphorylated p38 mitogen-activated protein kinase levels were increased in ASMCs from patients with severe asthma compared with those from patients with nonsevere asthma and nonasthmatic subjects, whereas TNF-α-induced phosphorylated c-Jun N-terminal kinase and phosphorylated extracellular signal-related kinase levels were increased in all asthmatic groups. A p38 inhibitor increased the inhibitory effect of dexamethasone.ConclusionsASMCs of patients with severe asthma are corticosteroid insensitive; this might be secondary to heightened p38 mitogen-activated protein kinase levels.
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Project description:Bitter taste receptors (TAS2Rs) are recognized as being expressed on multiple cell types and organs, including human airway smooth muscle (HASM) cells, where agonists promote significant relaxation to constrictive stimuli. Thus, the HASM TAS2Rs have been targeted as novel bronchodilators for the treatment of asthma and other obstructive lung diseases. The TAS2R5 subtype, a dominant receptor on HASM, has few known agonists, all with reported low potency and efficacy. We screened multiple compounds by measuring [Ca2+]i release in HASM (a consequence of receptor-G protein coupling) to establish structure-activity relationships and arrive at a potent agonist for TAS2R5. HASM physiological studies using magnetic twisting cytometry confirmed the relaxation effects of lead compounds. 1,10-Phenanthroline-5,6-dione had the greatest potency (EC50 ≈ 120 nM), amounting to a >1000-fold improvement over the other compounds, and displayed maximal efficacy. These studies revealed critical structural requirements for favorable potencies and efficacies for a potential first-in-class bronchodilator targeting TAS2R5 of the airway.
| S-EPMC7737212 | biostudies-literature
Project description:The mechanisms driving glucocorticoid (GC) insensitivity in patients with severe asthma are still unknown. Recent evidence suggests the existence of GC-insensitive pathways in airway smooth muscle (ASM) caused by a defect in GC receptor (GR?) function. We examined whether other mechanisms could potentially explain the reduced sensitivity of ASM cells to GC in severe asthmatics.Airway smooth muscle cells from healthy and severe asthmatic subjects were treated with TNF-? and responses to corticosteroids in both cohorts were compared by ELISA, immunoblot, immunohistochemistry and real-time PCR. Immunohistochemistry and flow cytometry assays were used to assess the expression of the protein phosphatase PP5 in endobronchial biopsies and ASM cells.The production of CCL11 and CCL5 by TNF-? was insensitive to both fluticasone and dexamethasone in ASM cells from severe asthmatic compared to that in healthy subjects. Fluticasone-induced GR? nuclear translocation, phosphorylation at serine 211 and expression of GC-induced leucine zipper (GILZ) were significantly reduced in ASM cells from severe asthmatics compared to responses in healthy subjects. Levels of PP5 were increased in ASM cells from severe asthmatics and PP5 knockdown using siRNA restored fluticasone repressive action on chemokine production and its ability to induce GR? nuclear translocation and GRE-dependent GILZ expression. In vivo PP5 expression was also increased in the ASM bundles in endobronchial biopsies in severe asthmatics.PP5-dependent impairment of GR? function represents a novel mechanism driving GC insensitivity in ASM in severe asthma.
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Project description:BackgroundCorticosteroids remain a key therapy for treating children with asthma. Patients with severe asthma are insensitive, resistant, or refractory to corticosteroids and have poorly controlled symptoms that involve airway inflammation, airflow obstruction, and frequent exacerbations. While the pathways that mediate corticosteroid insensitivity in asthma remain poorly defined, recent studies suggest that enhanced Th1 pathways, mediated by TNFα and IFNγ, may play a role. We previously reported that the combined effects of TNFα and IFNγ promote corticosteroid insensitivity in developing human airway smooth muscle (ASM).MethodsTo further understand the effects of TNFα and IFNγ on corticosteroid sensitivity in the context of neonatal and pediatric asthma, we performed RNA sequencing (RNA-seq) on human pediatric ASM treated with fluticasone propionate (FP), TNFα, and/or IFNγ.ResultsWe found that TNFα had a greater effect on gene expression (~ 1000 differentially expressed genes) than IFNγ (~ 500 differentially expressed genes). Pathway and transcription factor analyses revealed enrichment of several pro-inflammatory responses and signaling pathways. Interestingly, treatment with TNFα and IFNγ augmented gene expression with more than 4000 differentially expressed genes. Effects of TNFα and IFNγ enhanced several pro-inflammatory genes and pathways related to ASM and its contributions to asthma pathogenesis, which persisted in the presence of corticosteroids. Co-expression analysis revealed several gene networks related to TNFα- and IFNγ-mediated signaling, pro-inflammatory mediator production, and smooth muscle contractility. Many of the co-expression network hubs were associated with genes that are insensitive to corticosteroids.ConclusionsTogether, these novel studies show the combined effects of TNFα and IFNγ on pediatric ASM and implicate Th1-associated cytokines in promoting ASM inflammation and hypercontractility in severe asthma.
| S-EPMC9109364 | biostudies-literature
Project description:BackgroundThe prevalence of fungal disease in cystic fibrosis (CF) and non-CF bronchiectasis is increasing and the clinical spectrum is widening. Poor sensitivity and a lack of standard diagnostic criteria renders interpretation of culture results challenging. In order to develop effective management strategies, a more accurate and comprehensive understanding of the airways fungal microbiome is required. The study aimed to use DNA sequences from sputum to assess the load and diversity of fungi in adults with CF and non-CF bronchiectasis.MethodsNext generation sequencing of the ITS2 region was used to examine fungal community composition (n = 176) by disease and underlying clinical subgroups including allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, non-tuberculous mycobacteria, and fungal bronchitis. Patients with no known active fungal disease were included as disease controls.ResultsITS2 sequencing greatly increased the detection of fungi from sputum. In patients with CF fungal diversity was lower, while burden was higher than those with non-CF bronchiectasis. The most common operational taxonomic unit (OTU) in patients with CF was Candida parapsilosis (20.4%), whereas in non-CF bronchiectasis sputum Candida albicans (21.8%) was most common. CF patients with overt fungal bronchitis were dominated by Aspergillus spp., Exophiala spp., Candida parapsilosis or Scedosporium spp.ConclusionThis study provides a framework to more accurately characterize the extended spectrum of fungal airways diseases in adult suppurative lung diseases.
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Project description:Communication between the airway epithelium and stroma is evident during embryogenesis, and both epithelial shedding and increased smooth muscle proliferation are features of airway remodeling. Hence, we hypothesized that after injury the airway epithelium could modulate airway smooth muscle proliferation. Fully differentiated primary normal human bronchial epithelial (NHBE) cells at an air-liquid interface were co-cultured with serum-deprived normal primary human airway smooth muscle cells (HASM) using commercially available Transwells. In some co-cultures, the NHBE were repeatedly (x4) scrape-injured. An in vivo model of tracheal injury consisted of gently denuding the tracheal epithelium (x3) of a rabbit over 5 days and then examining the trachea by histology 3 days after the last injury. Our results show that HASM cell number increases 2.5-fold in the presence of NHBE, and 4.3-fold in the presence of injured NHBE compared with HASM alone after 8 days of in vitro co-culture. In addition, IL-6, IL-8, monocyte chemotactic protein (MCP)-1 and, more markedly, matrix metalloproteinase (MMP)-9 concentration increased in co-culture correlating with enhanced HASM growth. Inhibiting MMP-9 release significantly attenuated the NHBE-dependent HASM proliferation in co-culture. In vivo, the injured rabbit trachea demonstrated proliferation in the smooth muscle (trachealis) region and significant MMP-9 staining, which was absent in the uninjured control. The airway epithelium modulates smooth muscle cell proliferation via a mechanism that involves secretion of soluble mediators including potential smooth muscle mitogens such as IL-6, IL-8, and MCP-1, but also through a novel MMP-9-dependent mechanism.
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Project description:Our laboratory has held a long interest in the glycosylation changes seen on the surface of airway epithelia of patients with the disease cystic fibrosis (CF). Experiments from our laboratory have detailed a CF glycosylation phenotype of increased Fuca1,3/4 and decreased Fuca1,2 and sialic acid on the surfaces of immortalized and primary CF cells compared to non-CF cells. Further, we have shown that gene transfer and subsequent expression of a wild type CF plasmid in CF airway cells results in correction or reversal of this glycosylation phenotype. We hypothesize that the changes in glycosylation seen in CF cells are key in the pathophysiology of the cystic fibrosis airway disease. For example, it has been shown that Pseudomonas aeruginosa, a bacterium that has a predilection for colonizing CF airways, adheres to asialylated glycolipids and glycoconjugates with terminal Fuca1,3/4. One focus of our laboratory is to elucidate the etiology of the glycosylation changes seen in CF cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. We propose to study the gene expression of immortalized and primary CF and non-CF airway epithelial cells: 1. CF/T43 vs. BEAS-2B cells. These are two widely used immortalized airway cell lines that we have used extensively in the past. 2. C38 cells; C38 cells are IB3 cells expressing wtCFTR. The experimental focus is to elucidate the etiology of the glycosylation changes seen in Cystic Fibrosis (CF) cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. To do so, the gene expression of immortalized and primary CF and non-CF airway epithelial cells were compared and studied. Cell lines used were CF/T43 and BEAS-2B, both widely used immortalized airway cell lines. Other cell lines studied included C38 cell lines (clonal derivatives of IB3 cells expressing wtCFTR).
2011-01-07 | E-GEOD-26482 | biostudies-arrayexpress
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Project description:Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.
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Project description:Defining mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory in response to mechanical and environmental stress is crucial for determining the contribution of airway smooth muscle (ASM) to inflammatory responses that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype, but little is known about the expression and functions of miRNA in smooth muscle. In the present study we used microarrays to determine whether miRNAs in human ASM cells are altered by a proinflammatory stimulus. In ASM cells exposed to IL-1beta, TNF-alpha, and IFN-gamma, we found 11 miRNAs to be significantly down-regulated. We verified decreased expression of miR-25, miR-140*, mir-188, and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin, and TNF-alpha; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle-specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.
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