Project description:Background and purposeInhaled corticosteroids (ICS) are the cornerstone of asthma pharmacotherapy and, acting via the glucocorticoid receptor (GR), reduce inflammatory gene expression. While this is often attributed to a direct inhibitory effect of the GR on inflammatory gene transcription, corticosteroids also induce the expression of anti-inflammatory genes in vitro. As there are no data to support this effect in asthmatic subjects taking ICS, we have assessed whether ICS induce anti-inflammatory gene expression in subjects with atopic asthma.Experimental approachBronchial biopsies from allergen-challenged atopic asthmatic subjects taking inhaled budesonide or placebo were subjected to gene expression analysis using real-time reverse transcriptase-PCR for the corticosteroid-inducible genes (official gene symbols with aliases in parentheses): TSC22D3 [glucocorticoid-induced leucine zipper (GILZ)], dual-specificity phosphatase-1 (MAPK phosphatase-1), both anti-inflammatory effectors, and FKBP5 [FK506-binding protein 51 (FKBP51)], a regulator of GR function. Cultured pulmonary epithelial and smooth muscle cells were also treated with corticosteroids before gene expression analysis.Key resultsCompared with placebo, GILZ and FKBP51 mRNA expression was significantly elevated in budesonide-treated subjects. Budesonide also increased GILZ expression in human epithelial and smooth muscle cells in culture. Immunostaining of bronchial biopsies revealed GILZ expression in the airways epithelium and smooth muscle of asthmatic subjects.Conclusions and implicationsExpression of the corticosteroid-induced genes, GILZ and FKBP51, is up-regulated in the airways of allergen-challenged asthmatic subjects taking inhaled budesonide. Consequently, the biological effects of corticosteroid-induced genes should be considered when assessing the actions of ICS. Treatment modalities that increase or decrease GR-dependent transcription may correspondingly affect corticosteroid efficacy.

Project description:BackgroundMicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level. While they have been implicated in various diseases, the profile changes in allergen inhalation challenge are not clarified in human. We aimed to evaluate changes in the microRNA profiles in the peripheral blood of asthmatic subjects undergoing allergen inhalation challenge.ResultsSeven mild asthmatic subjects participated in the allergen inhalation challenge. In addition, four healthy control subjects (HCs) were recruited. MicroRNA profiles in peripheral blood samples (pre-challenge and 2 hours post-challenge) were measured by the NanoString nCounter assay to determine changes in miRNA levels as these asthmatic subjects underwent an allergen inhalation challenge. One common miRNA, miR-192, was significantly expressed in both comparisons; HCs vs. pre-challenge and pre- vs. post-challenge, showing that miR-192 was significantly under-expressed in asthmatics compared to HCs and decreased in post-challenge at an FDR of 1%. Cell-specific statistical deconvolution attributed miR-192 expression in whole blood to PBMCs. MiR-192 was technically validated using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showing that the level in asthmatics (pre-challenge) was significantly lower than HCs and that post-challenge was significantly lower than pre-challenge. The normalized relative miR-192 expression quantified using RT-qPCR specific to PBMCs was also validated. Ontology enrichment and canonical pathway analyses for target genes suggested several functions and pathways involved in immune response and cell cycle.ConclusionsThe miRNA profile in peripheral blood was altered after allergen inhalation challenge. Change in miR-192 levels may be implicated in asthma mechanisms. These results suggest that allergen inhalation challenge is a suitable method to characterize peripheral miRNA profiles and potentially elucidate the mechanism of human asthma.

Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene.

Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene. 26 array were analyzed. We first identified differentially expreed genes between post- and pre-challenge samples from 4 asthmatic subjects by using globin reduction samples and non-globin reduction samples (16 arrays). Then we replicated our findings using 10 arrays from 5 asthmatic subjects challenged with allergen. Includes 4 normal blood samples.

Project description:IL-10-differentiated dendritic cells (DC10) can reverse the asthma phenotype in mice, but how they suppress the asthmatic B cell response is unclear. Herein we assessed the mechanism(s) by which DC10 and DC10-induced Treg affect IgG1 production in asthma. We observed a rapid decline in lung-resident OVA-specific IgG1-secreting B cells on cessation of airway allergen challenge, and intraperitoneal DC10 therapy did not amplify that (p>0.05). It did however increase the loss of IgG1-B cells from the bone marrow (by 45+/-7.2%; p≤0.01) and spleen (by 65+/-17.8%; p≤0.05) over 2 wk. Delivery of OVA-loaded DC10 directly into the airways of asthmatic mice decreased the lung IgG1 B cell response assessed 2 dy later by 33+/-9.7% (p≤0.01), while their co-culture with asthmatic lung cell suspensions reduced the numbers of IgG1-secreting cells by 56.5+/-9.7% (p≤0.01). This effect was dependent on the DC10 carrying intact allergen on their cell surface; DC10 that had phagocytosed and fully processed their allergen were unable to suppress B cell responses, although they did suppress asthmatic Th2 cell responses. We had shown that therapeutic delivery of DC10-induced Treg can effectively suppress asthmatic T and B cell (IgE and IgG1) responses; herein CD4+ cells or Treg from the lungs of DC10-treated OVA-asthmatic mice suppressed in vitro B cell IgG1 production by 52.2+/-8.7% (p≤0.001) or 44.6+/-12.2% (p≤0.05), respectively, but delivery of DC10-induced Treg directly into the airways of asthmatic mice had no discernible impact over 2 dy on the numbers of lung IgG1-secreting cells (p≥0.05). In summary, DC10 treatment down-regulates OVA-specific B cell responses of asthmatic mice. While DC10 that carry intact allergen on their cell surface can dampen this response, DC10-induced Treg are critical for full realization of this outcome. This suggests that infectious tolerance is an essential element in regulatory DC control of the B cell response in allergic asthma.

Dataset Information

Gene-metabolite expression in blood can discriminate allergen-induced isolated early from dual asthmatic responses.

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Gene-metabolite expression in blood can discriminate allergen-induced isolated early from dual asthmatic responses.

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