Project description:This protocol outlines a single-site mechanistic study aiming to investigate long RNAs differentially expressed in the airway epithelium of asthma patients both at baseline and in response to segmental airway allergen challenges. Over approximately 14 days, the study spanned three visits: Visit 1: Comprehensive characterization of participants, encompassing lung function testing, methacholine challenge testing, and allergen skin prick testing. Visit 2: Participants underwent bronchoscopy wherein three procedures were performed a. Epithelial brushings were performed in a segmental airway (baseline sample) b. Diluent (inactive control) was instilled into another segmental airway c. A small dose of allergen was administered into a third segmental airway using standardized cat or dust mite allergen extracts. Visit 3 (24 hours or 7 days post Visit 2): Another bronchoscopy was carried out to collect epithelial brushings in the diluent challenged and allergen challenged segments The collected epithelial brush samples underwent analysis for mRNA expression in the epithelial brushings. The study successfully incorporated a total of 23 subjects, which included 18 asthmatics (with stable or well-controlled conditions), 2 allergic non-asthmatics, and 3 non-allergic non-asthmatics.
Project description:Allergen exposure induces the airway epithelium to produce chemoattractants, proallergic interleukins, matrix-modifying proteins, and proteins that influence the growth and activation state of airway structural cells. These proteins, in turn, contribute to the influx of inflammatory cells and changes in structure that characterize the asthmatic airway. To use the response of the airway epithelium to allergen to identify genes not previously associated with allergic responses, we compared gene expression in cytokeratin-positive cells before and after segmental allergen challenge. After challenge with concentrations of allergen in the clinically relevant range, 755 (6%) of the detectable sequences had geometric mean fold-changes in expression, with 95% confidence intervals that excluded unity. Using a prospectively defined conservative filtering algorithm, we identified 141 sequences as upregulated and eight as downregulated, with confirmation by conventional polymerase chain reaction in all 10 sequences studied. Using this approach, we identified asthma-associated sequences including interleukin (IL-)-3, IL-4, and IL-5 receptor subunits, the p65 component of nuclear factor-kappaB, and lipocortin. The genomic response of the human airway to concentrations of allergen in the clinically relevant range involves a greater number of genes than previously recognized, including many not previously associated with asthma that are differentially expressed after airway allergen exposure. Keywords: Before and after allergen challenge
Project description:Using a human model of asthma exacerbation, we compared the airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA-sequencing frameworks. In response to allergen challenge, the airway epithelium in asthmatics was highly dynamic and upregulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls. Asthmatics also had a unique mucosal immune profile, characterized by IL9-expressing pathogenic TH2 cells and enrichment of DC2 (CD1C) and CCR2-expressing monocyte-derived cells (MC) after allergen, with upregulation of genes that promote pathologic airway remodeling. In contrast, controls were enriched for macrophage-like MC that upregulated tissue repair programs after allergen challenge, suggesting these populations may protect against asthmatic airway remodeling. These findings reveal a novel TH2-mononuclear phagocyte-epithelial interactome unique to asthmatics, suggesting that pathogenic effector circuits and the absence of pro-resolution programs drive structural airway disease in response to type 2 inflammation.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Allergen challenge induced mucus metaplasia modify the expression of two transcription factors belonging to the FOXA family: FOXA2 and FOXA3. Foxa2 expression is decreased during allergic airway disease whereas, Foxa3 expression is increased by allergen. Therefore, we asked whether persistent expression of Foxa2 prevents mucus and whether absence of Foxa3 affects mucus or other features asociated with allergic airway disease. We analyzed the effects of these changes in FOXA transcription factor expression using Foxa2 transgenic mice and Foxa3-/- mice. We found that persistent expression of FOXA2 reduced mucus but the absence of FOXA3 had no effect on mucus production induced by allergen challenge. However, the absence of FOXA3 decreased airway hyperreactivity and increased IgE production and eosinophilic inflammation but none of these features were affected by persistent expression of FOXA2. These results indicate that FOXA3 has functions distinct from those of FOXA2 in the allergic response. Keywords: gene expression comparison between Foxa3-/- and littermate control mice both challenged with OVA
Project description:We have developed a new model of the human airway epithelial cell by deriving the cell-specific metabolic reactions identified from (i) a draft automated model by Wang et al. 2017 (ii) gene expression datasets of the human airway epithelial cell (Deprez et al., 2020; Braga et al., 2020). (iii) We obtained additional reactions, gene-to-reaction associations and pathways (that were not in the automated model) from HumanCyc (Trupp et al., 2010) and (iv) performed stochastic and dynamic simulations on the model generated including manual curations from primary literature and Recon3D (Brunk et al., 2018). (v) We added the viral biomass maintenance function into the model, previously developed for the macrophage cell (Renz et al. 2020) to develop the new integrated model of the human airway epithelial cell and the SARS-CoV-2 virus, (iBBEC4660).