Project description:The mast cell-specific metalloprotease CPA3 has been given important roles in lung tissue homeostasis and disease pathogenesis. However, the dynamics and spatial distribution of mast cells CPA3 expression in lung diseases remains unknown. Methods: Using a histology-based approach for spatial and quantitative simultaneous decoding of mRNA and protein expression at a single cell level, this study investigates the dynamics of CPA3 expression across mast cells residing in lungs from healthy controls and patients with severe chronic obstructive pulmonary disease (COPD) or idiopathic lung fibrosis (IPF). Results: Mast cells in COPD lungs had increased CPA3 mRNA (bronchioles p<0.001, pulmonary vessels p< 0.01, alveolar parenchyma p< 0.01) compared to controls, while granule stored CPA3 protein was unaltered. IPF lungs had a significant upregulation of both CPA3 mRNA (p<0.001) and protein (p<0.05) in the fibrotic alveolar tissue. IPF was also characterized by highest density of distal lung mast cells. As an indication of disease relevant increased CPA3 turnover, spatial expression maps revealed altered mast cell mRNA/protein quotients in lung areas subjected to disease-relevant histopathological alterations. Single cell RNA sequencing of bronchial mast cells confirmed CPA3 as a top expressed gene with potential links to both inflammatory and protective markers. Conclusion: This study shows that lung tissue mast cell populations in COPD and IPF-affected lungs have spatially complex and markedly up-regulated CPA3 expression profiles that correlates with sites of structural pathologies. Given the proposed roles of CPA3 in tissue homeostasis, remodeling and inflammation, these alterations are likely to have clinical consequences.
Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated. In brief, congestive heart failure (CHF) was induced in juvenile rats by supracoronary aortic banding and rats were analyzed 9 weeks thereafter when they had established left heart failure with preserved ejection fraction and secondary pulmonary hypertension. Three heart failure rats were untreated, and 3 received the mast cell stabilizer ketotifen (1 mg/kg-1 bodyweight/day-1) with the drinking water. Sham rats underwent the same surgical procedure but without placement of a clip on the supracoronary aorta. Microarray analyses: Lungs from banded and control rats were excised and total RNA was extracted (Stratagene Absolutely RNA Miniprep Kit; Stratagene, La Jolla, USA). Three µg total RNA from three control and three banded rats each were processed according to the One-Cycle Target Labeling protocol (GeneChip Expression Analysis, Affymetrix, Santa Clara, USA). Before and after amplifications, the total RNA/complementary RNA concentrations were checked with NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA) and quality was controlled (Experion electrophoresis station, BioRad; Hercules, USA). Samples were hybridized to GeneChip® Rat Genome 230 2.0 arrays (Affymetrix; Santa Clara, USA) containing 31,000 probe sets covering 28,000 rat genes.
Project description:We are interested in comparing expression patterns of hematopoletic stem cells, mast cell precursors and mature mast cells. Our group recently reported that murine mast cells express CD34, Sca-1 and c-kit. Microarray analysis may uncover other novel surface antigens useful in separating mast cells from stem cells. Keywords: other
Project description:Type 2 immune responses are crucial for protection against helminth infections and for the maintenance of tissue homeostasis, yet the underlying mechanisms of immune activation remain incompletely understood. Here, we study the role of mast cells in type 2 immunity. While the number of lamina propria residing mast cells (termed LPMCs hereinafter) remained stable, intraepithelial mast cells (termed IEMCs hereinafter) markedly expanded during the course of type 2 responses. We uncovered a new type 2 response mechanism depending on the crosstalk between mast cells and epithelial cells, and defined the function of intraepithelial mast cells.
Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated.