Identification of asthma associated microRNAs in bronchial biopsies

Background Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. Methods Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. Results In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. Conclusion Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma

Gene signatures from scRNA-seq accurately quantify mast cells in biopsies in asthma

Respiratory disease, characterized by changes in the cells of the lung, can affect molecular phenotype of cells and the intercellular interactions, resulting in a disbalance in the relative proportions of individual cell types. Understanding these changes is essential to understand the pathophysiology of lung disease. Conventional 'bulk' RNA-sequencing (RNA-seq), analyzing the entire transcriptome of the tissue sample, provides information about average expression levels of each gene in the mixed cell population; whereas it does not consider the cellular heterogeneity in samples composed of more than one cell type 1 . Single-cell RNA-seq (scRNA-seq) assesses the transcriptome of a complex biological sample with single-cell resolution, allowing identification of the relative frequency of discrete cell-types and analysis of their transcriptomes 1 . Nevertheless, analyzing the transcriptomic signature in large numbers of patients by scRNA-Seq is currently limited by its high costs. Mast cells are key regulatory cells driving the inflammatory process in asthma2 . Since they can be quantified by immunohistochemical staining for validation purposes, we used mast cells as an example of a rare cell population to assess the validity of our deconvolution approach. Recently, a number of bulk RNA-seq deconvolution methods have become available 3 , for instance of two deconvolution methods, namely support vector regression (SVR) 4 , the machine-learning method implemented in CYBERSORT, and Non-Negative Least Square (NNLS) 5 , using a matrix of cell-type selective genes identified with AutoGeneSc 6 . Both approaches are designed to estimate relative proportion of the main, common cell types present in the sample. When we used these methods to estimate the number of mast cells, we found a poor correlation with the number of mast cells stained by immunohistochemistry in the biopsies, suggesting the CIBERSORT and NNLS are less reliable in the case of rare cell types. We explored the possibility to use scRNA-Seq data from small numbers of subjects to specifically interrogate the relative cell type frequency of a rare cell population in a bulk RNA-Seq dataset obtained from a large asthma cohort

Cholinergic neuroplasticity in asthma driven by TrkB signaling

Parasympathetic neurons in the airways control bronchomotor tone. Increased activity of cholinergic neurons are mediators of airway hyperresponsiveness (AHR) in asthma, however, mechanisms are not elucidated. We describe remodeling of the cholinergic neuronal network in asthmatic airways driven by brain-derived neurotrophic factor (BDNF) and Tropomyosin receptor kinase B (TrkB). Human bronchial biopsies were stained for cholinergic marker vesicular acetylcholine transporter (VAChT). Human lung gene expression and single nucleotide polymorphisms (SNP) in neuroplasticity-related genes were compared between asthma and healthy patients. Wild-type (WT) and mutated TrkB knock-in mice (Ntrk2tm1Ddg/J) with impaired BDNF signaling were chronically exposed to ovalbumin (OVA). Neuronal VAChT staining and airway narrowing in response to electrical field stimulation in precision cut lung slices (PCLS) were assessed. Increased cholinergic fibers in asthmatic airway biopsies was found, paralleled by increased TrkB gene expression in human lung tissue, and SNPs in the NTRK2 [TrkB] and BDNF genes linked to asthma. Chronic allergen exposure in mice resulted in increased density of cholinergic nerves, which was prevented by inhibiting TrkB. Increased nerve density resulted in AHR in vivo and in increased nerve-dependent airway reactivity in lung slices mediated via TrkB. These findings show cholinergic neuroplasticity in asthma driven by TrkB signaling and suggest that the BDNF-TrkB pathway may be a potential target

QTL mapping of inbreeding-related cold sensitivity and conditional lethality in <i>Drosophila melanogaster</i>

Inbreeding depression is a central theme within genetics, and is of specific interest for researchers within evolutionary and conservation genetics and animal and plant breeding. Inbreeding effects are thought to be caused by the joint expression of conditional and unconditional deleterious alleles. Whenever the expression of deleterious alleles is conditional, this can result in extreme environmental sensitivity in certain inbred lineages. Analysis of conditional lethal effects can reveal some of the loci that are sensitive to inbreeding. We performed a QTL (quantitative trait locus) mapping study of inbreeding-related and conditionally expressed lethality in Drosophila melanogaster. The lethal effect was triggered by exposure to a cold shock. We used a North Carolina crossing Design 3 to establish the mapping population, as well as to estimate the average dominance ratio and heritability. We found two QTL on the major autosomes carrying recessive lethals that caused male mortality, one of which also affected female mortality. More detailed study of these loci will provide information on the mechanistic basis and environmental sensitivity of inbreeding depression

Profiling of healthy and asthmatic airway smooth muscle cells following interleukin-1 beta treatment:A novel role for CCL20 in chronic mucus hypersecretion

Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroidresistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1 beta, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1 beta-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitro. IL-1 beta-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air liquid interface (AM-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed. The IL-1 beta-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls. 1L-10 induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients. Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the antiinflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma