Nasal airway transcriptome-wide association study of asthma reveals genetically driven mucus pathobiology.

To identify genetic determinants of airway dysfunction, we performed a transcriptome-wide association study for asthma by combining RNA-seq data from the nasal airway epithelium of 681 children, with UK Biobank genetic association data. Our airway analysis identified 95 asthma genes, 58 of which were not identified by transcriptome-wide association analyses using other asthma-relevant tissues. Among these genes were MUC5AC, an airway mucin, and FOXA3, a transcriptional driver of mucus metaplasia. Muco-ciliary epithelial cultures from genotyped donors revealed that the MUC5AC risk variant increases MUC5AC protein secretion and mucus secretory cell frequency. Airway transcriptome-wide association analyses for mucus production and chronic cough also identified MUC5AC. These cis-expression variants were associated with trans effects on expression; the MUC5AC variant was associated with upregulation of non-inflammatory mucus secretory network genes, while the FOXA3 variant was associated with upregulation of type-2 inflammation-induced mucus-metaplasia pathway genes. Our results reveal genetic mechanisms of airway mucus pathobiology.

Type 2 and interferon inflammation regulate SARS-CoV-2 entry factor expression in the airway epithelium.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

Single-Cell and Population Transcriptomics Reveal Pan-epithelial Remodeling in Type 2-High Asthma.

The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.

Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium.

Coronavirus disease 2019 (COVID-19) outcomes vary from asymptomatic infection to death. This disparity may reflect different airway levels of the SARS-CoV-2 receptor, ACE2, and the spike protein activator, TMPRSS2. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci (eQTL) for both ACE2 and TMPRSS2, that vary in frequency across world populations. Importantly, we find TMPRSS2 is part of a mucus secretory network, highly upregulated by T2 inflammation through the action of interleukin-13, and that interferon response to respiratory viruses highly upregulates ACE2 expression. Finally, we define airway responses to coronavirus infections in children, finding that these infections upregulate IL6 while also stimulating a more pronounced cytotoxic immune response relative to other respiratory viruses. Our results reveal mechanisms likely influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.

Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium.

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.

COVID-19-related Genes in Sputum Cells in Asthma. Relationship to Demographic Features and Corticosteroids.

Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

Genome-Wide Analysis Reveals Mucociliary Remodeling of the Nasal Airway Epithelium Induced by Urban PM2.5.

Air pollution particulate matter <2.5 µm (PM2.5) exposure is associated with poor respiratory outcomes. Mechanisms underlying PM2.5-induced lung pathobiology are poorly understood but likely involve cellular and molecular changes to the airway epithelium. We extracted and chemically characterized the organic and water-soluble components of air pollution PM2.5 samples, then determined the whole transcriptome response of human nasal mucociliary airway epithelial cultures to a dose series of PM2.5 extracts. We found that PM2.5 organic extract (OE), but not water-soluble extract, elicited a potent, dose-dependent transcriptomic response from the mucociliary epithelium. Exposure to a moderate OE dose modified the expression of 424 genes, including activation of aryl hydrocarbon receptor signaling and an IL-1 inflammatory program. We generated an OE-response gene network defined by eight functional enrichment groups, which exhibited high connectivity through CYP1A1, IL1A, and IL1B. This OE exposure also robustly activated a mucus secretory expression program (>100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF and FOXA3). Exposure to a higher OE dose modified the expression of 1,240 genes and further exacerbated expression responses observed at the moderate dose, including the mucus secretory program. Moreover, the higher OE dose significantly increased the MUC5AC/MUC5B gel-forming mucin expression ratio and strongly downregulated ciliated cell expression programs, including key ciliating cell transcription factors (e.g., FOXJ1 and MCIDAS). Chronic OE stimulation induced mucus metaplasia-like remodeling characterized by increases in MUC5AC+ secretory cells and MUC5AC mucus secretions. This epithelial remodeling may underlie poor respiratory outcomes associated with high PM2.5 exposure.

IL1RL1 asthma risk variants regulate airway type 2 inflammation.

Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 - rs1420101 and rs11685480 - are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33-driven type 2 inflammation.

Alternative splicing of interleukin-33 and type 2 inflammation in asthma.

Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an "alarmin" during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.