A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

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.

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.

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.

Expression and function of the ectopic olfactory receptor OR10G7 in patients with atopic dermatitis.

BACKGROUND: Ectopic olfactory receptors (ORs) are found in the skin, but their expression and biological function in normal skin and skin form patients with atopic dermatitis (AD) are unknown. OBJECTIVES: We sought to characterize the expression of ORs in the skin and assess OR-mediated biological responses of primary human keratinocytes in the presence of odorant ligands. METHODS: OR expression was examined by using whole-transcriptome sequencing of skin tape strips collected from patients with AD and healthy control (HC) subjects. OR10G7 and filaggrin 1 (FLG-1) expression was analyzed by using RT-PCR and immunostaining in skin biopsy specimens and primary human keratinocytes from patients with AD and HC subjects. ATP and cyclic AMP production by control and OR10G7 small interfering RNA-transfected keratinocytes in response to odorant stimulation with acetophenone and eugenol was assessed. RESULTS: A total of 381 OR gene transcripts were detected in the skin samples, with the greatest OR expression detected in the skin tape strips corresponding to the upper granular layer of the skin. OR10G7 expression was significantly increased in skin biopsy specimens from patients with AD compared with those from HC subjects (P = .01) and inversely correlated with FLG-1 expression (P = .009). OR10G7 expression was greatest in undifferentiated keratinocytes from patients with AD and was downregulated with progressive differentiation. Primary human keratinocytes produced ATP, an essential neurotransmitter in sensory pathways, in response to acetophenone and eugenol, odorants previously identified as potential ligands for this receptor. This response was abolished in OR10G7 small interfering RNA-transfected keratinocytes. CONCLUSIONS: OR10G7 is expressed at significantly greater levels in undifferentiated keratinocytes from patients with AD compared with HC subjects. OR10G7 is likely involved in transmission of skin-induced chemosensory responses to odorant stimulation, which might modulate differential nociceptive responses in AD skin.

Utilization of Air-Liquid Interface Cultures as an In Vitro Model to Assess Primary Airway Epithelial Cell Responses to the Type 2 Cytokine Interleukin-13.

The airway epithelium lines the respiratory tract and provides the primary protective barrier against inhalational insults including toxic environmental substances and microorganisms. The airway epithelium also plays a critical role in regulating airway immune responses. The airway epithelial response to the type 2 cytokine, interleukin-13 (IL-13), is critical to airway inflammation, mucus production, and airway hyperresponsiveness present in asthma. Relevant primary cell models of the human airway epithelium are needed to investigate the biology of IL-13-mediated airway epithelial effects. Here, we describe the generation of a differentiated mucociliary human airway epithelium using an in vitro air-liquid interface (ALI) culture model system. We also describe methods to stimulate this culture model with IL-13 and harvest cells and biomolecules to interrogate cellular and molecular aspects of the airway epithelial IL-13 response.

Lipid abnormalities in atopic skin are driven by type 2 cytokines.

Lipids in the stratum corneum of atopic dermatitis (AD) patients differ substantially in composition from healthy subjects. We hypothesized that hyperactivated type 2 immune response alters AD skin lipid metabolism. We have analyzed stratum corneum lipids from nonlesional and lesional skin of AD subjects and IL-13 skin-specific Tg mice. We also directly examined the effects of IL-4/IL-13 on human keratinocytes in vitro. Mass spectrometric analysis of lesional stratum corneum from AD subjects and IL-13 Tg mice revealed an increased proportion of short-chain (N-14:0 to N-24:0) NS ceramides, sphingomyelins, and 14:0-22:0 lysophosphatidylcholines (14:0-22:0 LPC) with a simultaneous decline in the proportion of corresponding long-chain species (N-26:0 to N-32:0 sphingolipids and 24:0-30:0 LPC) when compared with healthy controls. An increase in short-chain LPC species was also observed in nonlesional AD skin. Similar changes were observed in IL-4/IL-13-driven responses in Ca2+-differentiated human keratinocytes in vitro, all being blocked by STAT6 silencing with siRNA. RNA sequencing analysis performed on stratum corneum of AD as compared with healthy subjects identified decreased expression of fatty acid elongases ELOVL3 and ELOVL6 that contributed to observed changes in atopic skin lipids. IL-4/IL-13 also inhibited ELOVL3 and ELOVL6 expression in keratinocyte cultures in a STAT6-dependent manner. Downregulation of ELOVL3/ELOVL6 expression in keratinocytes by siRNA decreased the proportion of long-chain fatty acids globally and in sphingolipids. Thus, our data strongly support the pathogenic role of type 2 immune activation in AD skin lipid metabolism.

Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.

The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2-high atopic dermatitis disease endotype.

BACKGROUND: Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual-level AD pathobiological mechanisms (endotypes) in large research studies. OBJECTIVE: We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. METHODS: We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean ± SE], 36.3 ± 2.2 years) and healthy control subjects (9 male and 16 female subjects; age [mean ± SE], 34.8 ± 2.2 years). AmpliSeq whole-transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. RESULTS: Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis-related genes (false discovery rate < 5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2-high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change = 5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2-high group was enriched for inflammatory, type 2-skewed dendritic cells expressing FcεRI. The type 2-high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. CONCLUSION: Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.

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.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.