Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.

The Type 2 Asthma Mediator IL-13 Inhibits Severe Acute Respiratory Syndrome Coronavirus 2 Infection of Bronchial Epithelium.

Asthma is associated with chronic changes in the airway epithelium, a key target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many epithelial changes, including goblet cell metaplasia, are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We found that IL-13 stimulation of differentiated human bronchial epithelial cells (HBECs) cultured at air-liquid interface reduced viral RNA recovered from SARS-CoV-2-infected cells and decreased double-stranded RNA, a marker of viral replication, to below the limit of detection in our assay. An intact mucus gel reduced SARS-CoV-2 infection of unstimulated cells, but neither a mucus gel nor SPDEF, which is required for goblet cell metaplasia, were required for the antiviral effects of IL-13. Bulk RNA sequencing revealed that IL-13 regulated 41 of 332 (12%) mRNAs encoding SARS-CoV-2-associated proteins that were detected in HBECs (>1.5-fold change; false discovery rate < 0.05). Although both IL-13 and IFN-α each inhibit SARS-CoV-2 infection, their transcriptional effects differed markedly. Single-cell RNA sequencing revealed cell type-specific differences in SARS-CoV-2-associated gene expression and IL-13 responses. Many IL-13-induced gene expression changes were seen in airway epithelium from individuals with type 2 asthma and chronic obstructive pulmonary disease. IL-13 effects on airway epithelial cells may protect individuals with type 2 asthma from COVID-19 and could lead to identification of novel strategies for reducing SARS-CoV-2 infection.

A simple method to generate human airway epithelial organoids with externally orientated apical membranes.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 105 cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.

The type 2 asthma mediator IL-13 inhibits SARS-CoV-2 infection of bronchial epithelium.

RATIONALE: Asthma is associated with chronic changes in the airway epithelium, a key target of SARS-CoV-2. Many epithelial changes are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. OBJECTIVES: We sought to discover how IL-13 and other cytokines affect expression of genes encoding SARS-CoV-2-associated host proteins in human bronchial epithelial cells (HBECs) and determine whether IL-13 stimulation alters susceptibility to SARS-CoV-2 infection. METHODS: We used bulk and single cell RNA-seq to identify cytokine-induced changes in SARS-CoV-2-associated gene expression in HBECs. We related these to gene expression changes in airway epithelium from individuals with mild-moderate asthma and chronic obstructive pulmonary disease (COPD). We analyzed effects of IL-13 on SARS-CoV-2 infection of HBECs. MEASUREMENTS AND MAIN RESULTS: Transcripts encoding 332 of 342 (97%) SARS-CoV-2-associated proteins were detected in HBECs (≥1 RPM in 50% samples). 41 (12%) of these mRNAs were regulated by IL-13 (>1.5-fold change, FDR < 0.05). Many IL-13-regulated SARS-CoV-2-associated genes were also altered in type 2 high asthma and COPD. IL-13 pretreatment reduced viral RNA recovered from SARS-CoV-2 infected cells and decreased dsRNA, a marker of viral replication, to below the limit of detection in our assay. Mucus also inhibited viral infection. CONCLUSIONS: IL-13 markedly reduces susceptibility of HBECs to SARS-CoV-2 infection through mechanisms that likely differ from those activated by type I interferons. Our findings may help explain reports of relatively low prevalence of asthma in patients diagnosed with COVID-19 and could lead to new strategies for reducing SARS-CoV-2 infection.

Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets.

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low-abundance transcripts and microRNAs that are challenging to analyze with current single-cell RNA-sequencing methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

An airway epithelial IL-17A response signature identifies a steroid-unresponsive COPD patient subgroup.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous smoking-related disease characterized by airway obstruction and inflammation. This inflammation may persist even after smoking cessation and responds variably to corticosteroids. Personalizing treatment to biologically similar "molecular phenotypes" may improve therapeutic efficacy in COPD. IL-17A is involved in neutrophilic inflammation and corticosteroid resistance, and thus may be particularly important in a COPD molecular phenotype. METHODS: We generated a gene expression signature of IL-17A response in bronchial airway epithelial brushings from smokers with and without COPD (n = 238), and validated it using data from 2 randomized trials of IL-17 blockade in psoriasis. This IL-17 signature was related to clinical and pathologic characteristics in 2 additional human studies of COPD: (a) SPIROMICS (n = 47), which included former and current smokers with COPD, and (b) GLUCOLD (n = 79), in which COPD participants were randomized to placebo or corticosteroids. RESULTS: The IL-17 signature was associated with an inflammatory profile characteristic of an IL-17 response, including increased airway neutrophils and macrophages. In SPIROMICS the signature was associated with increased airway obstruction and functional small airways disease on quantitative chest CT. In GLUCOLD the signature was associated with decreased response to corticosteroids, irrespective of airway eosinophilic or type 2 inflammation. CONCLUSION: These data suggest that a gene signature of IL-17 airway epithelial response distinguishes a biologically, radiographically, and clinically distinct COPD subgroup that may benefit from personalized therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT01969344. FUNDING: Primary support from the NIH, grants K23HL123778, K12HL11999, U19AI077439, DK072517, U01HL137880, K24HL137013 and R01HL121774 and contracts HHSN268200900013C, HHSN268200900014C, HHSN268200900015C, HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C and HHSN268200900020C.

IFN-stimulated Gene Expression, Type 2 Inflammation, and Endoplasmic Reticulum Stress in Asthma.

RATIONALE: Quantification of type 2 inflammation provided a molecular basis for heterogeneity in asthma. Non-type 2 pathways that contribute to asthma pathogenesis are not well understood. OBJECTIVES: To identify dysregulated pathways beyond type 2 inflammation. METHODS: We applied RNA sequencing to airway epithelial brushings obtained from subjects with stable mild asthma not on corticosteroids (n = 19) and healthy control subjects (n = 16). Sequencing reads were mapped to human and viral genomes. In the same cohort, and in a separate group with severe asthma (n = 301), we profiled blood gene expression with microarrays. MEASUREMENTS AND MAIN RESULTS: In airway brushings from mild asthma on inhaled corticosteroids, RNA sequencing yielded 1,379 differentially expressed genes (false discovery rate < 0.01). Pathway analysis revealed increased expression of type 2 markers, IFN-stimulated genes (ISGs), and endoplasmic reticulum (ER) stress-related genes. Airway epithelial ISG expression was not associated with type 2 inflammation in asthma or with viral transcripts but was associated with reduced lung function by FEV1 (ρ = -0.72; P = 0.0004). ER stress was confirmed by an increase in XBP1 (X-box binding protein 1) splicing in mild asthma and was associated with both type 2 inflammation and ISG expression. ISGs were also the most activated genes in blood cells in asthma and were correlated with airway ISG expression (ρ = 0.55; P = 0.030). High blood ISG expression in severe asthma was similarly unrelated to type 2 inflammation. CONCLUSIONS: ISG activation is prominent in asthma, independent of viral transcripts, orthogonal to type 2 inflammation, and associated with distinct clinical features. ER stress is associated with both type 2 inflammation and ISG expression.

Airway epithelial miRNA expression is altered in asthma.

RATIONALE: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. OBJECTIVES: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. METHODS: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. MEASUREMENTS AND MAIN RESULTS: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. CONCLUSIONS: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).

Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells.

We investigated how cystic fibrosis (CF) alters the relationship between Cl(-) and mucin secretion in cultures of non-CF and CF human tracheobronchial gland mucous (HTGM and CFTGM, respectively) cells. Biochemical studies showed that HTMG cells secreted typical airway mucins, and immunohistochemical studies showed that these cells expressed MUC1, MUC4, MUC5B, MUC8, MUC13, MUC16, and MUC20. Effects of cumulative doses of methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP on mucin and Cl(-) secretion were studied on HTGM and CFTGM cultures. Baseline mucin secretion was not significantly altered in CFTGM cells, and the increases in mucin secretion induced by mediators were unaltered (Iso, Phe) or slightly decreased (MCh, ATP). Across mediators, there was no correlation between the maximal increases in Cl(-) secretion and mucin secretion. In HTGM cells, the Cl(-) channel blocker, diphenylamine-2-carboxylic acid, greatly inhibited Cl(-) secretion but did not alter mucin release. In HTGM cells, mediators (10(-5) M) increased mucin secretion in the rank order ATP > Phe = Iso > MCh. They increased Cl(-) secretion in the sequence ATP > MCh ≈ Iso > Phe. The responses in Cl(-) secretion to MCh, ATP, and Phe were unaltered by CF, but the response to Iso was greatly reduced. We conclude that mucin secretion by cultures of human tracheobronchial gland cells is independent of Cl(-) secretion, at baseline, and is unaltered in CF; that the ratio of Cl(-) secretion to mucus secretion varies markedly depending on mediator; and that secretions induced by stimulation of ß-adrenergic receptors will be abnormally concentrated in CF.

Cultures of human tracheal gland cells of mucous or serous phenotype.

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced "mucous" cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced "serous" cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both "serous" and "mucous" cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.