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.

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.

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.

The Endoplasmic Reticulum Resident Protein AGR3. Required for Regulation of Ciliary Beat Frequency in the Airway.

Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

Epithelial interleukin-25 is a key mediator in Th2-high, corticosteroid-responsive asthma.

RATIONALE: Activation of type 2 cytokine pathways plays a central role in a large subset of subjects with asthma. Th2-high and Th2-low asthma have distinct clinical, pathologic, and molecular phenotypes and respond differently to therapy. The factors that initiate type 2 responses in some subjects with asthma are unknown. OBJECTIVES: To determine whether expression of epithelial cytokines IL-25, IL-33, and thymic stromal lymphopoietin are associated with type 2 responses and predict response to inhaled corticosteroid (ICS) in asthma. METHODS: We analyzed pulmonary function tests, blood, and bronchoscopic biopsies from 21 healthy control subjects and 43 subjects with asthma. Subjects with asthma underwent an 8-week treatment with inhaled budesonide. MEASUREMENTS AND MAIN RESULTS: Epithelial expression of IL-25, but not IL-33 or thymic stromal lymphopoietin, was increased in a subset of subjects with asthma. The IL-25-high subset had greater airway hyperresponsiveness, more airway and blood eosinophils, higher serum IgE, more subepithelial thickening, and higher expression of Th2 signature genes. ICS improved FEV1 and hyperresponsiveness in the IL-25-high but not the IL-25-low subset. Plasma IL-25 levels correlated with epithelial IL-25 expression, airway eosinophilia, and beneficial responses to ICS treatment. CONCLUSIONS: IL-25 measurements identify two subsets of subjects with distinct asthma phenotypes and different responses to ICS. Because IL-25 has a major role in triggering type 2 responses, bronchial epithelial IL-25 expression is likely a key determinant of type 2 response activation in asthma. Plasma IL-25 level reflects airway IL-25/type 2 response activation and may be useful for predicting responses to asthma therapy.

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.

Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2.

The coronavirus SARS-CoV-2 causes the severe disease COVID-19. SARS-CoV-2 infection is initiated by interaction of the viral spike protein and host receptor angiotensin-converting enzyme 2 (ACE2). We report an improved bright and reversible fluorogenic reporter, named SURF (split UnaG-based reversible and fluorogenic protein-protein interaction reporter), that we apply to monitor real-time interactions between spike and ACE2 in living cells. SURF has a large dynamic range with a dark-to-bright fluorescence signal that requires no exogenous cofactors. Utilizing this reporter, we carried out a high-throughput screening of small-molecule libraries. We identified three natural compounds that block replication of SARS-CoV-2 in both Vero cells and human primary nasal and bronchial epithelial cells. Cell biological and biochemical experiments validated all three compounds and showed that they block the early stages of viral infection. Two of the inhibitors, bruceine A and gamabufotalin, were also found to block replication of the Delta and Omicron variants of SARS-CoV-2. Both bruceine A and gamabufotalin exhibited potent antiviral activity in K18-hACE2 and wild-type C57BL6/J mice, as evidenced by reduced viral titres in the lung and brain, and protection from alveolar and peribronchial inflammation in the lung, thereby limiting disease progression. We propose that our fluorescent assay can be applied to identify antiviral compounds with potential as therapeutic treatment for COVID-19 and other respiratory diseases.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

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.

Epithelial miR-141 regulates IL-13-induced airway mucus production.

IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator.

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.

The airway epithelium in asthma.

Asthma is a genetically and phenotypically complex disease that has a major impact on global health. Signs and symptoms of asthma are caused by the obstruction of airflow through the airways. The epithelium that lines the airways plays a major role in maintaining airway patency and in host defense. The epithelium initiates responses to inhaled or aspirated substances, including allergens, viruses, and bacteria, and epithelial-derived cytokines are important in the recruitment and activation of immune cells in the airway. Changes in the structure and function of the airway epithelium are a prominent feature of asthma. Approximately half of individuals with asthma have evidence of active type 2 immune responses in the airway. In these individuals, epithelial cytokines promote type 2 responses, and responses to type 2 cytokines result in increased epithelial mucus production and other effects that cause airway obstruction. Recent work also implicates other epithelial responses, including interleukin-17, interferon and ER stress responses, that may contribute to asthma pathogenesis and provide new targets for therapy.

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.

Airway Mucus and Asthma: The Role of MUC5AC and MUC5B.

Asthma is characterized by mucus abnormalities. Airway epithelial hyperplasia and metaplasia result in changes in stored and secreted mucin and the production of a pathologic mucus gel. Mucus transport is impaired, culminating in mucus plugging and airway obstruction-a major cause of morbidity in asthma. The polymeric mucins MUC5AC and MUC5B are integral components of airway mucus. MUC5AC and MUC5B gene expression is altered in asthma, and recent work sheds light on their contribution to asthma pathogenesis. Herein, we review our current understanding of the role of MUC5AC and MUC5B in mucus dysfunction in asthma.

MUC5AC and a Glycosylated Variant of MUC5B Alter Mucin Composition in Children With Acute Asthma.

BACKGROUND: Diffuse airway mucus obstruction is an important feature of severe and fatal asthma. MUC5AC and MUC5B are the principal gel-forming mucins found in airway mucus. The mucin composition of airway mucus likely affects its functional properties. METHODS: We quantified the principal airway mucins MUC5AC and MUC5B in the sputum of age-matched children with acute and stable asthma and healthy control subjects by using Western blotting. RESULTS: Sputum samples from 38 children (13 with acute asthma, 15 with stable asthma, 10 control subjects) were obtained. Sputum MUC5AC concentrations were 7.6 µg/mL in control subjects, 22.4 µg/mL in those with stable asthma (P = .17), and 44.7 µg/mL in those with acute asthma (P < .05). MUC5B concentrations showed less variation, with 238.5, 208.4 and 165.9 µg/mL in control subjects, those with stable asthma, and those with acute asthma, respectively. The greater MUC5AC concentration in those with acute asthma resulted in a significantly altered MUC5B:MUC5AC ratio between control subjects and those with acute asthma (P < .05). Significant differences in MUC5B glycoforms were present between the groups, with the low-charge-only glycoform being found uniquely in those with acute asthma. CONCLUSIONS: Increased MUC5AC and the presence of a low-charge-only MUC5B glycoform significantly altered mucin composition in children with acute asthma. These changes may be important contributory factors to the airway mucus obstruction observed during acute asthma.

Epithelial tethering of MUC5AC-rich mucus impairs mucociliary transport in asthma.

The development of pathologic mucus, which is not readily cleared from the airways, is an important contributor to the morbidity and mortality associated with asthma. It is not clear how the major airway mucins MUC5AC and MUC5B are organized within the mucus gel or how this gel contributes to airway obstruction in asthma. Here, we demonstrated that mucus plugs from individuals with fatal asthma are heterogeneous gels with distinct MUC5AC- and MUC5B-containing domains. Stimulation of cultured human bronchial epithelial cells with IL-13, a key mediator in asthma, induced the formation of heterogeneous mucus gels and dramatically impaired mucociliary transport. Impaired transport was not associated with defects in ciliary function but instead was related to tethering of MUC5AC-containing mucus gel domains to mucus-producing cells in the epithelium. Replacement of tethered mucus with untethered mucus restored mucociliary transport. Together, our results indicate that tethering of MUC5AC-containing domains to the epithelium causes mucostasis and likely represents a major cause of mucus plugging in asthma.