Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase.

Toll-like receptor 7 (TLR7) is known for eliciting immunity against single-stranded RNA viruses, and is increased in both human and cigarette smoke (CS)-induced, experimental chronic obstructive pulmonary disease (COPD). Here we show that the severity of CS-induced emphysema and COPD is reduced in TLR7-deficient mice, while inhalation of imiquimod, a TLR7-agonist, induces emphysema without CS exposure. This imiquimod-induced emphysema is reduced in mice deficient in mast cell protease-6, or when wild-type mice are treated with the mast cell stabilizer, cromolyn. Furthermore, therapeutic treatment with anti-TLR7 monoclonal antibody suppresses CS-induced emphysema, experimental COPD and accumulation of pulmonary mast cells in mice. Lastly, TLR7 mRNA is increased in pre-existing datasets from patients with COPD, while TLR7+ mast cells are increased in COPD lungs and associated with severity of COPD. Our results thus support roles for TLR7 in mediating emphysema and COPD through mast cell activity, and may implicate TLR7 as a potential therapeutic target.

ACE2 expression is elevated in airway epithelial cells from older and male healthy individuals but reduced in asthma.

BACKGROUND AND OBJECTIVE: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD. METHODS: We obtained lower AEC from 145 people from two independent cohorts, aged 2-89 years, Newcastle (n = 115) and Perth (n = 30), Australia. The Newcastle cohort was enriched with people with asthma (n = 37) and COPD (n = 38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC. RESULTS: Increased gene expression of ACE2 was associated with older age (P = 0.03) and male sex (P = 0.03), but not with pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients (P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma (P = 0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface, was increased (P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients. CONCLUSION: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

PAT in the ER for Transmembrane Protein Folding.

Integral membrane proteins (IMPs) have crucial roles in many cellular processes. A novel intramembrane chaperone complex, recently elucidated by Chitwood and Hedge, provides mechanistic insight of IMP biogenesis and folding, illustrating how IMPs with multiple transmembrane domains (TMDs) are assembled within the endoplasmic reticulum (ER) membrane.

Human coronaviruses 229E and OC43 replicate and induce distinct antiviral responses in differentiated primary human bronchial epithelial cells.

The recurrent emergence of novel, pathogenic coronaviruses (CoVs) severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1; 2002), Middle East respiratory syndrome (MERS)-CoV (2012), and most recently SARS-CoV-2 (2019) has highlighted the need for physiologically informative airway epithelial cell infection models for studying immunity to CoVs and development of antiviral therapies. To address this, we developed an in vitro infection model for two human coronaviruses; alphacoronavirus 229E-CoV (229E) and betacoronavirus OC43-CoV (OC43) in differentiated primary human bronchial epithelial cells (pBECs). Primary BECs from healthy subjects were grown at air-liquid interface (ALI) and infected with 229E or OC43, and replication kinetics and time-course expression of innate immune mediators were assessed. OC43 and 229E-CoVs replicated in differentiated pBECs but displayed distinct replication kinetics: 229E replicated rapidly with viral load peaking at 24 h postinfection, while OC43 replication was slower peaking at 96 h after infection. This was associated with diverse antiviral response profiles defined by increased expression of type I/III interferons and interferon-stimulated genes (ISGs) by 229E compared with no innate immune activation with OC43 infection. Understanding the host-virus interaction for previously established coronaviruses will give insight into pathogenic mechanisms underpinning SARS-CoV-2-induced respiratory disease and other future coronaviruses that may arise from zoonotic sources.

Understanding the Unfolded Protein Response in the Pathogenesis of Asthma.

Asthma is a heterogeneous, chronic inflammatory disease of the airways. It is a complex disease with different clinical phenotypes and results in a substantial socioeconomic burden globally. Poor understanding of pathogenic mechanisms of the disease hinders the investigation into novel therapeutics. Emerging evidence of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) has demonstrated previously unknown functions of this response in asthma development. A worsening of asthmatic condition can be brought on by stimuli such as oxidative stress, pathogenic infections, and allergen exposure. All of which can induce ER stress and activate UPR leading to activation of different inflammatory responses and dysregulate the innate immune functions in the airways. The UPR as a central regulator of asthma pathogenesis may explain several unknown mechanism of the disease onset, which leads us in new directions for future asthma treatments. In this review, we summarize and discuss the causes and impact of ER-UPR in driving the pathogenesis of asthma and highlight its importance in clinical implications.

Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

INTRODUCTION: The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. MATERIALS AND METHODS: Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. RESULTS: HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. CONCLUSIONS: Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD.

Influenza A virus (IAV) infections lead to severe inflammation in the airways. Patients with chronic obstructive pulmonary disease (COPD) characteristically have exaggerated airway inflammation and are more susceptible to infections with severe symptoms and increased mortality. The mechanisms that control inflammation during IAV infection and the mechanisms of immune dysregulation in COPD are unclear. We found that IAV infections lead to increased inflammatory and antiviral responses in primary bronchial epithelial cells (pBECs) from healthy nonsmoking and smoking subjects. In pBECs from COPD patients, infections resulted in exaggerated inflammatory but deficient antiviral responses. A20 is an important negative regulator of NF-κB-mediated inflammatory but not antiviral responses, and A20 expression was reduced in COPD. IAV infection increased the expression of miR-125a or -b, which directly reduced the expression of A20 and mitochondrial antiviral signaling (MAVS), and caused exaggerated inflammation and impaired antiviral responses. These events were replicated in vivo in a mouse model of experimental COPD. Thus, miR-125a or -b and A20 may be targeted therapeutically to inhibit excessive inflammatory responses and enhance antiviral immunity in IAV infections and in COPD.

The genetic and epigenetic landscapes of the epithelium in asthma.

Asthma is a global health problem with increasing prevalence. The airway epithelium is the initial barrier against inhaled noxious agents or aeroallergens. In asthma, the airway epithelium suffers from structural and functional abnormalities and as such, is more susceptible to normally innocuous environmental stimuli. The epithelial structural and functional impairments are now recognised as a significant contributing factor to asthma pathogenesis. Both genetic and environmental risk factors play important roles in the development of asthma with an increasing number of genes associated with asthma susceptibility being expressed in airway epithelium. Epigenetic factors that regulate airway epithelial structure and function are also an attractive area for assessment of susceptibility to asthma. In this review we provide a comprehensive discussion on genetic factors; from using linkage designs and candidate gene association studies to genome-wide association studies and whole genome sequencing, and epigenetic factors; DNA methylation, histone modifications, and non-coding RNAs (especially microRNAs), in airway epithelial cells that are functionally associated with asthma pathogenesis. Our aims were to introduce potential predictors or therapeutic targets for asthma in airway epithelium. Overall, we found very small overlap in asthma susceptibility genes identified with different technologies. Some potential biomarkers are IRAKM, PCDH1, ORMDL3/GSDMB, IL-33, CDHR3 and CST1 in airway epithelial cells. Recent studies on epigenetic regulatory factors have further provided novel insights to the field, particularly their effect on regulation of some of the asthma susceptibility genes (e.g. methylation of ADAM33). Among the epigenetic regulatory mechanisms, microRNA networks have been shown to regulate a major portion of post-transcriptional gene regulation. Particularly, miR-19a may have some therapeutic potential.

Fibulin-1 regulates the pathogenesis of tissue remodeling in respiratory diseases.

Airway and/or lung remodeling, involving exaggerated extracellular matrix (ECM) protein deposition, is a critical feature common to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Fibulin-1 (Fbln1), an important ECM protein involved in matrix organization, may be involved in the pathogenesis of these diseases. We found that Fbln1 was increased in COPD patients and in cigarette smoke-induced (CS-induced) experimental COPD in mice. Genetic or therapeutic inhibition of Fbln1c protected against CS-induced airway fibrosis and emphysema-like alveolar enlargement. In experimental COPD, this occurred through disrupted collagen organization and interactions with fibronectin, periostin, and tenascin-c. Genetic inhibition of Fbln1c also reduced levels of pulmonary inflammatory cells and proinflammatory cytokines/chemokines (TNF-α, IL-33, and CXCL1) in experimental COPD. Fbln1c-/- mice also had reduced airway remodeling in experimental chronic asthma and pulmonary fibrosis. Our data show that Fbln1c may be a therapeutic target in chronic respiratory diseases.

Impaired Antiviral Stress Granule and IFN-ß Enhanceosome Formation Enhances Susceptibility to Influenza Infection in Chronic Obstructive Pulmonary Disease Epithelium.

Chronic obstructive pulmonary disease (COPD) is a serious lung disease that progressively worsens lung function. Those affected are highly susceptible to influenza virus infections that result in exacerbations with exaggerated symptoms with increased mortality. The mechanisms underpinning this increased susceptibility to infection in COPD are unclear. In this study, we show that primary bronchial epithelial cells (pBECs) from subjects with COPD have impaired induction of type I IFN (IFN-ß) and lead to heightened viral replication after influenza viral infection. COPD pBECs have reduced protein levels of protein kinase (PK) R and decreased formation of PKR-mediated antiviral stress granules, which are critical in initiating type I IFN inductions. In addition, reduced protein expression of p300 resulted in decreased activation of IFN regulatory factor 3 and subsequent formation of IFN-ß enhanceosome in COPD pBECs. The decreased p300 induction was the result of enhanced levels of microRNA (miR)-132. Ectopic expression of PKR or miR-132 antagomiR alone failed to restore IFN-ß induction, whereas cotreatment increased antiviral stress granule formation, induction of p300, and IFN-ß in COPD pBECs. This study reveals that decreased induction of both PKR and p300 proteins contribute to impaired induction of IFN-ß in COPD pBECs upon influenza infection.

Innate Immunity and Immune Evasion by Enterovirus 71.

Enterovirus 71 (EV71) is a major infectious disease affecting millions of people worldwide and it is the main etiological agent for outbreaks of hand foot and mouth disease (HFMD). Infection is often associated with severe gastroenterological, pulmonary, and neurological diseases that are most prevalent in children. Currently, no effective vaccine or antiviral drugs exist against EV71 infection. A lack of knowledge on the molecular mechanisms of EV71 infection in the host and the virus-host interactions is a major constraint to developing specific antiviral strategies against this infection. Previous studies have identified and characterized the function of several viral proteins produced by EV71 that interact with the host innate immune proteins, including type I interferon signaling and microRNAs. These interactions eventually promote efficient viral replication and increased susceptibility to the disease. In this review we discuss the functions of EV71 viral proteins in the modulation of host innate immune responses to facilitate viral replication.

Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD.

BACKGROUND: Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls. METHODS: Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication. RESULTS: COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-ß/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-ß expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines. CONCLUSIONS: COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD.

Innate immunity to influenza in chronic airways diseases.

Influenza presents a unique human infectious disease that has a substantial impact on the public health, in general, and especially for those with chronic airways diseases. People with asthma and chronic obstructive pulmonary disease (COPD) are particularly vulnerable to influenza infection and experience more severe symptoms with the worsening of their pre-existing conditions. Recent advances in reverse genetics and innate immunity has revealed several influenza virulence factors and host factors involved in influenza pathogenesis and the immune responses to infection. Early innate immunity plays a critical role of limiting viral infection and spread; however, the underlying mechanisms that lead to enhanced susceptibility to influenza infection and severe symptoms in those with asthma and COPD to infection remain un-investigated. This review will explore the importance of early innate antiviral responses to influenza infection and how these responses are altered by influenza virus and in those with chronic airways diseases.

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection.

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-ß. However it was found that there was constitutive release of IFN-ß by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-ß release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.