Parkin, an E3 ubiquitin ligase, enhances airway mitochondrial DNA release and inflammation.

INTRODUCTION: Parkin (Park2), an E3 ubiquitin ligase, is critical to maintain mitochondrial function by regulating mitochondrial biogenesis and degradation (mitophagy), but recent evidence suggests the involvement of Parkin in promoting inflammation. In the present study, we determined if Parkin regulates airway mitochondrial DNA (mtDNA) release and inflammatory responses to type 2 cytokine interleukin (IL)-13 and allergens. METHODS: We measured Parkin mRNA expression in brushed bronchial epithelial cells and mtDNA release in the paired bronchoalveolar lavage fluid (BALF) from normal subjects and asthmatics. Parkin-deficient primary human tracheobronchial epithelial (HTBE) cells generated using the CRISPR-Cas9 system were stimulated with IL-13. To determine the in vivo function of Parkin, Parkin knockout (PKO) and wild-type (WT) mice were treated with IL-13 or allergen (house dust mite, HDM) in the presence or absence of mtDNA isolated from normal mouse lungs. RESULTS: Parkin mRNA expression in asthmatic airway epithelium was upregulated, which positively correlated with the levels of released mtDNA in BALF. IL-13-stimulated HTBE cells increased Parkin expression. Moreover, IL-13 induced mtDNA release in Parkin-sufficient, but not in Parkin-deficient HTBE cells. PKO (vs WT) mice attenuated airway mtDNA release and inflammation following IL-13 or HDM treatments. mtDNA amplified airway inflammation in mice treated with IL-13 or HDM. Notably, Parkin also mediated mtDNA-induced exacerbation of airway inflammation. CONCLUSION: Our research findings suggest that Parkin promotes mtDNA release and inflammation in airways, thus improving our understanding of the complex role of Parkin and mitochondrial dysfunction in asthma pathogenesis.

Interleukin 1 Receptor-Like 1 (IL1RL1) Promotes Airway Bacterial and Viral Infection and Inflammation.

Interleukin 1 receptor-like 1 (IL1RL1), also known as suppression of tumorigenicity 2 (ST2), is the receptor for interleukin 33 (IL-33) and has been increasingly studied in type 2 inflammation. An increase in airway IL-33/ST2 signaling in asthma has been associated with eosinophilic inflammation, but little is known about the role of ST2 in neutrophilic inflammation. Airway Mycoplasma pneumoniae and human rhinovirus (HRV) infections are linked to neutrophilic inflammation during acute exacerbations of asthma. However, whether ST2 contributes to M. pneumoniae- and HRV-mediated airway inflammation is poorly understood. The current study sought to determine the functions of ST2 during airway M. pneumoniae or HRV infection. In cultured normal human primary airway epithelial cells, ST2 overexpression (OE) increased the production of neutrophilic chemoattractant IL-8 in the absence or presence of M. pneumoniae or HRV1B infection. ST2 OE also enhanced HRV1B-induced IP-10, a chemokine involved in asthma exacerbations. In the M. pneumoniae-infected mouse model, ST2 deficiency, in contrast to sufficiency, significantly reduced the levels of neutrophils following acute (≤24 h) infection, while in the HRV1B-infected mouse model, ST2 deficiency significantly reduced the levels of proinflammatory cytokines KC, IP-10, and IL-33 in bronchoalveolar lavage (BAL) fluid. Overall, ST2 overexpression in human epithelial cells and ST2 sufficiency in mice increased the M. pneumoniae and HRV loads in cell supernatants and BAL fluid. After pathogen infection, ST2-deficient mice showed a higher level of the host defense protein lactotransferrin in BAL fluid. Our data suggest that ST2 promotes proinflammatory responses (e.g., neutrophils) to airway bacterial and viral infection and that blocking ST2 signaling may broadly attenuate airway infection and inflammation.

Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus.

The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if Tollip regulates the airway inflammatory response to RV infection in vivo under IL-13 and IL-33 treatment. Following IL-13, IL-33, and RV treatment, Tollip-deficient (vs. -sufficient) HTBE cells produced excessive IL-8, accompanied by decreased sST2 production but increased IRAK1 activation. IL-8 production following IL-13/IL-33/RV exposure was markedly attenuated in IRAK1-deficient HTBE cells, as well as in Tollip KO HTBE cells treated with an IRAK1 inhibitor or a recombinant sST2 protein. Tollip KO (vs. wild-type) mice developed exaggerated airway neutrophilic responses to RV in the context of IL-13 and IL-33 treatment. Collectively, these data demonstrate that Tollip restricts excessive IL-8 production in type 2 cytokine-exposed human airways during RV infection by promoting sST2 production and inhibiting IRAK1 activation. sST2 and IRAK1 may be therapeutic targets for attenuating excessive neutrophilic airway inflammation in asthma, especially during RV infection.

Nicotine-Free e-Cigarette Vapor Exposure Stimulates IL6 and Mucin Production in Human Primary Small Airway Epithelial Cells.

PURPOSE: Electronic cigarettes (e-cigs) are relatively new devices that allow the user to inhale a heated and aerosolized solution. At present, little is known about their health effects in the human lung, particularly in the small airways (<2 mm in diameter), a key site of airway obstruction and destruction in chronic obstructive pulmonary disease and other acute and chronic lung conditions. The aim of this study was to investigate the effect of e-cigarettes on human distal airway inflammation and remodeling. METHODS: We isolated primary small airway epithelial cells from donor lungs without known lung disease. Small airway epithelial cells were cultured at air-liquid interface and exposed to 15 puffs vapor obtained by heating a commercially available e-cigarette solution (e-vapor) with or without nicotine. After 24 hrs of e-vapor exposure, basolateral and apical media as well as cell lysates were collected to measure the pleiotropic cytokine interleukin 6 (IL6) and MUC5AC, one of the major components in mucus. RESULTS: Unlike the nicotine-containing e-vapor, nicotine-free e-vapor significantly increased the amount of IL6, which was coupled with increased levels of intracellular MUC5AC protein. Importantly, a neutralizing IL6 antibody (vs an IgG isotype control) significantly inhibited the production of MUC5AC induced by nicotine-free e-vapor. CONCLUSION: Our results suggest that human small airway epithelial cells exposed to nicotine-free e-vapor increase the inflammatory response and mucin production, which may contribute to distal lung airflow limitation and airway obstruction.

The effect of BPIFA1/SPLUNC1 genetic variation on its expression and function in asthmatic airway epithelium.

Bacterial permeability family member A1 (BPIFA1), also known as short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is a protein involved in the antiinflammatory response. The goal of this study was to determine whether BPIFA1 expression in asthmatic airways is regulated by genetic variations, altering epithelial responses to type 2 cytokines (e.g., IL-13). Nasal epithelial cells from patients with mild to severe asthma were collected from the National Heart, Lung, and Blood Institute Severe Asthma Research Program centers, genotyped for rs750064, and measured for BPIFA1. To determine the function of rs750064, cells were cultured at air-liquid interface and treated with IL-13 with or without recombinant human BPIFA1 (rhBPIFA1). Noncultured nasal cells with the rs750064 CC genotype had significantly less BPIFA1 mRNA expression than the CT and TT genotypes. Cultured CC versus CT and TT cells without stimulation maintained less BPIFA1 expression. With IL-13 treatment, CC genotype cells secreted more eotaxin-3 than CT and TT genotype cells. Also, rhBPIFA1 reduced IL-13-mediated eotaxin-3. BPIFA1 mRNA levels negatively correlated with serum IgE and fractional exhaled nitric oxide. Baseline FEV1% levels were lower in the asthma patients with the CC genotype (n = 1,016). Our data suggest that less BPIFA1 in asthma patients with the CC allele may predispose them to greater eosinophilic inflammation, which could be attenuated by rhBPIFA1 protein therapy.