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.

IntroductionParkin (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.MethodsWe 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.ResultsParkin 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.ConclusionOur 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.

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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.

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 (Յ24 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.