Effect of cigarette smoke extract on allergen and viral responses in human bronchial epithelial cells

Abstract

Background: Asthma is a heterogeneous lung disorder characterized by airway inflammation, hyperresponsiveness, and remodeling. Airway inflammation is a primary characteristic of asthma, and the endotypes of asthma are classified into two main types based on inflammatory profiles, namely Type 2-high allergic asthma (T2-high) and non-Type 2 asthma (Non-T2). Cigarette smoke combined with asthmatic inflammation may induce important changes in asthma pathogenesis. Cigarette smoke extract (CSE) can modify the inflammatory responses in human airway smooth muscle cells (HASMCs), promoting a shift from steroid-sensitive type 2 (T2- high) eosinophilic inflammatory responses to steroid-insensitive (Non-T2) neutrophilic inflammatory responses, at least in part through a COX-2/PGE2-dependent mechanism. Human bronchial epithelial cells (HBECs) are the first line of defence against inhaled insults, including viruses and allergens, and are an important source of a group of cytokines termed alarmins, which play a key role in the initiation of allergen- and viral-induced T2 inflammatory responses in asthma. Cell-free DNA (cfDNA), refers to all non-encapsulated DNA in the bloodstream or other fluids that occurs as active secretion from the cells and during normal apoptotic and necrotic processes, and is known to be released from virally infected HBECs. Whether CSE can modify allergen and viral-induced T2 inflammatory responses and promote non-T2 neutrophilic inflammatory responses, and modulate cfDNA release from HBECs that can alter HASMCs function is unclear. Methods: CSE was prepared from the smoke of (3R4F) research-grade cigarettes bubbled into 20 ml of culture medium. Immortalised HBECs (iHBECs) were treated with and without CSE (3%) and/or house dust mite (Der p1) 10 μg/ml for 24 hours, and/or viral mimic Poly(I:C) 10 μg/ml for 24 hours and 48 hours. The concentrations of alarmins (IL-25, IL-33, and TSLP), Th2 cytokines (IL-4, IL-5, and IL-13), eosinophilic chemokines (Eotaxin, RANTES, and IP-10), pleiotropic cytokine IL-6, and neutrophilic chemokine IL-8 were measured in conditioned media using a Luminex® Multiplex assay. Quantitative real-time PCR (qPCR) for telomerase reverse transcript (TERT) and the TapeStation System were used to investigate the concentration of cfDNA released. BrdU Cell Proliferation ELISA and PrestoBlueTM Cell Viability Reagent were used to assess HASMCS proliferation. A cell Collagen-based Contraction assay was used to assess HASMCs contraction, and transcriptome profiling using RNA-Sequence technology was used identify gene expression changes in HASMCs. Results: I found that CSE did not modulate the inflammatory mediator response of iHBECs to Der P1 allergen, with no interaction between CSE and Der P1 exposure on the production of alarmins (IL-25, IL-33, TSLP), Th2 cytokines (IL-4, IL-5, IL-13), eosinophilic chemokines (Eotaxin, IP-10, RANTES). However, CSE significantly increased the production of neutrophilic chemokine IL-8 in response to Der P1. Notably, I found that CSE modulates the response of iHBECs to viral mimic poly (I:C) through altering inflammatory responses, suppressing epithelial-derived alarmins and eosinophil responses, and inducing neutrophilic responses. cfDNA was released in response to Poly (I:C) but not CSE. Interestingly, co-stimulation of CSE and Poly (I:C) significantly increased the concentration of released cfDNA, relative to Poly (I:C) alone, (0.03901 and 0.03696 Cq respectively p<0.05, n=3). Functional studies of iHBEC cfDNA revealed that cfDNA from poly (I:C) and CSE+poly (I:C)-stimulated iHBECs had no effect on HASMC proliferation but significantly modulated gene expression in HASMCs. Conclusion: This thesis showed that CSE may have a minimal effect on allergen-induced inflammatory mediator response of iHBECs but did have profound effects on the inflammatory response to the viral mimic poly (I:C) through altering inflammatory responses, which may suggest a shift from eosinophilic to neutrophilic airway inflammation. cfDNA released from poly (I:C) and co-stimulation of CSE and poly (I:C)-stimulated iHBECs had no effect on remodelling of HASMCs but significantly modulated gene expression specifically in pathways related to chemokine activity and defence response to virus.