Summary: Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke. The main goal of this study was to explore the effects of cigarette smoke extracts (CSE) on Toll‐like receptor (TLR) expression and activation in a human bronchial epithelial cell line (16‐HBE). The CSE increased the expression of TLR4 and the lipopolysaccharide (LPS) binding, the nuclear factor‐κB (NF‐κB) activation, the release of interleukin‐8 (IL‐8) and the chemotactic activity toward neutrophils. It did not induce TLR2 expression or extracellular signal‐regulated signal kinase 1/2 (ERK1/2) activation. The LPS increased the expression of TLR4 and induced both NF‐κB and ERK1/2 activation. The combined exposure of 16‐HBE to CSE and LPS was associated with ERK activation rather than NF‐κB activation and with a further increase of IL‐8 release and of chemotactic activity toward neutrophils. Furthermore, CSE decreased the constitutive interferon‐inducible protein‐10 (IP‐10) release and counteracted the effect of LPS in inducing both the IP‐10 release and the chemotactic activity toward lymphocytes. In conclusion, cigarette smoke, by altering the expression and the activation of TLR4 via the preferential release of IL‐8, may contribute to the accumulation of neutrophils within the airways of smokers.

Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE₂is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE₂concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE₂receptor expression as well as on PGE₂release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE₂and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE₂levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers ( P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE₂immunoprecipitation. In vitro experiments confirmed that CSE increased PGE₂release and COX-2 and PGE₂receptor expression in neutrophils and AM; PGE₂enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP₄) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE₂in the airways of COPD subjects.

Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms. The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13). In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter. This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.

Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M₁, M₂, and M₃ receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) κB activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1β, TNF-α, and CSE. ChAT, M₁, and M₃ expression and ERK1/2 and NFκB activation were increased, whereas M₂ was reduced, in COPD and smoker subjects compared with controls. IL-1β increased the ChAT and M₃, TNF-α down-regulated M₂, and CSE increased ChAT and M₃ expression while down-regulating the expression of M₂ in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1β- and TNF-α-stimulated HFL-1 cells. Tiotropium (Spiriva; [1α,2β,4β,5α,7β-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.0²⁴], C₁₉H₂₂ NO₄S₂Br·H₂O), gallamine triethiodide (C₁₉H₂₂N₄O₂S·2HCl·H₂O), telenzepine [4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C₃₀H₆₀I₃N₃O₃], 4-diphenylacetoxy-N-methylpiperidine, PD098059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C₁₆H₁₃NO₃], and BAY 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C₁₀H₉NO₂C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFκB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.

In the early 1970s, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and named high-mobility group (HMG) proteins. High-mobility group box 1 (HMGB1) is the most studied HMG protein that detects and coordinates cellular stress response. The biological function of HMGB1 depends on its subcellular localization and expression. It plays a critical role in the nucleus and cytoplasm as DNA chaperone, chromosome gatekeeper, autophagy maintainer, and protector from apoptotic cell death. HMGB1 also functions as an extracellular alarmin acting as a damage-associated molecular pattern molecule (DAMP). Recent findings describe HMGB1 as a sophisticated signal of danger, with a pleiotropic function, which is useful as a clinical biomarker for several disorders. HMGB1 has emerged as a mediator in acute and chronic inflammation. Furthermore, HMGB1 targeting can induce beneficial effects on oxidative stress related diseases. This review focus on HMGB1 redox status, localization, mechanisms of release, binding with receptors, and its activities in different oxidative stress-related chronic diseases. Since a growing number of reports show the key role of HMGB1 in socially relevant pathological conditions, to our knowledge, for the first time, here we analyze the scientific literature, evaluating the number of publications focusing on HMGB1 in humans and animal models, per year, from 2006 to 2021 and the number of records published, yearly, per disease and category (studies on humans and animal models).