Abstract
Clinical studies suggest that smokers with chronic obstructive pulmonary disease who use menthol cigarettes may display more severe lung inflammation than those who smoke non-menthol cigarette. However, the mechanisms for this difference remain unclear. Menthol is a ligand of transient receptor potential melastatin-8 (TRPM8), a Ca2+-permeant channel sensitive to reactive oxygen species (ROS). We previously reported that exposure of human bronchial epithelial cells (HBECs) to non-menthol cigarette smoke extract (Non-M-CSE) triggers a cascade of inflammatory signaling leading to IL-8 induction. In this study, we used this in vitro model to compare the inflammatory effects of menthol cigarette smoke extract (M-CSE) and Non-M-CSE and delineate the mechanisms underlying the differences in their impacts. Compared with Non-M-CSE, M-CSE initially increased a similar level of extracellular ROS, suggesting the equivalent oxidant potency. However, M-CSE subsequently produced more remarkable elevations in intracellular Ca2+, activation of the mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling, and IL-8 induction. The extracellular ROS responses to both CSE types were totally inhibited by N-acetyl-cysteine (NAC; a ROS scavenger). The intracellular Ca2+ responses to both CSE types were also totally prevented by NAC, AMTB (a TRPM8 antagonist), or EGTA (an extracellular Ca2+ chelator). The activation of the MAPK/NF-κB signaling and induction of IL-8 to both CSE types were suppressed to similar levels by NAC, AMTB, or EGTA. These results suggest that, in addition to ROS generated by both CSE types, the menthol in M-CSE may act as another stimulus to further activate TRPM8 and induce the observed responses. We also found that menthol combined with Non-M-CSE induced greater responses of intracellular Ca2+ and IL-8 compared with Non-M-CSE alone. Moreover, we confirmed the essential role of TRPM8 in these responses to Non-M-CSE or M-CSE and the difference in these responses between the both CSE types using HBECs with TRPM8 knockdown and TRPM8 knockout, and using HEK293 cells transfected with hTRPM8. Thus, compared with exposure to Non-M-CSE, exposure to M-CSE induced greater TRPM8-mediated inflammatory responses in HBECs. These augmented effects may be due to a double-hit on lung epithelial TRPM8 by ROS generated from CSE and the menthol in M-CSE.
Highlights
Chronic obstructive pulmonary disease (COPD) is characterized by persistent lung inflammation, and cigarette smoking is its major etiologic factor (Chung and Adcock, 2008)
Further analysis revealed that increased IL-8 production induced by Non-menthol cigarette smoke extract (M-CSE) or M-CSE was significantly suppressed by scavenging reactive oxygen species (ROS) with NAC (Non-M-CSE vs. MCSE, 401% vs. 571% of the basal level), antagonizing transient receptor potential melastatin8 (TRPM8) with AMTB (Non-M-CSE vs. M-CSE, 439% vs. 573% of the basal level), or removing extracellular Ca2+ with ethylene glycol tetraacetic acid (EGTA) (Non-M-CSE vs. M-CSE, 432% vs. 534% of the basal level) to similar levels
The augmented IL-8 response induced by M-CSE may have resulted from a more rigorous activation of the mitogen-activated protein kinases (MAPKs)/nuclear factorκB (NF-κB) signaling (Figure 5), because this inflammatory signaling is vital for IL8 induction in human bronchial epithelial cells (HBECs) (Liu et al, 2014; Lin et al, 2015)
Summary
Chronic obstructive pulmonary disease (COPD) is characterized by persistent lung inflammation, and cigarette smoking is its major etiologic factor (Chung and Adcock, 2008). Increased ROS in the lung epithelial cells may activate ROS-sensitive signaling pathways, such as mitogen-activated protein kinases (MAPKs), and downstream transcriptional factors, such as nuclear factorκB (NF-κB) (Mossman et al, 2006) These events upregulate inflammatory gene expression (Mossman et al, 2006; Tang et al, 2011; Liu et al, 2014; Wu et al, 2014; Lin et al, 2015). These pathogenetic mechanisms have been relatively elucidated, the negative impact of menthol compared with those of non-menthol cigarette smoke on lung inflammation remains unknown
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