The TRPA1 cation channel is upregulated by cigarette smoke in mouse and human macrophages modulating lung inflammation

Abstract

Cigarette smoke (CS) is a well-known source of several inflammatory, cytotoxic and genotoxic compounds that cause chronic lung diseases. The transient receptor potential ankyrin 1 (TRPA1), a smoking-responsive, non-selective cation channel, is expressed by both capsaicin-sensitive peptidergic sensory nerves and non-neuronal cells of the lung, but there are few and controversial data on its expression and function on macrophages. Here, we investigated TRPA1 mRNA and protein expression in mouse and human lung tissues and human 3D spheroids, with a particular focus on its expression and potential regulatory effects on pro- and anti-inflammatory macrophage functions in response to CS. TRPA1 was stably expressed in both human and mouse alveolar macrophages, being upregulated after CS exposure and its functional activity was demonstrated in mouse macrophage culture. Moreover, besides CS, the TRPA1 genotype itself affected the expression of M1- (Il-1β, Il-23) and M2-type (Il-10, Tgfβ) macrophage cytokines. Furthermore, CS extract increased TRPA1 mRNA in human lung spheroids showing more prominent expression in macrophage-containing 3D aggregates, while CS extract influenced an elevated TGFβ expression specifically in macrophage-containing spheroids. These results suggest the fine-tuning role of TRPA1 activation in CS-induced airway inflammation, particularly in macrophages, but further studies are needed to draw precise conclusions.

Keywords: 3D human lung spheroid; Airway inflammation; Alveolar macrophage; Cigarette smoke exposure; TGFβ; TRPA1.

Fig. 1

TRPA1 mRNA expression in mouse and human alveolar macrophages. Representative images of hematoxylin–eosin staining of non-smoking healthy mouse (a) and human (d) lung tissue representing surface epithelium, supporting tissue and network of capillaries (Scale bar: 200 µm), RNAscope images of alveolar macrophages co-expressing Trpa1 (red signal) and Iba1 (green signal) in the mouse (b, c), and TRPA1 (red signal) and CD68 (green signal) in the human lung (e, f). Scale bars of (b, c, e, f): 10 µm.

Fig. 2

Effect of AITC (200 μM) on cultured mouse macrophages. (a) The percentage of responsive cells after AITC administration (200 μM) is presented in % of total number of examined cells, **g > 0.8, (Trpa1^+/+ vs. Trpa1^−/−, effect size analysis, n = 98 cells). (b) Change of the fluorescence ratio (R = F340/F380) after AITC administration (Trpa1^+/+ n = 24, Trpa1^−/− n = 1). Each column represents mean ± SEM. (c) Representative original recording showing the response induced by AITC on a wildtype mouse macrophage.

Fig. 3

Effects of chronic cigarette smoke exposure on Trpa1^+/+ and Trpa1^−/− mouse lungs. (a) Representative images of macrophage-specific CD68 immunostaining counterstained with hematoxylin of the intact and inflamed mouse lungs after 1, 2 and 3 months of CSE (Magnification: 200x, arrows: CD68 immunpositivity, a: alveolus, b: bronchiolus, v: vessel, red boxes with *: oedema) (b) TRPA1 mRNA expression changes of Trpa1^+/+ mouse lungs after 1, 2 and 3 months of CSE. Each column represents mean ± SEM, n = 5–9/group, *g > 0.5, **g > 0.8, vs. Trpa1^+/+ intact, effect size analysis. (c) Evaluation of CD68 positive cell density in Trpa1^+/+ and Trpa1^−/− mouse lungs after CSE. Each column represents mean ± SEM, n = 5 tissue sections/group, 20 images/section; *g > 0.5, **g > 0.8, * vs. respective control, #g > 0.5, ##g > 0.8, # vs. respective treatment, effect size analysis.

Fig. 4

Smoking-induced mRNA expression changes of M1 and M2 cytokines in whole mouse lung homogenates. Each column represents mean ± SEM, n = 5/group, *g > 0.8 vs. respective control, #g > 0.8 vs. respective treatment, effect size analysis.

Fig. 5

TRPA1 and TGF-β expressions in human lung spheroids. (a) Demonstrative pictures of TRPA1 immunopositivity of untreated and 24-h CS extract-treated SN (SAEC epithelial cells + NHLF fibroblasts) and SNM (+ primary macrophages) 3D human lung cultures. Red: TRPA1, green: cytokeratin7, blue: DAPI for nuclei. Scale bars, 100 µm, magnification 200x. (b) Quantification of TRPA1 protein with densitometry; each column represents mean ± SEM; n = 4–6 in the intact, 11–15 in the treated groups, **g > 0.8, effect size analysis. (c) TRPA1 and TGF-β mRNA expressions; n = 5, *g > 0.5, **g > 0.8, effect size analysis. Each column represents mean ± SEM.