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. 2020 Nov 17:11:596314.
doi: 10.3389/fphys.2020.596314. eCollection 2020.

Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice

Hsin-Kuo Ko  1   2 , An-Hsuan Lin  3   4 , Diahn-Warng Perng  1   2 , Tzong-Shyuan Lee  5 , Yu Ru Kou  3
Affiliations

Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice

Hsin-Kuo Ko et al. Front Physiol. .

Abstract

Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for lipopolysaccharide (LPS). Transient receptor potential ankyrin 1 (TRPA1), a Ca2+-permeant channel, has been suggested as a non-TLR receptor membrane-bound sensor of LPS. We recently reported that TRPA1 is expressed in lung epithelial cells (LECs) and mediates lung inflammation induced by cigarette smoke. However, the role of TRPA1 in LPS-induced lung inflammation has not been conclusively defined, and its underlying cellular mechanisms remain unclear. In this study, our in vitro results showed that LPS sequentially produced a cascade of events, including the elevation of intracellular Ca2+, the activation of NADPH oxidase, increase in intracellular reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kB (NF-κB) signaling, and the induction of IL-8. The increase in intracellular Ca2+ was inhibited by HC030031 (a TRPA1 antagonist) but was unaffected by TAK-242 (a TLR-4 inhibitor). The activation of NADPH oxidase was prevented by its inhibitor apocynin, EGTA (an extracellular Ca2+ chelator), and HC030031. The increase in intracellular ROS was attenuated by apocynin, N-acetyl-cysteine (NAC, a ROS scavenger), EGTA, and HC030031. The activation of the MAPK/NF-κB signaling was halted by NAC, EGTA, and HC030031. IL-8 induction was suppressed by HC030031 and TRPA1 siRNA, and further reduced by the combination of HC030031 and TAK-242. Our in vivo studies showed that trpa1-/- mice exhibited a reduced level of LPS-induced lung inflammation compared with wild-type mice as evidenced by the alleviations of increases in vascular permeability, inflammatory cell infiltration, inflammatory cytokine levels, oxidative stress, and MAPK signaling activation. Thus, in LECs, LPS may activate TRPA1 resulting in an increase in Ca2+ influx. The increased intracellular Ca2+ leads to NADPH oxidase activation, which causes an increase in intracellular ROS. The intracellular ROS activates the MAPK/NF-κB signaling resulting in IL-8 induction. This mechanism may possibly be at work to induce lung inflammation in mice.

Keywords: TRPA1; calcium; lipopolysaccharide; lung epithelial cell; lung inflammation; reactive oxygen species; signaling pathway.

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Figures

FIGURE 1
FIGURE 1
LPS increases the production of IL-8 (A,B), IL-1β (C), and IL-6 (D) in HBECs. (A) Cells were exposed to 0–2 μg/ml LPS for 24 h. (B–D) Cells were exposed to medium alone at time 0 for 24 h (unstimulated condition) or to 1 μg/ml LPS for the indicated times. Protein levels of IL-8 (A,B), IL-1β (C), and IL-6 (D) in the culture medium were determined by ELISA. Data in each group are mean ± SEM from four independent experiments. *P < 0.05 vs. time 0.
FIGURE 2
FIGURE 2
The LPS-induced IL-8 expression was alleviated by treatment with a TRPA1 antagonist or TRPA1 siRNA in HBECs. (A,B) Cells were exposed to medium alone or 1 μg/ml LPS for 24 h with or without pretreatment of HC030031 (HC, a TRPA1 antagonist, 9 μM). (C) Cells were treated with different concentrations of TRPA1 siRNA (0, 25, and 50 nM). (D) Cells were incubated with medium alone or 1 μg/ml LPS for 24 h with pretreatment of 50 nM siRNA (siTRPA1) or scramble siRNA. The protein levels in cell lysate (A,C,D) and cell medium (B) were measured using Western blot and ELISA, respectively. Data in each group are mean ± SEM from four independent experiments. *P < 0.05 vs. the control; #P < 0.05 vs. LPS alone.
FIGURE 3
FIGURE 3
TRPA1 mediates LPS-induced increase in intracellular ROS via the Ca2+-dependent activation of NADPH oxidase in HBECs. (A) Cells were exposed to medium alone, 1 μg/ml LPS, or the combination of LPS and HC030031 (HC, 9 μM) for 0, 10, 30, 60, 90, and 120 s and 3, 5, and 10 min. (B) Cells were treated with or without LPS (1 μg/ml) for indicated times. (C) Cells were exposed to medium alone or 1 μg/ml LPS for 15 min after pretreatment with EGTA (500 μM), apocynin (APO, 150 μM), NAC (1 mM), or HC (9 μM). HC, EGTA, APO, and NAC are a TRPA1 antagonist, an extracellular Ca2+ chelator, an inhibitor of NADPH oxidase, and a ROS scavenger, respectively. (D) Cells were exposed to medium alone or 1 μg/ml LPS for 10 min after pretreatment with EGTA (500 μM), APO (150 μM), or HC (9 μM). Intracellular levels of Ca2+ (A) and ROS (B,C) and NADPH oxidase activity (D) were measured by Fluo-8, HE/DTH, and NADP+/NADPH fluorescent probe assays, respectively. Data in each group are mean ± SEM from four independent experiments. *P < 0.05 vs. the control; #P < 0.05 vs. LPS alone.
FIGURE 4
FIGURE 4
TRPA, intracellular Ca2+, and intracellular ROS contribute to the LPS-induced activation of the ERK/JNK/NF-κB signaling in HBECs. Cells were exposed to medium alone or 1 μg/ml LPS for 3 (A,B) and 9 h (C) and pretreated with EGTA (an extracellular Ca2+ chelator, 500 μM), NAC (ROS scavenger, 1 mM), or HC030031 (HC, a TRPA1 antagonist, 9 μM). Protein expression was analyzed by Western blot. The increased phosphorylation of kinases indicates the activation of ERK and JNK. The increased presence of the p65 subunit in the nucleus indicates the activation of NF-κB. Data in each group are mean ± SEM from four independent experiments. *P < 0.05 vs. the control; #P < 0.05 vs. LPS alone.
FIGURE 5
FIGURE 5
Both TRPA1 and TLR4 contribute to LPS-induced IL-8 expression. (A) Cells were exposed to medium alone, 1 μg/ml LPS for 24 h, or 1 μg/ml LPS with pretreatment with TAK-242 (a TLR4 antagonist, 1 μM). Intracellular levels of Ca2 + were measured by Fluo-8 fluorescent probe assays. (B) Cells were exposed to medium alone or 1 μg/ml LPS for 24 h and pretreated with HC030031 (HC, a TRPA1 antagonist, 9 mM), TAK-242 (a TLR4 antagonist, 1 μM), or the combination of HC and TAK-242. The protein levels in cell lysate were measured using Western blot. Data in each group are mean ± SEM from four independent experiments. *P < 0.05 versus the control; #P < 0.05 versus LPS alone; @P < 0.05 versus LPS + HC or LPS + TAK-242.
FIGURE 6
FIGURE 6
LPS-induced lung inflammation and lung inflammatory scores are reduced in trpa1– /– mice. Mice were treated with an intraperitoneal injection of E. coli LPS (20 mg/kg) or PBS (the vehicle). (A) Representative images of H&E-stained lung sections obtained from PBS- or LPS-exposed wild-type and trpa1– /– mice. Circles indicate the areas of inflammatory cell infiltration. (B) Lung inflammatory scores were calculated according to the sum of the levels of cell infiltration and damage levels assessed from lung sections. Data in each group are mean ± SEM from eight mice. *P < 0.05 vs. the PBS exposure group in both genotypes; #P < 0.05 vs. the LPS-exposed wild-type group.
FIGURE 7
FIGURE 7
LPS-induced inflammatory responses in the lungs are alleviated in trpa1– /– mice. Mice were treated with an intraperitoneal injection of E. coli LPS (20 mg/kg) or PBS (the vehicle). Total protein contents (A), total cell count (B), and differential cell count (C) in the BALF at 24 h after PBS or LPS treatment as indications of lung inflammation. Levels of MIP-2 in BALF (D) and lung tissues (E) were analyzed by ELISA. Data in each group are mean ± SEM from eight mice. *P < 0.05 vs. the PBS-exposed group in both genotypes; #P < 0.05 vs. the LPS-exposed wild-type group.
FIGURE 8
FIGURE 8
LPS-induced ERK/JNK activation and oxidative stress in lung tissues are reduced in trpa1– /– mice. Mice received an intraperitoneal injection of E. coli LPS (20 mg/kg) or PBS (the vehicle). (A,B) The increased phosphorylation of kinases indicates the activation of ERK and JNK. (C) LPS-induced increase in oxidative stress was indicated by increased 4-HNE expression. Protein expression was analyzed by Western blot. Data in each group are mean ± SEM from eight mice. *P < 0.05 vs. the PBS-exposed group in both genotypes; #P < 0.05 vs. the LPS-exposed wild-type group.
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