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

Hsin-Kuo Ko,Tzong-Shyuan Lee,An-Hsuan Lin,Diahn-Warng Perng,Yu Ru Kou

doi:10.3389/fphys.2020.596314

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.

Highlights

Sepsis is a systemic inflammatory state manifested by a complex interaction between infective microorganisms and host immune responses (Cecconi et al, 2018)
We explored the role of Transient receptor potential ankyrin 1 (TRPA1) in the inflammatory responses of human bronchial epithelial cells (HBECs) to LPS
The exposure of HBECs to LPS for 24 h increased the intracellular (Figure 2A) and extracellular expression of interleukin 8 (IL-8) (Figure 2B), which were largely or nearly prevented by pretreatment with the TRPA1 antagonist, HC030031 (9 μM); this dose was adapted from our recent study (Lin et al, 2015)

Summary

Introduction

Sepsis is a systemic inflammatory state manifested by a complex interaction between infective microorganisms and host immune responses (Cecconi et al, 2018). The recognition of pathogens by lung epithelial cells plays an important role in the lung inflammation induced by LPS (Diamond et al, 2000; Bals and Hiemstra, 2004). Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for LPS (Neyen and Lemaitre, 2016), and its activation induces lung inflammation (Chen et al, 2019) or inflammatory responses in lung epithelial cells (Liu et al, 2018). TRPA1 mediates LPS-induced lung inflammation in mice (Liu et al, 2020) or inflammatory responses to LPS in vitro (Prandini et al, 2016; Yin et al, 2018). The role of TRPA1 in LPS-induced inflammation has not been conclusively defined

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