Benzo[ a]pyrene induces NLRP1 expression and promotes prolonged inflammasome signaling

Abstract

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon in the air, triggers pulmonary inflammation. This study focused on BaP-induced inflammation in the alveolar epithelium. A549 cells were stimulated with BaP for four days. BaP treatment markedly increased NLRP1 expression but slightly decreased NLRP3. Furthermore, aryl hydrocarbon receptor (AhR) knockdown displayed no increase in BaP-induced NLRP1 expression. Similar results were also observed by blocking reactive oxygen species (ROS), which is mediated through AhR, suggesting that the AhR-ROS axis operates in BaP-induced NLRP1 expression. p53 involvement in ROS-mediated NLRP1 induction has also been implied. When we confirmed inflammasome activation in cells treated with BaP for four days, while BaP transiently activated NLRP3, it predominantly activated the NLRP1 inflammasome. These findings have led to the conclusion that BaP could be a potential ligand for the NLRP1 inflammasome persistently observed in the lung epithelium. Our study may provide additional evidence for the sustained pulmonary inflammation caused by environmental air pollution.

Keywords: NLRP1; airborne pollutants; aryl hydrocarbon receptor; benzo[a]pyrene; inflammasome; lung epithelium; reactive oxygen species.

Figure 1

PM_2.5 enhances NLRP1 expression in A549 cells. Expression of inflammasome-related PRRs in A549 cells. A549 cells were exposed to PM_2.5 at 2 µg/mL for four days. PM_2.5 was dissolved in deionized water to produce 20 µg/mL stock solutions, which were then added to the cell culture at the final concentration. Data are presented as the mean ± standard error. n = 3 independent experiments. *p<0.05; N.D, not detected. Control vehicle-treated control.

Figure 2

BaP enhances NLRP1 expression in A549 cells. BaP was dissolved in DMSO (final concentration, 0.05%) and added to A549 cells. (A) Expression of inflammasome-related PRRs in A549 cells. The cells were treated with 2 μM BaP for four days. (B) NLRP1 expression followed by BaP (0–2000 nM) for 0–4 days. (C) NLRP1 protein expression levels after four days of BaP treatment (2000 nM). Data are presented as the mean ± standard error. n = 4–9 independent experiments. **p<0.01, ***p<0.001; Control vehicle-treated control.

Figure 3

AhR knockdown by siRNA abrogates the BaP-induced NLRP1 expression. AhR participation in the enhanced NLRP1 expression in BaP-treated A549 cells was determined using AhR KD cells and an AhR antagonist. (A) AhR mRNA and protein expression in response to specific siRNAs. (B) NLRP1 expression after 2 µM BaP exposure for two (mRNA) or four (protein) days in AhR-knockdown cells and control cells. (C) Pro-IL-1β, ASC, and pro-Caspase-1 expressions after 2 µM BaP exposure for two days in AhR-knockdown cells and control cells. Data are presented as the mean ± standard error. n = 4–7 independent experiments; *p<0.05, **p<0.01.

Figure 4

Effects of FICZ, a high-affinity ligand for AhR, on the NLRP1 expression in A549 cells. FICZ was dissolved in DMSO (final concentration, 0.05%) and added to A549 cells. NLRP-1, Pro-IL-1β, ASC, and pro-Caspase-1 expression at mRNA (A) and protein (B) levels. The cells were treated with 500 nM FICZ for 0–4 days. (C) CYP1A1 induction by BaP and FICZ treatment. Cells were treated with BaP (2 µM) and FICZ (500 nM) for four days. Data are presented as the mean ± standard error. n = 3–9 independent experiments. n.s., not significant.

Figure 5

ROS generation is involved in the BaP-induced NLRP1 expression. The involvement of ROS was measured in BaP-treated A549 cells using NAC as an antioxidant. (A, B) NAC was added to the cells at a concentration of 5 μM 1 h prior to BaP application. Abrogated BaP-induced NLRP1 and p20 expression after NAC treatment were analyzed. Densitometry analysis of the western blot data of NLRP1 and p20 is shown in the bar graphs. Expression of NLRP1 mRNA (C) and protein (D) under H₂O₂ treatment (1 mM). (E) Analysis of components of the inflammasome protein complex in the H₂O₂-treated A549. The cells were then exposed to H₂O₂ at 1 mM for 9 h. Representative images (scale bar = 10 μm) of confocal microscopic observations of H₂O₂-treated cells. (F, G) AhR KD and control cells were exposed to BaP (F) at 2 µM and FICZ (G) at 500 nM for four days. The induced ROS were analyzed using flow cytometry, and the fold production of ROS was represented as the mean fluorescence intensity of DCF. Data are presented as the mean ± standard error. n = 3–4 independent experiments. *p<0.05, **p<0.01; NAC, N-acetyl-l-cysteine.; gMFI, geometric mean fluorescence intensity; DCF, dichlorofluorescein.

Figure 6

Determination of the role of p53 in the BaP-induced NLRP1 expression. When p53 was knocked down using TP53-siRNA in A549 cells, BaP-induced NLRP1 expression was attenuated. (A) Phosphorylation of p53 in BaP-treated cells. Densitometry analysis of the western blot data of p53 phosphorylation is shown in the bar graph. (B) Expression levels of p53 in response to specific siRNAs. (C) Expression of NLRP1 mRNA and protein in p53 knockdown and control cells. BaP (2 µM) was added and incubated for four days. Data are presented as the mean ± standard error. n = 3–4 independent experiments. *p<0.05, **p<0.01, ***p<0.0001.

Figure 7

Detection of inflammasome formation using co-immunoprecipitation assay. Analysis of inflammasome protein complex components in BaP-treated A549 cells. Cells were exposed to 2 µM BaP for four days. (A) Representative images (scale bar = 10 μm) and quantitative bar graphs of confocal microscopy observations of BaP-treated cells. (B) Representative immunoblot of the co-immunoprecipitation assay using an anti-ASC antibody. (C) time-dependent increase in p20 expression in response to BaP treatment. Densitometry analysis of the western blot data of IL-1β is shown in the bar graph. (D) IL-1β production in BaP-treated cells. Data are presented as the mean ± standard error. n = 3–6 independent experiments. ***p<0.001.

Figure 8

Proposed scheme for regulating NLR proteins by BaP in the lung epithelial cells. We demonstrated that BaP enhances NLRP1 expression in lung epithelial A549 cells and leads to activation of the NLRP1 inflammasome cascade in the cells. At the same time, BaP also regulates the NLRP3 inflammasome through an independent pathway. There is a time gap between NLRP1 and the NLRP3 inflammasome caused by BaP.