Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR

Abstract

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca²⁺-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Keywords: IL-33; airway epithelium; asthma; cigarette smoke; cysteine oxidation; dual oxidase 1.

Fig. 1.

Acrolein (ACR) inhalation suppresses innate airway allergen responses. A: C57BL/6 mice, naive or ACR (5 ppm) exposed for 4 h, were challenged with HDM, and BAL levels of IL-33, IL-1α, and IL-25/IL-17E were measured by ELISA. Mean values ± SE from 3 independent experiments are shown. Naive n = 6, HDM n = 12, ACR n = 6, HDM/ACR n = 16; *P < 0.05. B: MTE cells were exposed to ACR for 30 min followed by exposure to HDM. At indicated time points, conditioned media were analyzed for IL-33, IL-1α, or IL-17E. C: MTE cells were exposed to ACR (30 min) and challenged with HDM, ALT, or ATP for 2 h, and production of IL-33, IL-1α, and IL-17E was measured. *P < 0.05 by 1-way ANOVA. D: HBE1 cells were pretreated with ACR (30 min) prior to stimulation with HDM, ALT, or ATP for 2 h. Production of IL-33 or IL-1α was measured after 2 h. Data represent average of 2 experiments with 2 replicates each. *P < 0.05.

Fig. 2.

Different thiol-reactive electrophiles inhibit allergen-induced cytokine production by MTE cells. MTE cells were treated with either ACR, curcumin (CUR), cinnemaldehyde (CINN), or sulforaphane (SFN) for 30 min and subsequently stimulated with HDM for 2 h. A: molecular structures of different electrophiles. B: ELISA analysis of cytokine production in conditioned media. *P < 0.05.

Fig. 3.

ACR inhibition of allergen-induced cytokine production is independent of Ca²⁺ signaling. HBE1 cells were preloaded with FluoForte AM and pretreated with ACR before stimulation with HDM for analysis of intracellular Ca²⁺ increase. A: representative kinetic increases in Ca²⁺-induced fluorescence. B: quantitative increase in Ca²⁺ fluorescence measured 10 min after HDM challenge. *P < 0.05 compared with corresponding control.

Fig. 4.

ACR and related thiol-reactive electrophiles inhibit allergen-induced activation of EGFR, ERK1/2, and STAT3. A and B: HBE1 cells were pretreated with ACR (30 min) prior to stimulation with HDM or ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3, as well as their unphosphorylated forms, were analyzed by Western blot. A: representative Western blots. B: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3, normalized to unphosphorylated proteins, from 3 experiments in duplicate. *P < 0.05. C and D: HBE1 cells were pretreated with ACR, CUR, CINN, or SFN (30 min) prior to stimulation with ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3 was determined by Western blot. C: representative Western blots. D: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3. *P < 0.05.

Fig. 5.

ACR directly inhibits EGFR activation by modification of C797. A and B: HBE1 cells were exposed to ACR (30 min) and stimulated with EGF (100 ng/ml) for 10 min for analysis of activation of EGFR, STAT3, and ERK 1/2 by Western blot as in Fig. 4. Representative blots (A) and densitometry data from 2 independent experiments (B) are shown. *P < 0.05 compared with untreated control; #P < 0.05 compared with control EGF treatment. C: effect of ACR treatment (30 min) of recombinant EGFR kinase domain on tyrosine kinase activity. D: kinase activity analysis of wild-type and C797S variant form of EGFR. The kinase activity of wild-type enzyme was significantly reduced due to the effect of ACR while C797S variant's activity remained unchanged. *P < 0.05.

Fig. 6.

ACR and other thiol-reactive electrophiles inhibit allergen-induced H₂O₂ production by HBE cells. A: HBE1 cells were pretreated with ACR (30 min) prior to stimulation with HDM, ALT, or ATP, and production of extracellular H₂O₂ in conditioned medium was measured after 15 min as described in materials and methods. B: dose-dependent inhibition of ATP-induced H₂O₂ production by ACR, SFN, or CINN. Data were expressed relative to ATP-stimulated H₂O₂ production in the absence of electrophiles and corrected for basal H₂O₂ production in the absence of ATP. C: inhibition of ionomycin-stimulated H₂O₂ production by ACR, SFN, or CINN (30 μM). *P < 0.05.

Fig. 7.

ACR exposure induces cysteine modification and carbonylation within DUOX1, EGFR, and Src. A and B: untreated or ACR-treated HBE1 cells (30 min) were lysed in the presence of the thiol-specific reagent EZ-Link Iodoacetyl-LC-Biotin, and avidin-purified proteins as well as whole cell lysates were analyzed by Western blot for DUOX1, EGFR, and Src. A: representative blots. B: densitometry analysis of relative thiol content of DUOX1, EGFR, or Src from 2 experiments in duplicate. *P < 0.05. C: untreated or ACR-treated HBE1 cells (30 min) were derivatized with ARP, and avidin-purified proteins or whole cell lysates (input controls) were evaluated by Western blot for DUOX1, EGFR, or Src. D: mice were exposed to ACR (5 ppm; 4 h) or filtered air, and lung homogenates were derivatized with biotin hydrazide for similar analysis of carbonylated proteins. Representative blots from 2–3 separate experiments are shown.