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Acrolein exposure suppresses antigen-induced pulmonary inflammation.

Spiess PC, Kasahara D, Habibovic A, Hristova M, Randall MJ, Poynter ME, van der Vliet A - Respir. Res. (2013)

Bottom Line: Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling.Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, College of Medicine, D205 Given Building, 89 Beaumont Ave, Burlington, VT 05405, USA. albert.van-der-vliet@uvm.edu.

ABSTRACT

Background: Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma.

Methods: C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14-16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14-17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity.

Results: Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.

Conclusion: Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.

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Acrolein exposure activates the Nrf2 pathway. Lung tissue homogenates or lysis lavage samples were collected 6 hrs after a single OVA challenge and/or acrolein exposure. (A) Analysis of HO-1 protein levels in lysis lavage samples by Western blot.(B) PCR analysis of Gclm and Gclc in lung tissues. (C) Evaluation of biotin hydrazide-labeled proteins and whole lung lysates for the Nrf2 repressor Keap1. Band densities were quantified using Image J software. Results are expressed as mean ± SEM (n = 3-6/group) (*, p < 0.05).
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Figure 6: Acrolein exposure activates the Nrf2 pathway. Lung tissue homogenates or lysis lavage samples were collected 6 hrs after a single OVA challenge and/or acrolein exposure. (A) Analysis of HO-1 protein levels in lysis lavage samples by Western blot.(B) PCR analysis of Gclm and Gclc in lung tissues. (C) Evaluation of biotin hydrazide-labeled proteins and whole lung lysates for the Nrf2 repressor Keap1. Band densities were quantified using Image J software. Results are expressed as mean ± SEM (n = 3-6/group) (*, p < 0.05).

Mentions: The transcription factor Nrf2 plays a key role in redox homeostasis by regulating the activation of glutathione synthesis and antioxidant defense genes, such as HO-1 [47]. To determine if Nrf2 was activated within the airway epithelium following acrolein exposure, lysis lavage samples from animals exposed to a single OVA challenge and acrolein exposure were analyzed for Nrf2-target protein HO-1. Indeed, acrolein exposure resulted in significant induction of HO-1 in airway epithelial cells (Figure 6A). Nrf2 also transcriptionally regulates the catalytic (Gclc) and regulatory (Gclm) subunits of glutamate-cysteine ligase, the rate limiting enzyme in GSH synthesis [48] and acrolein exposure was found to increase lung tissue mRNA expression of Gclm and Gclc, reaching statistical significance in acrolein-exposed OVA/OVA mice vs non-exposed OVA/OVA mice in case of Gclm (Figure 6B). We next determined Nrf2 protein accumulation in whole lung tissue lysates, as an indicator of Nrf2 activation, and observed significant increases in Nrf2 in lungs from acrolein-exposed mice, in both sham- and OVA-sensitized animals (Figure 6C). Consistent with the observed accumulation and apparent activation of Nrf2, we observed increased carbonylation of Keap1 in lung homogenates following acrolein exposure as detected by biotin hydrazide labeling and Western blot analysis (Figure 6C), indicating direct alkylation of Keap1 by acrolein through Michael addition to its cysteine residues [49]. Interestingly, the extent of acrolein-induced Nrf2 accumulation and Keap1 alkylation appeared to be reduced in OVA-sensitized and challenged mice compared to sham-sensitized mice, although this did not reach statistical significance. Together, these results indicate that acrolein exposure results in alkylation of Keap1 and thereby leads to activation of the Nrf2 pathway in airway epithelial cells, resulting in increased expression of antioxidant and anti-inflammatory genes, which could contribute to the inhibitory effects of acrolein on OVA-induced allergic inflammation.


Acrolein exposure suppresses antigen-induced pulmonary inflammation.

Spiess PC, Kasahara D, Habibovic A, Hristova M, Randall MJ, Poynter ME, van der Vliet A - Respir. Res. (2013)

Acrolein exposure activates the Nrf2 pathway. Lung tissue homogenates or lysis lavage samples were collected 6 hrs after a single OVA challenge and/or acrolein exposure. (A) Analysis of HO-1 protein levels in lysis lavage samples by Western blot.(B) PCR analysis of Gclm and Gclc in lung tissues. (C) Evaluation of biotin hydrazide-labeled proteins and whole lung lysates for the Nrf2 repressor Keap1. Band densities were quantified using Image J software. Results are expressed as mean ± SEM (n = 3-6/group) (*, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852782&req=5

Figure 6: Acrolein exposure activates the Nrf2 pathway. Lung tissue homogenates or lysis lavage samples were collected 6 hrs after a single OVA challenge and/or acrolein exposure. (A) Analysis of HO-1 protein levels in lysis lavage samples by Western blot.(B) PCR analysis of Gclm and Gclc in lung tissues. (C) Evaluation of biotin hydrazide-labeled proteins and whole lung lysates for the Nrf2 repressor Keap1. Band densities were quantified using Image J software. Results are expressed as mean ± SEM (n = 3-6/group) (*, p < 0.05).
Mentions: The transcription factor Nrf2 plays a key role in redox homeostasis by regulating the activation of glutathione synthesis and antioxidant defense genes, such as HO-1 [47]. To determine if Nrf2 was activated within the airway epithelium following acrolein exposure, lysis lavage samples from animals exposed to a single OVA challenge and acrolein exposure were analyzed for Nrf2-target protein HO-1. Indeed, acrolein exposure resulted in significant induction of HO-1 in airway epithelial cells (Figure 6A). Nrf2 also transcriptionally regulates the catalytic (Gclc) and regulatory (Gclm) subunits of glutamate-cysteine ligase, the rate limiting enzyme in GSH synthesis [48] and acrolein exposure was found to increase lung tissue mRNA expression of Gclm and Gclc, reaching statistical significance in acrolein-exposed OVA/OVA mice vs non-exposed OVA/OVA mice in case of Gclm (Figure 6B). We next determined Nrf2 protein accumulation in whole lung tissue lysates, as an indicator of Nrf2 activation, and observed significant increases in Nrf2 in lungs from acrolein-exposed mice, in both sham- and OVA-sensitized animals (Figure 6C). Consistent with the observed accumulation and apparent activation of Nrf2, we observed increased carbonylation of Keap1 in lung homogenates following acrolein exposure as detected by biotin hydrazide labeling and Western blot analysis (Figure 6C), indicating direct alkylation of Keap1 by acrolein through Michael addition to its cysteine residues [49]. Interestingly, the extent of acrolein-induced Nrf2 accumulation and Keap1 alkylation appeared to be reduced in OVA-sensitized and challenged mice compared to sham-sensitized mice, although this did not reach statistical significance. Together, these results indicate that acrolein exposure results in alkylation of Keap1 and thereby leads to activation of the Nrf2 pathway in airway epithelial cells, resulting in increased expression of antioxidant and anti-inflammatory genes, which could contribute to the inhibitory effects of acrolein on OVA-induced allergic inflammation.

Bottom Line: Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling.Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, College of Medicine, D205 Given Building, 89 Beaumont Ave, Burlington, VT 05405, USA. albert.van-der-vliet@uvm.edu.

ABSTRACT

Background: Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma.

Methods: C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14-16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14-17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity.

Results: Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.

Conclusion: Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.

Show MeSH
Related in: MedlinePlus