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Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production.

Chen J, Kinter M, Shank S, Cotton C, Kelley TJ, Ziady AG - PLoS ONE (2008)

Bottom Line: We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized.The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2.We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA.

ABSTRACT
Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

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Inhibition of CFTR activity decreases Nrf-2 activity and increases H2O2 levels.Normal and CF matched cell pairs incubated with 20 µM CFTRinh-172 are co-transfected with either a plasmid coding for Firefly luciferase driven by a Nrf-2 (Panel A) or Nrf-1 (Panel B) promoter, and one coding for Renilla luciferase driven by the CMV promoter. Three days following transfection, normalized luciferase activity is measured. Panel C: H2O2 levels in cells incubated with CFTRinh-172 for 72 hrs and stimulated with TNFα/IL-1β (10 ng/ml each). * connotes significant difference (p<0.05) from respective uninhibited controls. Each data bar represents the average of 8 replicate wells in 3 experiments for A, B, and C.
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pone-0003367-g005: Inhibition of CFTR activity decreases Nrf-2 activity and increases H2O2 levels.Normal and CF matched cell pairs incubated with 20 µM CFTRinh-172 are co-transfected with either a plasmid coding for Firefly luciferase driven by a Nrf-2 (Panel A) or Nrf-1 (Panel B) promoter, and one coding for Renilla luciferase driven by the CMV promoter. Three days following transfection, normalized luciferase activity is measured. Panel C: H2O2 levels in cells incubated with CFTRinh-172 for 72 hrs and stimulated with TNFα/IL-1β (10 ng/ml each). * connotes significant difference (p<0.05) from respective uninhibited controls. Each data bar represents the average of 8 replicate wells in 3 experiments for A, B, and C.

Mentions: Since the loss of CFTR function (9HTEo− cell pair, and CFTR inhibited wdHPTE) or expression (16HBEo− cell pair) is the defining difference in our CF cell pairs, inhibiting CFTR in the normal cell pairs should produce the aberrations in H2O2 processing and Nrf-2 activity we observed in previous experiments. Therefore, to test this hypothesis, we used a pharmacological agent, CFTRinh-172, to inhibit CFTR activity in normal cell line controls. In CF cells, inhibition of CFTR for 72 hrs did not affect Nrf-2 activity (Figure 5a). In normal cells however, inhibition of CFTR significantly decreased Nrf-2 activity vs. non-inhibited control, by ∼80% in the 16HBEo− and ∼70% in the 9HTEo− cells, in the presence or absence of inflammatory stimulation. These findings were Nrf-2 specific as no significant differences were observed for Nrf-1 activity (Figure 5b). Decreases in Nrf-2 activity in CFTR inhibited normal cells correlates with a significant 3- to 4-fold increase in H2O2 versus non-inhibited controls (Figure 5c). No significant increase is observed in the CF cells lines, consistent with an already existing lack of CFTR function in these cells.


Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production.

Chen J, Kinter M, Shank S, Cotton C, Kelley TJ, Ziady AG - PLoS ONE (2008)

Inhibition of CFTR activity decreases Nrf-2 activity and increases H2O2 levels.Normal and CF matched cell pairs incubated with 20 µM CFTRinh-172 are co-transfected with either a plasmid coding for Firefly luciferase driven by a Nrf-2 (Panel A) or Nrf-1 (Panel B) promoter, and one coding for Renilla luciferase driven by the CMV promoter. Three days following transfection, normalized luciferase activity is measured. Panel C: H2O2 levels in cells incubated with CFTRinh-172 for 72 hrs and stimulated with TNFα/IL-1β (10 ng/ml each). * connotes significant difference (p<0.05) from respective uninhibited controls. Each data bar represents the average of 8 replicate wells in 3 experiments for A, B, and C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563038&req=5

pone-0003367-g005: Inhibition of CFTR activity decreases Nrf-2 activity and increases H2O2 levels.Normal and CF matched cell pairs incubated with 20 µM CFTRinh-172 are co-transfected with either a plasmid coding for Firefly luciferase driven by a Nrf-2 (Panel A) or Nrf-1 (Panel B) promoter, and one coding for Renilla luciferase driven by the CMV promoter. Three days following transfection, normalized luciferase activity is measured. Panel C: H2O2 levels in cells incubated with CFTRinh-172 for 72 hrs and stimulated with TNFα/IL-1β (10 ng/ml each). * connotes significant difference (p<0.05) from respective uninhibited controls. Each data bar represents the average of 8 replicate wells in 3 experiments for A, B, and C.
Mentions: Since the loss of CFTR function (9HTEo− cell pair, and CFTR inhibited wdHPTE) or expression (16HBEo− cell pair) is the defining difference in our CF cell pairs, inhibiting CFTR in the normal cell pairs should produce the aberrations in H2O2 processing and Nrf-2 activity we observed in previous experiments. Therefore, to test this hypothesis, we used a pharmacological agent, CFTRinh-172, to inhibit CFTR activity in normal cell line controls. In CF cells, inhibition of CFTR for 72 hrs did not affect Nrf-2 activity (Figure 5a). In normal cells however, inhibition of CFTR significantly decreased Nrf-2 activity vs. non-inhibited control, by ∼80% in the 16HBEo− and ∼70% in the 9HTEo− cells, in the presence or absence of inflammatory stimulation. These findings were Nrf-2 specific as no significant differences were observed for Nrf-1 activity (Figure 5b). Decreases in Nrf-2 activity in CFTR inhibited normal cells correlates with a significant 3- to 4-fold increase in H2O2 versus non-inhibited controls (Figure 5c). No significant increase is observed in the CF cells lines, consistent with an already existing lack of CFTR function in these cells.

Bottom Line: We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized.The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2.We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA.

ABSTRACT
Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

Show MeSH
Related in: MedlinePlus