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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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Overexpression of Nrf-2 reduces H2O2 and increases total peroxidase levels.16HBEo− and 9HTEo− cell line pairs are co-transfected with either a mammalian expression plasmid for human Nrf-2 or the pCI-neo empty vector at 0.1 µg/5×105 cells/well. H2O2 and total peroxidase levels are measured 48 hrs following transfection, and normalized to total cell protein concentration. Measurements of cell line pairs in the absence or presence of TNFα/IL-1β (10 ng/ml each) are shown. Panel A: Verification of Nrf-2 expression by Western blot in cells transfected with the Nrf-2 expression plasmid. Band densities are used to calculate relative abundance. Panel B: H2O2 levels normalized to protein concentration. Panel C: Total peroxidase units normalized to protein concentration. * connotes significant difference compared with respective normal control (Sense or pCEP cells). Each data bar represents the average of 6 replicate wells in 4 experiments for B and C.
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pone-0003367-g006: Overexpression of Nrf-2 reduces H2O2 and increases total peroxidase levels.16HBEo− and 9HTEo− cell line pairs are co-transfected with either a mammalian expression plasmid for human Nrf-2 or the pCI-neo empty vector at 0.1 µg/5×105 cells/well. H2O2 and total peroxidase levels are measured 48 hrs following transfection, and normalized to total cell protein concentration. Measurements of cell line pairs in the absence or presence of TNFα/IL-1β (10 ng/ml each) are shown. Panel A: Verification of Nrf-2 expression by Western blot in cells transfected with the Nrf-2 expression plasmid. Band densities are used to calculate relative abundance. Panel B: H2O2 levels normalized to protein concentration. Panel C: Total peroxidase units normalized to protein concentration. * connotes significant difference compared with respective normal control (Sense or pCEP cells). Each data bar represents the average of 6 replicate wells in 4 experiments for B and C.

Mentions: Previous experiments suggest that a potential mechanism for the elevation of the inflammatory cytokines IL-6 and IL-8 in CF cells is an elevation of H2O2, which is mediated through the dysregulation of Nrf-2. To further validate this mechanist data, we tested the hypothesis that correcting Nrf-2 activity in CF cells would reverse the misprocessing of H2O2 and decrease excessive cytokine production. We used two approaches. First, we transfected CF cell pairs with an expression plasmid for Nrf-2 to increase expression to normal levels (Figure 6a). When the CF cells pairs over express Nrf-2, H2O2 decreases to levels not different from normal controls (Figure 6b), in contrast to non-transfected cell values (Figure 1). This decrease, in transfected CF cells pairs, is accompanied by a marked increase in peroxidase activity, consistent with an increase in antioxidant proteins expression (Figure 6c). No increase in Nrf-2 expression was observed in cells transfected with the pCI-neo vector (empty vector). Furthermore, transfection with an pCI-neo did not alter either peroxidase activity, or H2O2 levels in the CF 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)

Overexpression of Nrf-2 reduces H2O2 and increases total peroxidase levels.16HBEo− and 9HTEo− cell line pairs are co-transfected with either a mammalian expression plasmid for human Nrf-2 or the pCI-neo empty vector at 0.1 µg/5×105 cells/well. H2O2 and total peroxidase levels are measured 48 hrs following transfection, and normalized to total cell protein concentration. Measurements of cell line pairs in the absence or presence of TNFα/IL-1β (10 ng/ml each) are shown. Panel A: Verification of Nrf-2 expression by Western blot in cells transfected with the Nrf-2 expression plasmid. Band densities are used to calculate relative abundance. Panel B: H2O2 levels normalized to protein concentration. Panel C: Total peroxidase units normalized to protein concentration. * connotes significant difference compared with respective normal control (Sense or pCEP cells). Each data bar represents the average of 6 replicate wells in 4 experiments for B and C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563038&req=5

pone-0003367-g006: Overexpression of Nrf-2 reduces H2O2 and increases total peroxidase levels.16HBEo− and 9HTEo− cell line pairs are co-transfected with either a mammalian expression plasmid for human Nrf-2 or the pCI-neo empty vector at 0.1 µg/5×105 cells/well. H2O2 and total peroxidase levels are measured 48 hrs following transfection, and normalized to total cell protein concentration. Measurements of cell line pairs in the absence or presence of TNFα/IL-1β (10 ng/ml each) are shown. Panel A: Verification of Nrf-2 expression by Western blot in cells transfected with the Nrf-2 expression plasmid. Band densities are used to calculate relative abundance. Panel B: H2O2 levels normalized to protein concentration. Panel C: Total peroxidase units normalized to protein concentration. * connotes significant difference compared with respective normal control (Sense or pCEP cells). Each data bar represents the average of 6 replicate wells in 4 experiments for B and C.
Mentions: Previous experiments suggest that a potential mechanism for the elevation of the inflammatory cytokines IL-6 and IL-8 in CF cells is an elevation of H2O2, which is mediated through the dysregulation of Nrf-2. To further validate this mechanist data, we tested the hypothesis that correcting Nrf-2 activity in CF cells would reverse the misprocessing of H2O2 and decrease excessive cytokine production. We used two approaches. First, we transfected CF cell pairs with an expression plasmid for Nrf-2 to increase expression to normal levels (Figure 6a). When the CF cells pairs over express Nrf-2, H2O2 decreases to levels not different from normal controls (Figure 6b), in contrast to non-transfected cell values (Figure 1). This decrease, in transfected CF cells pairs, is accompanied by a marked increase in peroxidase activity, consistent with an increase in antioxidant proteins expression (Figure 6c). No increase in Nrf-2 expression was observed in cells transfected with the pCI-neo vector (empty vector). Furthermore, transfection with an pCI-neo did not alter either peroxidase activity, or H2O2 levels in the CF 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