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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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Steady state hydrogen peroxide levels are elevated in CF in the absence and presence of inflammatory stimulation.H2O2 levels were assayed in: A) Two immortalized cell line pair models of CF, the 16 HBEo− (sense and antisense (AS)) and 9 HTEo− (pCEP and pCEP-R), and; B) one polarized primary cell model, the wd-HPTE (untreated or treated with 20 µM CFTRinh-172 for 72 hrs.). Unstimulated cells are compared to cells harvested 24 hrs following 1 hr. incubation with TNF-α/IL-1β (10 ng/ml each). * connotes significant difference from unstimulated normal control (p<0.05), while ** connotes significant difference from both unstimulated and stimulated normal control. Each data bar represents the average of 8 replicate wells in 4 experiments for (A), or 3 replicate wells from donors 1 and 2, or 2 replicate wells for donor 3 in 1 experiment for (B).
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pone-0003367-g001: Steady state hydrogen peroxide levels are elevated in CF in the absence and presence of inflammatory stimulation.H2O2 levels were assayed in: A) Two immortalized cell line pair models of CF, the 16 HBEo− (sense and antisense (AS)) and 9 HTEo− (pCEP and pCEP-R), and; B) one polarized primary cell model, the wd-HPTE (untreated or treated with 20 µM CFTRinh-172 for 72 hrs.). Unstimulated cells are compared to cells harvested 24 hrs following 1 hr. incubation with TNF-α/IL-1β (10 ng/ml each). * connotes significant difference from unstimulated normal control (p<0.05), while ** connotes significant difference from both unstimulated and stimulated normal control. Each data bar represents the average of 8 replicate wells in 4 experiments for (A), or 3 replicate wells from donors 1 and 2, or 2 replicate wells for donor 3 in 1 experiment for (B).

Mentions: In initial studies we compared the cultured CF model cell pairs 9HTEo− pCEP (normal) and pCEP-R (CF), and the 16HBEo− S (normal) and AS (CF). We examined H2O2 levels in CF versus normal cells. In immortalized cell lines, unstimulated CF cells produce significantly higher levels of H2O2 compared with normal matched pairs (Figure 1a). These levels are further increased 24 hrs following stimulation with a cocktail of the inflammatory cytokines TNF-α/IL-1β (Figure 1a). Our measurements reflect steady state H2O2 in the cells. Since significant increases were observed in the 9HTEo− pCEP-R which express CFTR but lack its function, we tested the effect of the inhibition of CFTR on steady state H2O2 levels in well differentiated human primary tracheal epithelia (wdHPTE) from three different donors. Levels of peroxide significantly increased following pharmacological inhibition of CFTR for 72 hrs with 20 µM CFTRinh-172 (Figure 1b), compared to “same donor” cells that were not treated with inhibitor. Stimulation with TNF-α/IL-1β further increased H2O2 levels (Figure 1b). These results were reproduced in cells from all three donors.


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)

Steady state hydrogen peroxide levels are elevated in CF in the absence and presence of inflammatory stimulation.H2O2 levels were assayed in: A) Two immortalized cell line pair models of CF, the 16 HBEo− (sense and antisense (AS)) and 9 HTEo− (pCEP and pCEP-R), and; B) one polarized primary cell model, the wd-HPTE (untreated or treated with 20 µM CFTRinh-172 for 72 hrs.). Unstimulated cells are compared to cells harvested 24 hrs following 1 hr. incubation with TNF-α/IL-1β (10 ng/ml each). * connotes significant difference from unstimulated normal control (p<0.05), while ** connotes significant difference from both unstimulated and stimulated normal control. Each data bar represents the average of 8 replicate wells in 4 experiments for (A), or 3 replicate wells from donors 1 and 2, or 2 replicate wells for donor 3 in 1 experiment for (B).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2563038&req=5

pone-0003367-g001: Steady state hydrogen peroxide levels are elevated in CF in the absence and presence of inflammatory stimulation.H2O2 levels were assayed in: A) Two immortalized cell line pair models of CF, the 16 HBEo− (sense and antisense (AS)) and 9 HTEo− (pCEP and pCEP-R), and; B) one polarized primary cell model, the wd-HPTE (untreated or treated with 20 µM CFTRinh-172 for 72 hrs.). Unstimulated cells are compared to cells harvested 24 hrs following 1 hr. incubation with TNF-α/IL-1β (10 ng/ml each). * connotes significant difference from unstimulated normal control (p<0.05), while ** connotes significant difference from both unstimulated and stimulated normal control. Each data bar represents the average of 8 replicate wells in 4 experiments for (A), or 3 replicate wells from donors 1 and 2, or 2 replicate wells for donor 3 in 1 experiment for (B).
Mentions: In initial studies we compared the cultured CF model cell pairs 9HTEo− pCEP (normal) and pCEP-R (CF), and the 16HBEo− S (normal) and AS (CF). We examined H2O2 levels in CF versus normal cells. In immortalized cell lines, unstimulated CF cells produce significantly higher levels of H2O2 compared with normal matched pairs (Figure 1a). These levels are further increased 24 hrs following stimulation with a cocktail of the inflammatory cytokines TNF-α/IL-1β (Figure 1a). Our measurements reflect steady state H2O2 in the cells. Since significant increases were observed in the 9HTEo− pCEP-R which express CFTR but lack its function, we tested the effect of the inhibition of CFTR on steady state H2O2 levels in well differentiated human primary tracheal epithelia (wdHPTE) from three different donors. Levels of peroxide significantly increased following pharmacological inhibition of CFTR for 72 hrs with 20 µM CFTRinh-172 (Figure 1b), compared to “same donor” cells that were not treated with inhibitor. Stimulation with TNF-α/IL-1β further increased H2O2 levels (Figure 1b). These results were reproduced in cells from all three donors.

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