Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression - PubMed (original) (raw)

Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression

Kieran A Ryan et al. Infect Immun. 2004 Apr.

Abstract

Toll-like receptor 4 (TLR4) has been identified as a transmembrane protein involved in the host innate immune response to gram-negative bacterial lipopolysaccharide (LPS). Upon activation by LPS recognition, the TIR domain of TLR4 signals through MyD88 to activate the nuclear factor kappa B (NF-kappa B) pathway, a critical regulator of many proinflammatory genes, including interleukin-8 (IL-8). Emerging evidence suggests that reactive oxygen species (ROS) can contribute to diverse signaling pathways, including the LPS-induced cascade. In the present study we investigated the role of ROS in TLR-mediated signaling. Purified Escherichia coli LPS, a highly specific TLR4 agonist, elicited an oxidative burst in the monocyte-like cell line THP-1 in a time- and dose-dependent manner. This oxidative burst was shown to be dependent on the presence of TLR4 through transfection studies in HEK cells, which do not normally express this protein, and with bone marrow-derived macrophages from C3H/HeJ mice, which express a mutated TLR4 protein. LPS-stimulated IL-8 expression could be blocked by the antioxidants N-acetyl-L-cysteine and dimethyl sulfoxide at both the protein and mRNA levels. These antioxidants also blocked LPS-induced IL-8 promoter transactivation as well as the nuclear translocation of NF-kappa B. These data provide evidence that ROS regulate immune signaling through TLR4 via their effects on NF-kappa B activation.

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Figures

FIG. 1.

LPS elicits a time- and dose-dependent oxidative burst. (A) THP-1 cells were incubated with the H2DCFDA dye, which is oxidized by hydroxyl radicals, peroxinitrite, and hydrogen peroxide. Subsequently, THP-1 cells were treated with various doses of LPS for 15 min. Dye oxidation was calculated by dividing the mean channel fluorescence of a treated sample by that of the untreated sample and multiplying by 100 to obtain the relative change expressed as a percentage. (B) H2DCFDA dye oxidation in THP-1 cells treated with 10 μg of LPS per ml for various times. Data shown are the medians of duplicates ± the upper and lower quartiles from five separate experiments. *, statistical differences between dye oxidation of LPS-treated and control cells were significant to an overall P value of ≤0.05 as determined with the Wilcoxon test with an adjusted Bonferroni correction.

FIG. 2.

LPS-induced oxidative burst was higher in cells expressing TLR4 compared to non-TLR4-expressing controls. The LPS had been purified to ensure that it was a highly specific TLR4 agonist and did not signal in the absence of TLR4. (A) Cells were incubated with H2DCFDA dye and then treated with various doses of LPS for 15 min. (B) Cells were incubated with H2DCFDA dye and then treated with 10 μg of LPS per ml for various times. •, TLR4-transfected HEK293 cells; ▴, empty vector-transfected HEK293cells. Data shown are from four separate experiments carried out in duplicate. (C) H2DCFDA dye oxidation in C57BL/6 and C3H/HeJ bone marrow-derived macrophages treated with 5 μg of LPS per ml for various times. •, C57BL/6; ▴, C3H/HeJ. Data shown are from three independent experiments. All data are expressed as the medians ± the upper and lower quartiles from three independent experiments. *, statistical differences between dye oxidation of LPS-treated and control cells were significant to an overall P value of ≤0.05, as determined with the Wilcoxon test with an adjusted Bonferroni correction.

FIG. 3.

LPS-induced IL-8 secretion is inhibited by the antioxidants NAC, DMSO, and MEG. THP-1 cells were incubated with various antioxidants and then treated with 10 μg of LPS per ml for 4 h. Supernatants were collected and tested for IL-8 protein by ELISA. Data are expressed as the means ± standard error of the mean of three independent experiments carried out in duplicate. Statistical differences between LPS alone and LPS plus antioxidant samples are indicated; *, overall P value of ≤0.05, as determined with Student's t test and an adjusted Bonferroni correction.

FIG. 4.

IL-8 secretion induced by sodium nitroprusside and xanthine oxidase is eliminated by NAC, DMSO, and MEG. THP-1 cells were incubated with various antioxidants and then treated with a combination of sodium nitroprusside and xanthine oxidase for 4 h. Supernatants were collected and tested for IL-8 protein by ELISA. Data are expressed as the means ± standard error of the mean of three independent experiments carried out in duplicate. Statistical differences between samples treated with sodium nitroprusside and xanthine oxidase alone and sodium nitroprusside and xanthine oxidase plus antioxidant are indicated; *, overall P value of ≤0.05, as determined with Student's t test and an adjusted Bonferroni correction.

FIG. 5.

IL-8 mRNA expression induced by LPS is inhibited by NAC and DMSO. THP-1 cells were incubated with various antioxidants and then treated with 10 μg of LPS per ml for 4 h. Total RNA was extracted and reverse transcribed. IL-8 mRNA expression was assessed by real-time PCR. Data are expressed as the means ± standard error of the mean of four (NAC and DMSO) or three (all other samples) independent experiments carried out in duplicate. Statistical differences between LPS alone and LPS plus antioxidant samples are indicated; *, overall P value of ≤0.05, as determined with the Wilcoxon test with an adjusted Bonferroni correction.

FIG. 6.

LPS-induced IL-8 promoter transactivation was eliminated by NAC and DMSO. To assess if antioxidants could block transactivation of the IL-8 gene, IL-8 promoter reporter constructs were transfected into HEK293 cells expressing TLR4 or not. Cells were incubated with various antioxidants and then treated with 10 μg of LPS per ml for 4 h. The IL-8 promoter construct was not transactivated by LPS in cells lacking TLR4 (open bars). (A) Full-length IL-8 promoter transactivation in TLR4-expressing HEK293 cells (solid bars) or non-TLR4-expressing cells (open bars). (B) Truncated IL-8 promoter (lacking an AP-1 binding site) transactivation in TLR4-expressing HEK293 cells (solid bars) or non-TLR4-expressing cells (open bars). Transactivation was not dependent on the presence of an AP-1 binding site. Data are expressed as the means ± standard error of the mean of three independent experiments carried out in triplicate. Statistical differences between LPS alone and LPS plus antioxidant samples are indicated; *, an overall P value of ≤0.05, as determined with the Wilcoxon test with an adjusted Bonferroni correction.

FIG. 7.

Nuclear translocation of NF-κB is sensitive to antioxidant treatment with NAC in THP-1 cells. (A) LPS induced the nuclear translocation of NF-κB protein after 15 min, which continued until 60 min, and NAC decreased NF-κB translocation at all time points. (B) Supershift analysis of nuclear protein from LPS-treated cells showed that NF-κB complexes contain c-rel, p50, and possibly p65 protein. The gels shown are representative of three independent experiments.

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Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression - PubMed (original) (raw)