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Epithelial expression of TLR4 is modulated in COPD and by steroids, salmeterol and cigarette smoke.

MacRedmond RE, Greene CM, Dorscheid DR, McElvaney NG, O'Neill SJ - Respir. Res. (2007)

Bottom Line: We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged.Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein.The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland. rmacredmond@mrl.ubc.ca

ABSTRACT
The toll-like receptors (TLRs) are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10-6 M) caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.

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Cigarette smoke downregulates TLR4 gene and protein expression in A549 cells resulting in relative hyporesponsiveness to LPS. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium or cigarette smoke condensates for 4 hours. Cigarette smoke condensates were prepared as described in the methods and numbers correspond to serial dilutions of the initial cigarette smoke extract. A. Following treatment, total RNA was extracted, reverse transcribed into cDNA and used as a template for semi-quantitative PCR reactions using TLR4 and GAPDH gene-specific primers. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P ≤ 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with anti-TLR4 antibody. Data are representative of three separate experiments. (CSE † cigarette smoke extract). Because actin is not compartmentalised to the membrane, equal protein loading is demonstrated with a panel from the Ponsceau Stain. C. Viability assay of A549 cells following treatment with CSE. Data are representative of three separate experiments. D. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in low-serum (1% FCS) medium and were left untreated or incubated with serial dilutions of CSE × 4 hours. Following treatment with CSE cells were stimulated with LPS 10 μg/ml for a further 24 hours. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as pg/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (* signifies P ≤ 0.05 of observed effect vs. control, † signifies P ≤ 0.05 of observed effect vs. control plus LPS).
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Figure 2: Cigarette smoke downregulates TLR4 gene and protein expression in A549 cells resulting in relative hyporesponsiveness to LPS. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium or cigarette smoke condensates for 4 hours. Cigarette smoke condensates were prepared as described in the methods and numbers correspond to serial dilutions of the initial cigarette smoke extract. A. Following treatment, total RNA was extracted, reverse transcribed into cDNA and used as a template for semi-quantitative PCR reactions using TLR4 and GAPDH gene-specific primers. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P ≤ 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with anti-TLR4 antibody. Data are representative of three separate experiments. (CSE † cigarette smoke extract). Because actin is not compartmentalised to the membrane, equal protein loading is demonstrated with a panel from the Ponsceau Stain. C. Viability assay of A549 cells following treatment with CSE. Data are representative of three separate experiments. D. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in low-serum (1% FCS) medium and were left untreated or incubated with serial dilutions of CSE × 4 hours. Following treatment with CSE cells were stimulated with LPS 10 μg/ml for a further 24 hours. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as pg/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (* signifies P ≤ 0.05 of observed effect vs. control, † signifies P ≤ 0.05 of observed effect vs. control plus LPS).

Mentions: We next examined the effect of cigarette smoke on expression of TLR4 in respiratory epithelium in vitro. There was a dose dependant downregulation in TLR4 mRNA (Figure 2A) and protein (Figure 2B) following exposure to the cigarette smoke extracts. To ensure that the effect was not caused by direct toxicity of the cigarette smoke, a viability assay was performed which demonstrated 50% reduction in viability with undiluted CSE, but no toxic effect following dilution of the extracts (Figure 2C) which showed no significant difference in viability compared to untreated cells. We went on to examine functional effect by IL-8 ELISA. As expected, CSE has some direct inflammatory effect resulting in IL-8 production at dilute concentrations. However, concordant with the reduced expression of TLR4, the respiratory epithelial cells have dose dependent reduced secretion of IL-8 following treatment with higher concentrations of CSE, both with and without additional LPS (Figure 2D). Failure of the cells to produce any IL-8 following exposure to undiluted CSE may be a result of the direct toxic effects demonstrated in the viability assay.


Epithelial expression of TLR4 is modulated in COPD and by steroids, salmeterol and cigarette smoke.

MacRedmond RE, Greene CM, Dorscheid DR, McElvaney NG, O'Neill SJ - Respir. Res. (2007)

Cigarette smoke downregulates TLR4 gene and protein expression in A549 cells resulting in relative hyporesponsiveness to LPS. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium or cigarette smoke condensates for 4 hours. Cigarette smoke condensates were prepared as described in the methods and numbers correspond to serial dilutions of the initial cigarette smoke extract. A. Following treatment, total RNA was extracted, reverse transcribed into cDNA and used as a template for semi-quantitative PCR reactions using TLR4 and GAPDH gene-specific primers. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P ≤ 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with anti-TLR4 antibody. Data are representative of three separate experiments. (CSE † cigarette smoke extract). Because actin is not compartmentalised to the membrane, equal protein loading is demonstrated with a panel from the Ponsceau Stain. C. Viability assay of A549 cells following treatment with CSE. Data are representative of three separate experiments. D. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in low-serum (1% FCS) medium and were left untreated or incubated with serial dilutions of CSE × 4 hours. Following treatment with CSE cells were stimulated with LPS 10 μg/ml for a further 24 hours. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as pg/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (* signifies P ≤ 0.05 of observed effect vs. control, † signifies P ≤ 0.05 of observed effect vs. control plus LPS).
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Related In: Results  -  Collection

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Figure 2: Cigarette smoke downregulates TLR4 gene and protein expression in A549 cells resulting in relative hyporesponsiveness to LPS. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium or cigarette smoke condensates for 4 hours. Cigarette smoke condensates were prepared as described in the methods and numbers correspond to serial dilutions of the initial cigarette smoke extract. A. Following treatment, total RNA was extracted, reverse transcribed into cDNA and used as a template for semi-quantitative PCR reactions using TLR4 and GAPDH gene-specific primers. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P ≤ 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with anti-TLR4 antibody. Data are representative of three separate experiments. (CSE † cigarette smoke extract). Because actin is not compartmentalised to the membrane, equal protein loading is demonstrated with a panel from the Ponsceau Stain. C. Viability assay of A549 cells following treatment with CSE. Data are representative of three separate experiments. D. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in low-serum (1% FCS) medium and were left untreated or incubated with serial dilutions of CSE × 4 hours. Following treatment with CSE cells were stimulated with LPS 10 μg/ml for a further 24 hours. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as pg/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (* signifies P ≤ 0.05 of observed effect vs. control, † signifies P ≤ 0.05 of observed effect vs. control plus LPS).
Mentions: We next examined the effect of cigarette smoke on expression of TLR4 in respiratory epithelium in vitro. There was a dose dependant downregulation in TLR4 mRNA (Figure 2A) and protein (Figure 2B) following exposure to the cigarette smoke extracts. To ensure that the effect was not caused by direct toxicity of the cigarette smoke, a viability assay was performed which demonstrated 50% reduction in viability with undiluted CSE, but no toxic effect following dilution of the extracts (Figure 2C) which showed no significant difference in viability compared to untreated cells. We went on to examine functional effect by IL-8 ELISA. As expected, CSE has some direct inflammatory effect resulting in IL-8 production at dilute concentrations. However, concordant with the reduced expression of TLR4, the respiratory epithelial cells have dose dependent reduced secretion of IL-8 following treatment with higher concentrations of CSE, both with and without additional LPS (Figure 2D). Failure of the cells to produce any IL-8 following exposure to undiluted CSE may be a result of the direct toxic effects demonstrated in the viability assay.

Bottom Line: We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged.Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein.The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland. rmacredmond@mrl.ubc.ca

ABSTRACT
The toll-like receptors (TLRs) are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10-6 M) caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.

Show MeSH
Related in: MedlinePlus