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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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Downregulation of TLR4 by dexamethasone results in relative hypo-responsiveness to LPS. 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 dexamethasone at dose of 10-9 to 10-6 Molar for 16 hours. Following treatment with dexamethasone, 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 4: Downregulation of TLR4 by dexamethasone results in relative hypo-responsiveness to LPS. 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 dexamethasone at dose of 10-9 to 10-6 Molar for 16 hours. Following treatment with dexamethasone, 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: In order to determine the functional relevance of this effect, we stimulated the cells with the TLR4 agonist LPS. Stimulation of the cells with LPS 10 μg/ml for 24 hours resulted in a significant induction of IL-8, as measured by ELISA of the cell culture supernatant (P < 0.05) (Figure 4). Pre-treatment of the cells with dexamethasone dose-dependently abrogated this effect, reaching statistical significant at a dose of 10-7 M (P < 0.05). A similar effect was seen on the induced expression of HBD2 mRNA in response to LPS (data not shown).


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)

Downregulation of TLR4 by dexamethasone results in relative hypo-responsiveness to LPS. 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 dexamethasone at dose of 10-9 to 10-6 Molar for 16 hours. Following treatment with dexamethasone, 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194695&req=5

Figure 4: Downregulation of TLR4 by dexamethasone results in relative hypo-responsiveness to LPS. 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 dexamethasone at dose of 10-9 to 10-6 Molar for 16 hours. Following treatment with dexamethasone, 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: In order to determine the functional relevance of this effect, we stimulated the cells with the TLR4 agonist LPS. Stimulation of the cells with LPS 10 μg/ml for 24 hours resulted in a significant induction of IL-8, as measured by ELISA of the cell culture supernatant (P < 0.05) (Figure 4). Pre-treatment of the cells with dexamethasone dose-dependently abrogated this effect, reaching statistical significant at a dose of 10-7 M (P < 0.05). A similar effect was seen on the induced expression of HBD2 mRNA in response to LPS (data not shown).

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