Limits...
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.

Show MeSH

Related in: MedlinePlus

Cigarette smoke potentiates hyporesponsiveness to LPS by Dexamethasone and Salmeterol. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium (1 and 2), CSE (10-1) × 4 hours [3], pretreated for 16 h with Dex (10-7 M) then for 4 h with CSE (10-1) [4], pretreated for 16 h with Sal (10-6 M) then for 4 h with CSE (10-1) [5] or pretreated for 16 h with Dex (10-7 M) AND (Sal 10-6 M) then for 4 h with CSE (10-1 M) × 4 hours. Following these treatments, 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 fold change compared to unstimulated controls. 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. LPS alone, † signifies P ≤ 0.05 of observed effect vs. CSE plus LPS).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2194695&req=5

Figure 7: Cigarette smoke potentiates hyporesponsiveness to LPS by Dexamethasone and Salmeterol. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium (1 and 2), CSE (10-1) × 4 hours [3], pretreated for 16 h with Dex (10-7 M) then for 4 h with CSE (10-1) [4], pretreated for 16 h with Sal (10-6 M) then for 4 h with CSE (10-1) [5] or pretreated for 16 h with Dex (10-7 M) AND (Sal 10-6 M) then for 4 h with CSE (10-1 M) × 4 hours. Following these treatments, 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 fold change compared to unstimulated controls. 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. LPS alone, † signifies P ≤ 0.05 of observed effect vs. CSE plus LPS).

Mentions: As previously demonstrated in figure 2D, IL-8 production in response to LPS was downregulated following exposure to CSE 10-1. IL-8 production was further inhibited by pre-treatment with dexamethasone consistent with an additive effect of downregulation of TLR4 expression by both treatments in isolation. Salmeterol treatment was not able to enhance LPS-induced IL-8 expression in the presence of CSE however, and similarly the "protective" effect of salmeterol on dexamethasone-induced inhibition of TLR4 signalling was lost in the presence of CSE. In fact there was further downregulation of IL-8 production (Figure 7). These findings are in keeping with recent report that combination of fluticasone and salmeterol potentiates the suppression of cigarette smoke-induced IL-8 production by macrophages [15]. Salmeterol was found to have no effect on CSE induced IL-8 production in airway smooth muscle cells [16], although the effect of LPS was not examined in these studies.


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 potentiates hyporesponsiveness to LPS by Dexamethasone and Salmeterol. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium (1 and 2), CSE (10-1) × 4 hours [3], pretreated for 16 h with Dex (10-7 M) then for 4 h with CSE (10-1) [4], pretreated for 16 h with Sal (10-6 M) then for 4 h with CSE (10-1) [5] or pretreated for 16 h with Dex (10-7 M) AND (Sal 10-6 M) then for 4 h with CSE (10-1 M) × 4 hours. Following these treatments, 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 fold change compared to unstimulated controls. 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. LPS alone, † signifies P ≤ 0.05 of observed effect vs. CSE plus LPS).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Cigarette smoke potentiates hyporesponsiveness to LPS by Dexamethasone and Salmeterol. A549 cells (3 × 105) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium (1 and 2), CSE (10-1) × 4 hours [3], pretreated for 16 h with Dex (10-7 M) then for 4 h with CSE (10-1) [4], pretreated for 16 h with Sal (10-6 M) then for 4 h with CSE (10-1) [5] or pretreated for 16 h with Dex (10-7 M) AND (Sal 10-6 M) then for 4 h with CSE (10-1 M) × 4 hours. Following these treatments, 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 fold change compared to unstimulated controls. 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. LPS alone, † signifies P ≤ 0.05 of observed effect vs. CSE plus LPS).
Mentions: As previously demonstrated in figure 2D, IL-8 production in response to LPS was downregulated following exposure to CSE 10-1. IL-8 production was further inhibited by pre-treatment with dexamethasone consistent with an additive effect of downregulation of TLR4 expression by both treatments in isolation. Salmeterol treatment was not able to enhance LPS-induced IL-8 expression in the presence of CSE however, and similarly the "protective" effect of salmeterol on dexamethasone-induced inhibition of TLR4 signalling was lost in the presence of CSE. In fact there was further downregulation of IL-8 production (Figure 7). These findings are in keeping with recent report that combination of fluticasone and salmeterol potentiates the suppression of cigarette smoke-induced IL-8 production by macrophages [15]. Salmeterol was found to have no effect on CSE induced IL-8 production in airway smooth muscle cells [16], although the effect of LPS was not examined in these studies.

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