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

Corticosteroids downregulate TLR4 expression in respiratory epithelial cells. 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 fluticasone propionate or dexamethasone over the dose ranges 10-9 to 10-6 Molar for 16 hours. A & B. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using, TLR4 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualised under UV. Gels are representative of three independent experiments. C. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4 or anti-Actin antibody. Equal protein loading and transfer efficiency was confirmed by Ponceau S staining. Data are representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Corticosteroids downregulate TLR4 expression in respiratory epithelial cells. 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 fluticasone propionate or dexamethasone over the dose ranges 10-9 to 10-6 Molar for 16 hours. A & B. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using, TLR4 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualised under UV. Gels are representative of three independent experiments. C. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4 or anti-Actin antibody. Equal protein loading and transfer efficiency was confirmed by Ponceau S staining. Data are representative of three separate experiments.

Mentions: To explore other potential modulators of TLR4 expression pertinent to COPD, we first examined the effect of Fluticasone on expression of, TLR4 mRNA by RT-PCR in the respiratory epithelial cell line A549 grown in culture (Figure 3A). A dose dependent downregulation of TLR4 compared to the housekeeping gene GAPDH was observed with an Inhibitory Concentration (IC) 50 between 10-9 and 10-8 M. Consistent with the data of Homma et, who found no upregulation of TLR2 in A549 cells treated with dexamethasone alone [13], we found no change in expression of TLR2 or of HBD2 (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)

Corticosteroids downregulate TLR4 expression in respiratory epithelial cells. 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 fluticasone propionate or dexamethasone over the dose ranges 10-9 to 10-6 Molar for 16 hours. A & B. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using, TLR4 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualised under UV. Gels are representative of three independent experiments. C. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4 or anti-Actin antibody. Equal protein loading and transfer efficiency was confirmed by Ponceau S staining. Data are representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Corticosteroids downregulate TLR4 expression in respiratory epithelial cells. 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 fluticasone propionate or dexamethasone over the dose ranges 10-9 to 10-6 Molar for 16 hours. A & B. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using, TLR4 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualised under UV. Gels are representative of three independent experiments. C. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4 or anti-Actin antibody. Equal protein loading and transfer efficiency was confirmed by Ponceau S staining. Data are representative of three separate experiments.
Mentions: To explore other potential modulators of TLR4 expression pertinent to COPD, we first examined the effect of Fluticasone on expression of, TLR4 mRNA by RT-PCR in the respiratory epithelial cell line A549 grown in culture (Figure 3A). A dose dependent downregulation of TLR4 compared to the housekeeping gene GAPDH was observed with an Inhibitory Concentration (IC) 50 between 10-9 and 10-8 M. Consistent with the data of Homma et, who found no upregulation of TLR2 in A549 cells treated with dexamethasone alone [13], we found no change in expression of TLR2 or of HBD2 (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