Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb 5;16(1):12.
doi: 10.1186/s12931-015-0179-5.

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Javier Milara  1   2   3 , Anselm Morell  4   5 , Bea Ballester  6 , Celia Sanz  7 , Jose Freire  8 , Xiaozhong Qian  9 , Maggie Alonso-Garcia  10 , Esteban Morcillo  11 , Julio Cortijo  12   13   14
Affiliations

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Javier Milara et al. Respir Res. .

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists.

Methods: Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was measured in cell cultures after TLR3 stimulation with poly I:C 10 μg/mL.

Results: We found that TLR3 expression was increased by 1.95 fold (protein) and 2.5 fold (mRNA) in lung tissues from smokers with COPD and inversely correlated with lung function. The TLR3 agonist poly I:C 10 μg/mL increased the IL-8 release in HBECs that was poorly inhibited by dexamethasone in smokers (24.5%) and smokers with COPD (21.6%). In contrast, roflumilast showed similar inhibitory effects on IL-8 release in healthy (58.8%), smokers (56.6%) and smokers with COPD (50.5%). The combination of roflumilast N-oxide and dexamethasone showed additive inhibitory effects. Mechanistically, roflumilast N-oxide when combined with dexamethasone increased the expression of MKP1, and enhanced the inhibitory effects on phospho-p38, AP1 and NFκB activities which may explain the additive anti-inflammatory effects.

Conclusions: Altogether, our data provide in vitro evidence for a possible clinical utility to add roflumilast on top of inhaled corticosteroid in COPD.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of TLR3 in lung tissues of non-smokers, smokers, and COPD patients. Total protein and mRNA was obtained from lung tissue of non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15). TLR3 protein and mRNA expression was determined by western blot (A) and real time PCR (B), respectively, in lung parenchyma. (A) Representative images of western blot for TLR3 and corresponding densitometry expressed as ratio of β-actin. (B) TLR3 mRNA expression expressed as the ratio to GAPDH. (C) Spearman “ρ” correlation of the protein expression of TLR3 in COPD patients and lung function, FEV1% predicted. (D, E, F) Lung sections were immunostained for TLR3 and quantified by means of immunohistochemical score of TLR3 in alveolar macrophages (D) and bronchial epithelial cells (E). (F) Representative immunohistochemistry images are shown. The control IgG isotype signal was negative. Data are presented as individual values and mean ± standard deviation. Exact P values were obtained using Kruskal-Wallis and Dunn’s post-hoc tests.
Figure 2
Figure 2
Expression of TLR7 in lung tissues of non-smokers, smokers, and COPD patients. Total protein and mRNA were obtained from lung tissues of non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15). TLR7 protein and mRNA expression were determined by western blot (A) and real time PCR (B) in lung parenchyma. (A) Representative images of western blot for TLR7 and corresponding densitometry expressed as ratio to β-actin. (B) TLR7 mRNA expression given as the ratio to GAPDH. (C, D, E) Lung sections were immunostained for TLR7 and quantified by means of immunohistochemical score of TLR7 in alveolar macrophages (C) and bronchial epithelial cells (D). (E) Representative immunohistochemistry images are shown. The control IgG isotype showed negative staining. Data are presented as individual values and mean ± standard deviation. Exact P values were obtained using Kruskal-Wallis and Dunn’s post-hoc tests.
Figure 3
Figure 3
Expression of TLR8 in lung tissues of non-smokers, smokers, and COPD patients. Total protein and mRNA were obtained from lung tissues of non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15). TLR8 protein and mRNA expression were determined by western blot (A) and real time PCR (B) in lung parenchyma. (A) Representative images of western blot for TLR8 and corresponding densitometry expressed as ratio to β-actin. (B) TLR8 mRNA expression given as the ratio to GAPDH. (C, D, E) Lung sections were immunostained for TLR8 and quantified by means of immunohistochemical score of TLR8 in alveolar macrophages (C) and bronchial epithelial cells (D). (E) Representative immunohistochemistry images are shown. The control IgG isotype showed negative staining. Data are presented as individual values and mean ± standard deviation. Exact P values were obtained using Kruskal-Wallis and Dunn’s post-hoc tests.
Figure 4
Figure 4
Roflumilast N-oxide inhibited IL-8 release in human bronchial epithelial cells and demonstrated additive anti-inflammatory properties in corticosteroid resistance conditions. Human bronchial epithelial cells (HBECs) from non-smokers (n = 4), smokers (n = 4), and COPD patients (n = 5) were isolated from lung tissues. (A) HBECs from different patients were stimulated with TLR3 agonist poly I:C or the TLR7/8 dual agonist CL097 for 24 hours and IL-8 levels in the supernatants were measured by ELISA. (B, C, D) HBECs from different patients were incubated with roflumilast N-oxide (RNO) or dexamethasone (DEX) at different concentrations for 1 hour followed by the stimulation with poly I:C 10 μg/ml for 24 hours to measure IL-8 release. (D) In COPD patients, fixed concentrations of DEX 10 nM were combined with different RNO concentrations. Results are expressed as means ± SEM of n = 4–5 (4 non-smoker, 4 smokers, and 5 COPD patients) run in triplicate experiments. Two-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with solvent controls. #P < 0.05 compared with monotherapy.
Figure 5
Figure 5
TLR3 is overexpressed in primary bronchial epithelial cells from current smokers and COPD patients and downregulated by roflumilast N-oxide. (A) Human bronchial epithelial cells (HBECs) from non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15) were isolated from lung tissues. (A) mRNA expression of TLR3 in HBECs from different patients was determined by real time PCR as the ratio to GAPDH. (B, C, D) HBECs from different patients were incubated in the presence or absence of roflumilast N-oxide (RNO) or dexamethasone (DEX) for 1 hour and stimulated with TLR3 agonist poly I:C for 24 hours. Results are expressed as means ± SEM of n = 4–5 (4 non-smokers, 4 smokers, and 5 COPD patients) run in triplicate. Two-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with non-smoker group or with solvent controls. #P < 0.05 compared with poly I:C stimulus.
Figure 6
Figure 6
TRIF is overexpressed in primary bronchial epithelial cells from current smokers and COPD patients and downregulated by roflumilast N-oxide. (A) Human bronchial epithelial cells (HBECs) from non-smokers (n = 15), smokers (n = 12), and COPD patients (n = 15) were isolated from lung tissue. (A) mRNA expression of TRIF in HBECs from different patients was determined by real time PCR as the ratio of GAPDH. (B, C, D) HBECs from different patients were incubated in the presence or absence of roflumilast N-oxide (RNO) or dexamethasone (DEX) for 1 hour and stimulated with TLR3 agonist poly I:C for 24 hours to measure TRIF mRNA expression. Results are expressed as means ± SEM of n = 3 (3 non-smokers, 3 smokers, and 3 COPD patients) run in triplicate. Two-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with non-smoker group or with solvent controls. #P < 0.05 compared with poly I:C stimulus.
Figure 7
Figure 7
BEAS2B bronchial epithelial cells exposed to cigarette smoke and stimulated with TLR3 agonist release IL-8 secretion that is inhibited by roflumilast N-oxide but not by dexamethasone. BEAS2B cells were pretreated (A, B) without or (A, C) with cigarette smoke extract (CSE) 1% for 1 h followed by the incubation in the presence or absence of different concentrations of roflumilast N-oxide (RNO) or dexamethasone (DEX) for 1 h. After drug incubation cells were stimulated with the TLR3 agonist poly I:C for 24 h and IL-8 release was measured by ELISA. Each graph represents the mean ± SEM of 3–4 independent experiments. One-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with cells exposed to air; #P < 0.05 compared with monotherapy.
Figure 8
Figure 8
Roflumilast N-oxide regulation of TLR3 expression and molecular pathway implicated in corticosteroid efficacy. BEAS2B cells were pretreated with or without cigarette smoke extract (CSE) 1% for 1 hour followed by the incubation in the presence or absence of different concentrations of roflumilast N-oxide (RNO) or dexamethasone (DEX) for 1 hour. After drug incubation cells were stimulated with the TLR3 agonist poly I:C for 24 hours and total mRNA was extracted to quantify the expression of (A) TLR3, (B) TRIF, (C) HDAC2, (D) MIF, and (E) MKP1 genes by real time PCR. Each graph represents the mean ± SEM of 3–4 independent experiments. One-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with control; #P < 0.05 compared with stimulus; ┸ P < 0.05 compared with monotherapy.
Figure 9
Figure 9
Roflumilast N-oxide shows additive or synergistic effects with dexamethasone in inhibiting p38, AP1 and NF-κB induced by TLR3 stimulation. BEAS2B cells were pretreated with or without cigarette smoke extract (CSE) 1% for 1 hour followed by the incubation in the presence or absence of different concentrations of roflumilast N-oxide (RNO) or dexamethasone (DEX) for 1 hour. After drug incubation cells were stimulated with the TLR3 agonist poly I:C for 30 minutes (A), 45 minutes (B), or 1 hour (C), and total protein (A) or nuclear protein (B, C) was extracted to measure p38 phosphorylation, AP1 nuclear activation, or NF-κB (p65) nuclear expression. Each graph represents the mean ± SEM of 3–4 independent experiments. One-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with control; #P < 0.05 compared with stimulus; ┸P < 0.05 compared with monotherapy.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sethi S, Mahler DA, Marcus P, Owen CA, Yawn B, Rennard S. Inflammation in COPD: implications for management. Am J Med. 2012;125(12):1162–70. doi: 10.1016/j.amjmed.2012.06.024. - DOI - PubMed
    1. Roche N, Marthan R, Berger P, Chambellan A, Chanez P, Aguilaniu B, et al. Beyond corticosteroids: future prospects in the management of inflammation in COPD. Eur Respir Rev. 2011;20(121):175–82. doi: 10.1183/09059180.00004211. - DOI - PMC - PubMed
    1. Hatzelmann A, Morcillo EJ, Lungarella G, Adnot S, Sanjar S, Beume R, et al. The preclinical pharmacology of roflumilast–a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease. Pulm Pharmacol Ther. 2010;23(4):235–56. doi: 10.1016/j.pupt.2010.03.011. - DOI - PubMed
    1. Wedzicha JA, Seemungal TA. COPD exacerbations: defining their cause and prevention. Lancet. 2007;370(9589):786–96. doi: 10.1016/S0140-6736(07)61382-8. - DOI - PMC - PubMed
    1. Farr BM, Gwaltney JM, Jr, Hendley JO, Hayden FG, Naclerio RM, McBride T, et al. A randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection. J Infect Dis. 1990;162(5):1173–7. doi: 10.1093/infdis/162.5.1173. - DOI - PubMed

Publication types

MeSH terms