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. 2017 Apr 18;18(1):61.
doi: 10.1186/s12931-017-0539-4.

Mechanisms of corticosteroid insensitivity in COPD alveolar macrophages exposed to NTHi

Rana M Khalaf  1   2 , Simon R Lea  3   4 , Hannah J Metcalfe  1   2 , Dave Singh  1   2
Affiliations

Mechanisms of corticosteroid insensitivity in COPD alveolar macrophages exposed to NTHi

Rana M Khalaf et al. Respir Res. .

Abstract

Background: Non-typeable Haemophilus influenza (NTHi) infection is common in COPD. Corticosteroids can have limited therapeutic effects in COPD patients. NTHi causes corticosteroid insensitive cytokine production from COPD alveolar macrophages. We investigated the mechanisms by which NTHi causes corticosteroid insensitive inflammatory responses, and the effects of NTHi exposure on COPD macrophage polarisation.

Method: Alveolar macrophages from COPD patients and controls were exposed to NTHi in conjunction with the corticosteroid dexamethasone and/or the p38 MAPK inhibitor BIRB-796. Cytokine release, GR phosphorylation and modulation and macrophage phenotype were analysed.

Results: Dexamethasone significantly inhibited NTHi induced TNF-α, IL-6 and IL-10 from COPD macrophages but, CXCL8 was not suppressed. BIRB-796 combined with dexamethasone caused significantly greater inhibition of all cytokines than either drug alone (p < 0.05 all comparisons). NTHi caused phosphorylation of GR S226 reducing GR nuclear localisation, an effect regulated by p38 MAPK. NTHi altered macrophage polarisation by increasing IL-10 and decreasing CD36, CD206, CD163 and HLA-DR.

Conclusion: NTHi exposure causes p38 MAPK dependent GR phosphorylation associated with decreased GR function in COPD alveolar macrophages. Combining a p38 MAPK inhibitor with corticosteroids can enhance anti-inflammatory effects during NTHi exposure of COPD alveolar macrophages. NTHi causes macrophage polarisation that favours bacterial persistence.

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Figures

Fig. 1
Fig. 1
Effect of dexamethasone on NTHi induced TNF-α, IL-6, CXCL8 and IL-10 in alveolar macrophages from COPD and smokers. Alveolar macrophages from COPD (a, c, e and g) and smokers (b, d, f and h) were pre-treated with dexamethasone (0.0001–1 μM) or with vehicle (DMSO 0.05%) for 1 h before exposure to NTHi either at 10:1 MOI (12 COPD and 8 smokers) or at 100:1 MOI (13 COPD and 10 smokers). Supernatants were collected after 24 h and assayed for TNF-α (a and b), IL-6 (c and d), CXCL8 (e and f) and IL-10 (g and h) release by ELISA. Data presented as Mean ± SEM absolute mediator levels with mean percentage inhibition stated above the bar. *, **, *** represent significant inhibition bellow DMSO control (p < 0.05, 0.01, 0.001 respectively, Repeated measures ANOVA)
Fig. 2
Fig. 2
Effect of NTHi on glucocorticoid receptor phosphorylation in COPD alveolar macrophages: COPD alveolar macrophages (n = 5) were either stimulated with NTHi (10:1MOI) for 20 min with or without 1 h pre-treatment with dexamethasone (1 μM) or BIRB-796 (1 μM). Cells were lysed and assessed for phosphorylation of p38 MAPK (a), GR at ser226 (b) or GR at ser211 (c) by Western blotting. Band density was normalised to β-actin. Representative blots are shown under corresponding conditions. Data presented as Mean ± SEM, *, **, *** represent significance above time matched basal controls (p < 0.05, 0.01, 0.001 respectively, Repeated measures ANOVA)
Fig. 3
Fig. 3
Effect of NTHi on dexamethasone induced nuclear localisation of glucocorticoid receptor. Alveolar macrophages were left untreated (a) or treated with dexamethasone (1 μM) (b), dexamethasone (1 μM) and NTHi (10:1 MOI) (c) or pre-treated with BIRB-796 (1 μM) followed by dexamethasone (1 μM) and NTHi (10:1 MOI) (d). Cells were fixed and immunostained for glucocorticoid receptors (green) and counterstained with 4', 6-diamidino-2-phenylindole nuclear stain (blue). Cells were imaged using fluorescent microscope (X20). Yellow arrows show cells with both cytoplasmic and nuclear localisation of GR, white arrows show cells with nuclear only localisation of GR. Cells expressing nuclear only GR are expressed as percentage of total cells (e). Data represents 4 individual patients with median. Representative images shown for a-d. *represents significantly above basal (p < 0.05). # represents significantly below dexamethasone alone (p < 0.05, Repeated measures two ANOVA with Bonferoni post-test)
Fig. 4
Fig. 4
Combination effect of dexamethasone and p38 MAPK inhibitor (BIRB-796) on NTHi-induced cytokine release from COPD alveolar macrophages. Alveolar macrophages from 6 COPD patients were pre-treated with either dexamethasone (0.01 and 1 μM) or BIRB-796 (1 μM) alone or in combination, for 1 h before NTHi (10:1 MOI) stimulation for 24 h. DMSO 0.05% was used as vehicle control. TNF-α (a), IL-6 (b), CXCL8 (c) and IL-10 (d) levels were measured by ELISA. Data presented as Mean ± SEM. All data analysed by Repeated measures ANOVA with Dunnett multiple comparison post-test. *, ** represent significant inhibition below DMSO control (p < 0.05, 0.01 respectively). #, ##, ### represent significantly higher inhibition than corresponding dexamethasone concentration (p < 0.05, 0.01, 0.001 respectively). $, $$ represent significantly higher inhibition than BIRB-796 alone (p < 0.05, 0.01 respectively)
Fig. 5
Fig. 5
COPD alveolar macrophage functional polarization in NTHi infection: Alveolar macrophages from COPD patients (n = 6) were infected with NTHi at MOI of 10:1 or 100:1, cells were lysed at 2, 6 and 24 h of exposure, RNA was extracted and gene expression of TNF-α (a), CXCL8 (b), CD38 (c), HLA-DR (d), IL-10 (e), CD36 (f), Mannose receptor (CD206) (g), CD14 (h) and CD163 (i) was assessed by RT-PCR. Data presented as median with range. *, ** represent significant difference of from time-matched basal control; (p < 0.05, <0.01 respectively, Repeated measures ANOVA or Friedman test). Relative expression levels were determined using the ΔΔCt method normalizing to the house keeping gene (GAPDH)
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