Corticosteroid-resistant inflammatory signalling in Pseudomonas-infected bronchial cells

Abstract

Decreasing the inflammatory response that leads to tissue damage during cystic fibrosis (CF) lung disease has been a long-standing goal of CF therapy. While corticosteroids are widely used anti-inflammatory drugs, their efficacy in CF lung disease remains debated. The complex interaction between the colonising bacteria and the host environment may impact corticosteroid responsiveness. In this study, sputum samples from adult CF patients were collected at baseline and during pulmonary exacerbation episodes. Lung function measurements and sputum microbiological analyses were performed. In parallel, the inflammatory response and corticosteroid sensitivity of airway epithelial cells to Pseudomonas-derived exoproducts was investigated. We report that adult CF patients colonised with mucoid Pseudomonas aeruginosa have higher levels of baseline inflammation, more frequent exacerbations and worse lung function compared with patients colonised with nonmucoid P. aeruginosa. Moreover, mucoid P. aeruginosa activates NF-κB via Toll-like receptor (TLR) 2, which acts in an additive manner to TLR5 to drive inflammation in airway epithelial cells. Furthermore, TLR2-mediated intracellular signalling is more resistant to the anti-inflammatory effects of corticosteroid when compared with other TLR signalling pathways. Overall, these results suggest that airway inflammation triggered by mucoid P. aeruginosa is less responsive to the anti-inflammatory action of corticosteroids. Whether this translates into a diminished response of CF patients to corticosteroid therapy should be examined in future clinical studies.

FIGURE 1

Higher circulating calprotectin levels are associated with lower lung function and the presence of mucoid Pseudomonas aeruginosa. a) Calprotectin circulating levels were measured by ELISA in the plasma collected from 49 adult cystic fibrosis (CF) patients of the Montreal Chest Institute Adult CF Clinic during a stable clinic visit (i.e. the patient had not experienced a pulmonary exacerbation within the previous 2 months). During the visit spirometry measurements were performed to determine forced expiratory volume in 1 s (FEV₁) % pred. A Poisson regression model was used to estimate the interactions between baseline continuous calprotectin values and FEV₁ % pred. b, c) Frequency distribution of sputum microbiology status in b) low (<150 ng·mL⁻¹) versus c) high (≥150 ng·mL⁻¹) calprotectin groups. MPA: mucoid P. aeruginosa; MR: multidrug-resistant P. aeruginosa; NF: normal flora; PA: nonmucoid P. aeruginosa; SA: Staphylococcus aureus.

FIGURE 2

Additive activation of NF-κB upon dimerisation of Toll-like receptor (TLR) 5 and TLR2. BEAS-2B epithelial cells stably transfected with an NF-κB luciferase reporter were treated with increasing concentrations of flagellin (40, 400 and 4000 ng·mL⁻¹) in the absence or presence of 40 ng·mL⁻¹ FSL-1 for 3 h. Cells were lysed using reporter lysis buffer and luciferase activity was measured using a Tecan plate. AU: arbitrary unit. *: p<0.05 flagellin compared with flagellin+FSL-1; ^#: p<0.05 FSL-1 compared with flagellin+FSL-1.

FIGURE 3

Toll-like receptor (TLR) 2-induced inflammatory signalling is more resistant to dexamethasone than TLR5-driven signalling. a) BEAS-2B NF-κB luciferase reporter cells were pre-treated for 1 h with increasing doses of dexamethasone (0.1–1000 nM), then treated for 3 h with 1 µg·mL⁻¹ Pam3CSK4, 100 ng·mL⁻¹ flagellin, 40 ng·mL⁻¹ FSL-1 or 1 µg·mL⁻¹ polyinosinic–polycytidylic acid (poly(I:C)) and luciferase activity was measured. b, c) BEAS-2B cells were pre-treated for 1 h with increasing doses of dexamethasone (0.1–1000 nM), then treated for 30 min with either b) 1 µg·mL⁻¹ Pam3CSK4 or c) 100 ng·mL⁻¹ flagellin. Following stimulation, cells were lysed and 20 µg of Triton-soluble material was subjected to SDS-PAGE. After transfer to nitrocellulose, the membranes were probed with antibodies recognising only the phosphorylated forms of p38 mitogen-activated protein kinase (MAPK) or antibodies that recognise all forms of p38 MAPK. Quantitative analysis of the signals from each antibody was performed using a LI-COR infrared Odyssey imaging system (LI-COR, Lincoln, NE, USA). AU: arbitrary unit. d, e) BEAS-2B glucocorticoid response element (GRE) luciferase reporter cells were treated for 1 h with increasing doses of dexamethasone (0.1–1000 nM). Cells were lysed and d) luciferase activity was measured as in figure 2 or e) mRNA levels of MAPK phosphatase-1 (MKP-1) were quantified by quantitative real-time PCR. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.