Burkholderia cenocepacia ET12 strain activates TNFR1 signalling in cystic fibrosis airway epithelial cells

Abstract

Burkholderia cenocepacia is an important pulmonary pathogen in individuals with cystic fibrosis (CF). Infection is often associated with severe pulmonary inflammation, and some patients develop a fatal necrotizing pneumonia and sepsis ('cepacia syndrome'). The mechanisms by which this species causes severe pulmonary inflammation are poorly understood. Here, we demonstrate that B. cenocepacia BC7, a potentially virulent representative of the epidemic ET12 lineage, binds to tumour necrosis factor receptor 1 (TNFR1) and activates TNFR1-related signalling pathway similar to TNF-alpha, a natural ligand for TNFR1. This interaction participates in stimulating a robust IL-8 production from CF airway epithelial cells. In contrast, BC45, a less virulent ET12 representative, and ATCC 25416, an environmental B. cepacia strain, do not bind to TNFR1 and stimulate only minimal IL-8 production from CF cells. Further, TNFR1 expression is increased in CF airway epithelial cells compared with non-CF cells. We also show that B. cenocepacia ET12 strain colocaizes with TNFR1 in vitro and in the lungs of CF patients who died due to infection with B. cenocepacia, ET12 strain. Together, these results suggest that interaction of B. cenocepacia, ET12 strain with TNFR1 may contribute to robust inflammatory responses elicited by this organism.

Fig. 1

Identification of cellular receptor for B. cenocepacia (A) Binding of bacteria to 55kDa protein. A fraction enriched in membrane proteins prepared from CF airway epithelial cells differentiated into a mucociliary phenotype was subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, blocked with gelatin, incubated with ³⁵S-labeled bacteria, and bound bacteria were detected by autoradiography. (B and C) Binding of TNF-α to denatured TNFR1. Denatured plasma membrane fraction or TNFR1/Fc chimera was subjected to SDS-PAGE and proteins were transferred to nitrocellulose. Blots were either subjected to Western blot analysis with monoclonal antibody to TNFR1 (panel B) or incubated with TNF-α and the bound protein was detected by using polyclonal antibody to TNF-α (Panel C). (D) Inhibition of BC7 binding to 55 kDa protein by TNF-α. Blots of plasma membrane proteins were pre-incubated with TNF-α or IL-1β followed by incubation with ³⁵S-labeled bacteria. Bound bacteria were detected by autoradiography and semi-quantitated by densitometry.s

Fig. 2

Expression of TNFR1 in CF and non-CF airway epithelial cells. (A)Western blot analysis: Cell lysates from CF and non-CF primary airway epithelial cells differentiated into a mucociliary phenotype were probed with monoclonal antibody to TNFR1. N1 and N2 are normal airway epithelial cells from two different donors; CF1 and CF2 are airway epithelial cells from two different CF patients. (B – E) Immunofluorescence detection of TNFR1: Paraffin embedded sections prepared from CF (panel B) and non-CF mucociliary (panel C) cultures were stained with monoclonal antibody to TNFR1 and the bound antibody was detected by antimouse IgG conjugated with AlexaFluor 488. Arrowheads represent TNFR1 expression on the apical surface. Sections stained with normal IgG are shown in panels D and E.

Fig. 3

Binding of bacteria to TNFR1 in intact cells. (A) Expression of TNFR1: Non-permeabilized IB3 cells were incubated with monoclonal antibody to TNFR1 and bound antibody was detected with antimouse IgG conjugated with Alexafluor 488 and analyzed by FACS. Data presented are representative of three independent experiments. (B) Binding of bacteria to cells: IB3 cells were lightly fixed with 0.5% paraformaldehyde, washed and incubated with FITC-labeled BC7 or ATCC 25416. Cells were harvested after removal of unbound bacteria and analyzed by FACS. Data presented are representative of three independent experiments. (C and D) Effect of TNF-α or TNFR1 antibody on binding of bacteria to IB3 cells: Lightly fixed IB3 cells were incubated with TNF-α or TNFR1 antibody for 30 min followed by incubation with FITC-labeled BC7. Bacterial binding was then assessed by FACS. Data represent mean ± SEM of 4 independent experiments carried out in triplicates; *p<0.05, ANOVA.

Fig 4

Colocalization of B. cenocepacia BC7 with TNFR1 in non-CF and CF cell cultures: Paraffin sections of non-CF (A and C) and CF (B and D) primary cultures infected with BC7 were incubated with antibodies to TNFR1 and B. cenocepacia and the bound antibodies were detected by Alexa Fluor-488 (for detection of TNFR1) and anti-rabbit Alexa Flour-598 (for detection of bacteria). Arrow heads in panels C and D indicate colocalization of bacteria with TNFR1. Panels A and B are phase-contrast micrographs and correspond to panels C and D respectively. The inset in panel D is a digital magnification of an area marked in rectangle to show co localization of bacteria with TNFR1 (yellow).

Fig. 5

Colocalization of B. cenocepacia with TNFR1 in lungs of a CF patient. Lung sections from normal donor (panels A and B) or a CF patient who died due to B. cenocepacia infection (panels C to F) were incubated with antibodies to TNFR1 and B. cenocepacia and the bound antibody was detected as described in Fig. 4. Panels A, C and E are phase-contrast micrographs and correspond to panels B, D and F respectively. Arrow heads indicate colocalization of bacteria with TNFR1. The inset in panels D and F is a digital magnification of an area marked in rectangle to demonstrate co localization of bacteria with TNFR1 (yellow) in the lung sections

Fig. 6

(A) Binding of BCM132 to 55 kDa TNFR1. Western blots of plasma membrane proteins was either incubated with 35S-labeled BCM132 or BC7. Bound bacteria were detected by autoradiography. (B) Stimulation of IL-8 response in IB3 cells by BCM132. IB3 cells were incubated with BCM132 or BC7 at MOI of 0.1 for 24 h and the IL-8 in cell culture media was measured by ELISA.

Fig 7

Effect of TNF-α neutralizing antibody on B. cenocepacia-stimulated IL-8 response in IB3 cells (A) IL-8 response: IB3 cells were incubated with B. cenocepacia BC7 or ATCC 25416 for 1, 3, 6 or 24 h, and IL-8 in the cell culture media was quantified by ELISA. (B) Binding of bacteria to cells. Cells incubated with bacteria for 3, 6 or 24 h were fixed and bacteria associated were immunostained with Bcc antibody and quantitated by immunofluorescence microscopy. (C) IL-8 response to UV- or heat-killed BC7: IB3 cells were incubated with live, UV-killed or heat-killed BC7 for 24 h, and IL-8 in the cell culture media was quantified by ELISA. (E and F) Effect of TNF-α neutralizing antibody: IB3 cells were incubated with 3 ng of TNF-α (panel B) or B. cenocepacia BC7 at MOI of 0.1 (panel C) in the presence or absence of varying concentrations of TNF-α neutralizing antibody or normal IgG for 24h. IL-8 in cell culture supernatants was quantified by ELISA. Bars represent mean ± SEM of 4 independent experiments carried out in triplicate; *p<0.05, ANOVA.

Fig. 8

Activation of TNFR1-related signaling pathway by B. cenocepacia. (A) Western blot analysis of immunoprecipitates: IB3 cells were incubated with either TNF-α (3ng/ml) or B. cenocepacia, BC7 or ATCC 25416 at MOI of 10 for 5, 15 or 30 min and lysed. Cell lysates were immunoprecipitated with TNFR1 antibody and subjected to western blot analysis with either TRADD or RIP1 antibody. (B and C) Phosphorylation of p38 and JNK: Lysates from the same experiment were subjected to immunoblot analysis with antibodies to phosho-p38, or total p38 (B) phospho-JNK1/2, or total JNK1/2 (C). (D) Phosphorylation of of p38 and JNK in IB3 cells incubated with heat-killed or UV-killed BC7: Cell lysates from IB3 cells incubated with live, UV- or heat-killed bacteria for 30 min were subjected to immunoblot analysis with antibodies to phosho-p38, or total p38 (B) phospho-JNK1/2, or total JNK1/2. Std- molecular mass standards; Cell lysate- total cell lysate was used to confirm the band positions of TRADD or RIP1 observed in immunoprecipitates; Con-untreated IB3 cells. Data presented are representative of three independent experiments.

Fig 9

Effect of TNFR1 siRNA on transactivation of NF-κB by B. cenocepacia BC7. (A) Transactivation of NF-κB by bacteria: IB3 cells were co-transfected with NF-κB luciferase and renilla luciferase, and then incubated with BC7, ATCC 25146 at MOI of 0.1 or TNF-α (3 ng/ml) for 24 hours. Cells were lysed and luciferase activity was measured and expressed as fold increase over control. (B) Western blot analysis: IB3 cells were transfected with TNFR1-specific or non-targeting siRNA, and incubated for 72 h. Cell lysates were subjected to Western blot analysis with TNFR1 antibody. (C) Inhibition of transactivation of NF-κB by TNFR1 siRNA: IB3 cells were co-transfected with TNFR1-specific or non-targeting siRNA, NF-κB luciferase and renilla luciferase, and incubated for 24h. Cells were serum starved overnight and then incubated with bacteria at MOI of 0.1 or TNF-α (3 ng/ml) for 24h and luciferase activity was measured as before. Bars represent mean ± SEM of 6 independent experiments carried out in duplicates; *p<0.05, ANOVA.