An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke

Figures & data

Figure 1 CSE increases PAFr expression in BEAS-2B bronchial epithelial cells.

Notes: (A) Untreated BEAS-2B cells. (B) Microscopy of BEAS-2B cells exposed to CSE (1%, 4 hours). All micrographs show BEAS-2B cells with PAFr expression (anti-PAFr monoclonal antibody; 1:100, red) and nuclei stained with 4′,6-diamidino-2-phenylindole (1:5,000, blue). Magnification =×400. (C) Percentage area of PAFr compared to cell nuclei area was measured using area of interest cell-tracing calculations. (D) PAFr mRNA expression relative to housekeeping genes. Bars represent standard error of the mean.
Abbreviations: CSE, cigarette smoke extract; h, hours; HK, housekeeping; PAFr, platelet-activating factor receptor.

Figure 2 CSE increases the adhesion of the NTHi strains and S. pneumoniae to BEAS-2B cells.

Notes: (A) Control BEAS-2B cells showing few FITC-tagged S. pneumoniae 132 cells adhering to BEAS-2B cells. (B) BEAS-2B cells exposed to CSE (1%) show increased attachment of FITC-tagged S. pneumoniae to BEAS-2B cells. All micrographs show BEAS-2B cells with PAFr expression (anti-PAFr monoclonal antibody; 1:100, red) and nuclei stained with 4′,6-diamidino-2-phenylindole (1:5,000, blue). Magnification =×400. (C) S. pneumoniae clinical isolate 132. (D) NTHi reference strain NCTC-4560. (E) NTHi clinical isolate RHH3. Bars represent standard error of the mean.

Abbreviations: CSE, cigarette smoke extract; FITC, fluorescein isothiocyanate; h, hours; NTHi, non-typeable Haemophilus influenzae; PAFr, platelet-activating factor receptor; S. pneumoniae, Streptococcus pneumoniae.

Figure 3 Correlation between change in PAFr protein expression and variation in the number of adherent bacteria to BEAS-2B cells.

Notes: (A) S. pneumoniae clinical isolate 132. (B) NTHi reference strain NCTC-4560. (C) NTHi clinical isolate RHH3.
Abbreviations: NTHi, non-typeable Haemophilus influenzae; PAFr, platelet-activating factor receptor; S. pneumoniae, Streptococcus pneumoniae.

Figure 4 Inhibitory effect of WEB-2086 on the adhesion of bacterial strains to CSE-treated BEAS-2B cells.

Notes: (A) BEAS-2B cells exposed to CSE (1%) showing attachment of FITC-tagged Streptococcus pneumoniae clinical isolate 132. (B) Reduced attachment of S. pneumoniae bacterial cells to CSE-treated BEAS-2B cells due to application of PAFr antagonist, WEB-2086. (C) Significant reductions in S. pneumoniae adhesion to CSE-treated cells were observed in the presence of 100 nM, 1 μM, and 10 μM WEB-2086. (D) A significant reduction in NTHi reference strain (NCTC-4560) adherence to CSE-treated cells was observed in the presence of 10 μM WEB-2086. (E) Significant reductions in NTHi clinical isolate (RHH3) adherence to CSE-treated cells were observed in the presence of 1 and 10 μM WEB-2086. Bars represent standard error of the mean.
Abbreviations: CSE, cigarette smoke extract; FITC, fluorescein isothiocyanate; NTHi, non-typeable Haemophilus influenzae; PAFr, platelet-activating factor receptor; S. pneumoniae, Streptococcus pneumoniae.

Figure 5 In silico analyses of PAFr structure and ligand binding.

Notes: (A) Ribbon representation of the molecular model of the PAFr receptor (residues 8–309), based on the template structures of NTR1 (PDB 4buo) and CCR5 (PDB 4mbs). The expected extracellular face is toward the top of the page. The predicted docking site for WEB-2086 is a deep cleft in the extracellular side of the receptor. (B) Surface representation of the central ligand-binding cleft in the PAFr model. WEB-2086 is shown in stick representation.
Abbreviation: PAFr, platelet-activating factor receptor.