Fig 2: Effect of 20 µg/mL London Underground particulate matter less than 10 microns from microns from Bakerloo (B-PM10) and Jubilee lines (J-PM10) on A549 cells, (a) dose-response of London Underground particulate matter less than 10 microns in aerodynamic diameter from the Bakerloo (B-PM10) and Jubilee (J-PM10) lines on pneumococcal adhesion. Increased pneumococcal adhesion is reflected by increased colony forming unit (CFU) count, and is significant at 20 µg/mL, (B-PM10 and J-PM10, n=8, technical replicates (TR)=3, =0.0003 and p=0.0005, respectively, (b) effect of 20 µg/mL PM10 on intracellular pneumococcal CFU count, assessed after treatment of cells with antibiotics to kill cell surface bacteria (B-PM10 and J-PM10, n=8, TR=3, p=0.007 and 0.006 respectively), (c) effect of B-PM10 and J-PM10 on platelet-activating factor receptor (PAFR) expression (B-PM10 and J-PM10, n=6, TR=2, p=0.002 and 0.017 respectively), (d) effect of the PAFR blocker CV3988 on pneumococcal adhesion (B-PM10 and J-PM10, n=8, TR=3, p=0.001 and p=0.004 respectively), (e) effect of the antioxidant N-acetyl cysteine on pneumococcal adhesion, (B-PM10 and J-PM10, n=7, TR=3, p=0.014 and 0.034, respectively), (f) comparison of adhesion stimulated by 20 µg/mL B-PM10 and 20 µg/mL overground Marylebone Road PM10, (n=5, TR=3, p=0.007) Expression was determined by flow cytometry and expressed as median fluorescence intensity (MFI). Columns represent medians and p values are calculated by either Kruskal-Wallis with post hoc multiple comparison testing, or by Mann Whitney test.

Fig 3: Schematic model of the synergistic effect of coinfection by bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM) in cattle with severe pneumonia. Epithelial cells in the upper respiratory tract (trachea) capture PM via intercellular adhesion molecule-1 (ICAM-1) under normal conditions and serve as a biological gateway (Sudaryatma et al., 2020). BRSV infection abrogates this gateway function by downregulating ICAM-1 on the epithelial cells of the upper respiratory tract and upregulating the platelet-activating factor receptor (PAFR) on the epithelial cells of the lower respiratory tract. This change permits PM to invade the lower respiratory tract, stimulating inflammation in the lung (Sudaryatma et al., 2019).

Fig 4: Ginkgolide B inhibits Pasteurella multocida (PM) adherence to HEK293T cells overexpressing bovine platelet-activating factor receptor (PAFR). (A) Cells overexpressing bovine PAFR were treated with a range of concentrations of ginkgolide B for 1 h. The cells then were incubated with PM for 1 h. (B) HEK293T cells overexpressing bovine PAFR were treated with a range of concentrations of ginkgolide B for 1 h. Cells then were incubated with fluorescein isothiocyanate-labeled PM for 1 h. Cell-associated PM was measured by flow cytometry. The average of three independent experiments conducted in triplicate is shown in (A,B). Error bars indicate SEM; *p < 0.05, statistical significance. PM adherence to cells treated with DMSO served as reference in (A,B).

Fig 5: Specific blocking of platelet-activating factor receptor (PAFR) reduces Pasteurella multocida (PM) adherence to bovine respiratory syncytial virus (BRSV)-infected bovine respiratory epithelial cells. (A) Bronchus (bBECs) and lung (bLECs) epithelial cells were infected with BRSV for 3 days. The cells then were treated with a range of concentrations of the anti-PAFR antibody before being incubated with PM. The numbers of cell-associated bacteria were counted. (B) Observation of bBECs and bLECs under the fluorescence microscope. BRSV-infected cells treated with the anti-PAFR antibody were incubated with fluorescein isothiocyanate-labeled PM (green) (bar = 30 µm). The average of three independent experiments using cells derived from three independent animals conducted in triplicate is shown in (A). Error bars indicate SEM; *p < 0.05, statistical significance. PM adherence to BRSV-infected cells that were not treated with antibody served as reference in (A).