Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding
Fig 1
IFI16 binds to LPS of different bacterial origin and inflammatory activity.
Coomassie brilliant blue staining of pull-down assays performed with 3 μg of recombinant IFI16 (A) or GST (B) in the presence or absence of biotin-labeled lipopolysaccharide (LPS) from E. coli O111:B4 (biotin-LPS-EB). (C) Saturation binding experiments performed with 2 μg/ml of IFI16 (red circles) and increasing amount of biotin-LPS-EB. Binding was detected by ELISA using HRP-conjugated streptavidin. Optical density (OD) of samples was measured at 450 nm. An excess of recombinant GST (blue circles) or BSA (green circles) and pre-treatment of biotin-LPS-EB with polymyxin B (PMB, empty circles) were used as negative controls. Data are expressed as mean values ± SD of three independent experiments. (D) Surface plasmon resonance (SPR) analysis of LPS-EB binding to immobilized IFI16. After immobilization of IFI16 on the CM5 sensor chip surface, increasing concentration of LPS-EB (3.125–100 nM) diluted in running buffer were injected over immobilized IFI16. Data are representative of three independent experiments. (E) Ex-vivo interaction analysis between increasing amount of recombinant IFI16 and formalin-fixed gram-negative (E. coli and K. pneumonia; pink circles and pink squares, respectively) or gram-positive (S. aureus, S. epidermidis, S. pyogenes; blue circles, squares and triangles, respectively) bacteria. Data are expressed as mean values ± SD of three independent experiments. (F) Lipid A structures of LPS derived from E. coli O111:B4 or F583 LPS (LPS-EB and LPS-F583, respectively; strong TLR4 agonists), P. gingivalis (LPS-PG; weak TLR4 agonist) and R. sphaeroides (LPS-RS, TLR4 antagonist). For LPS-PG, which harbors a mixture of di-, mono- and de-phosphorylated penta- or tetra-acylated lipid A moieties, a single isoform is represented for simplicity. PS-outer chain = polysaccharide outer chain. (G) Saturation binding experiments with increasing amount of recombinant IFI16 (8 to 12,288 ng/ml) and 10 μg/ml of LPS-EB (red line), LPS-F583 (green line), LPS-PG (blue line) or LPS-RS (purple line). Anti-IFI16 antibodies against the N-terminus of the protein and HRP-labelled anti-rabbit IgG were added as primary and secondary antibody, respectively, and binding was detected by ELISA at 450 nm. Data are expressed as mean values ± SD of three independent experiments.