Fig 2: IFI16 proinflammatory activity is potentiated by the strong TLR4 activator LPS-EB.(A) qRT-PCR analysis of IL-6, IL-8, TNF-α and IL-1β mRNA expression levels in 786-O or THP-1 cells stimulated for 24 h with IFI16 (25 μg/ml), IFI16ΔHINB (25 μg/ml), LPS from E. coli O111:B4 (LPS-EB, 10 ng/ml or 1 μg/ml), IFI16/LPS-EB complex (preincubated O/N at 4°C), IFI16ΔHINB/LPS-EB (preincubated O/N at 4°C), or left untreated (mock). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SD (*P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA followed by Dunnett’s test). (B) Protein concentration of IL-6, IL-8 and TNF-α evaluated by ELISA in supernatants derived from 786-O or THP-1 cells stimulated for 24 h as described in A. Data are expressed as mean values ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA followed by Dunnett’s test).

Fig 3: 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.

Fig 4: Endogenous IFI16 is released by UVB-exposed U2OS cells and triggers proinflammatory cytokines production in a TLR4-dependent fashion.(A) Western blot analysis of IFI16 in culture supernatants and total cell extracts of UVB-exposed (0 or 800 Jm-2) U2OS or U2OS-IFI16-/- cells at 16 h after treatment. β-actin cellular expression was used for protein loading control. Data are representative of three independent experiments with similar results. (B) Protein concentration of IL-6, IL-8 and TNF-α evaluated by ELISA in supernatants derived from THP-1 cells stimulated for 24 h in the presence or absence of anti-TLR4 neutralizing antibodies (10 μg/ml) using conditioned medium from UVB-exposed (0 or 800 Jm-2) U2OS and U2OS-IFI16-/- cells, or complete medium (mock), preincubated (O/N at 4°C), or not, with LPS from E. coli O111:B4 (LPS-EB, 10 ng/ml), or LPS from R. sphaeroides (LPS-RS, 10 ng/ml). Values were normalized to the initial protein concentration of the analyzed cytokines in the supernatants used for the treatment. Data are expressed as mean values ± SD of three independent experiments. The P values refer to comparison in each group with cells treated only with the medium without any addiction (white bar and black border; *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA followed by Dunnett’s test).

Fig 5: Proposed model depicting the inflammatory activities and binding kinetics to TLR4 of LPS and IFI16, alone or in combination.The relative inflammatory activities, from low to high, are reported in the upper part of the scheme (orange arrow). The relative binding kinetics to TLR4 are reported in the lower part of the scheme (green arrow). The thickness of the black arrows is directly proportional to the ability of the pathway to induce NF-κB activation.