Annexin A5 suppresses HMGB1-mediated proinflammatory response by inhibiting TLR4 signaling in immune cells (A) delayed treatment model: C57BL/6 mice were intraperitoneally injected with 50 mg/kg LPS. Three h after the LPS challenge, each mouse was intravenously injected with 500 µg (in 100 µL) annexin A5 and monitored for 50 h. The line graph illustrates survival in different treatment groups over time. Each group consisted of 6 mice and the experiments were performed twice. (B) TLR agonists + Annexin A5 treatment: Bone marrow DCs (1 × 106) from C57BL6 mice were stimulated with TLR agonists: 10 µg/mL Pam3CSK4 (TLR1/2), 10 µg/mL Poly(l:C) (TLR3), 50 ng/mL LPS(TLR4), 5 µg/mL Imiquimod (TLR7) and 50 µg/mL ODN1826 (TLR9). Each group was incubated with 50 µg/mL annexin A5 protein, along with designated TLR agonist at 37°C for 18 h. Eighteen hours after the treatment, CD40 on DCs were measured using flow cytometry. The bar graph illustrates the mean fluorescence intensity (MFI) of surface marker (above). Eighteen hours after the treatment, concentrations of proinflammatory cytokines IL6 and TNFα were assessed in supernatants using ELISA. Each cytokine levels are illustrated using bar graphs (below). Each TLR agonist treatment was used as a positive control and immature DC culture supernatant served as a negative control. (C) In vitro HMGB1-mediated cytokine level with TLR4 Ab treatment: Bone marrow DCs (1 × 106) from C57BL6 mice were pre-incubated with 40 µg/mL of TLR4 Ab at 37°C for 1 h. DCs were stimulated with 5 µg/mL HMGB1, followed by 50 µg annexin A5 treatment. All groups were incubated at 37°C for 18 h. Eighteen h after the treatment, concentrations of proinflammatory cytokines IL6 and TNFα were assessed in supernatants using ELISA. Each cytokine levels are illustrated using bar graphs. HMGB1 treatment was used as a positive control and immature DC culture supernatant served as a negative control. TLR4 Ab designates monoclonal antibody for TLR4/MD2. (D) In vivo cytokine level: C57BL/6 mice were intravenously injected with the mixture of 300 µg HMGB1 and 500 µg annexin A5 in 100 µL PBS. Whole blood was collected by cardiac puncture and serum was extracted 18 h after the treatment. Serum concentrations of proinflammatory cytokines IL6 and TNFα were determined using ELISA. (E) In vivo measurement of HMGB1 release: C57BL/6 mice were challenged with 50 mg/kg LPS, followed by intravenous injection of 500 µg (in 100 µL PBS) annexin A5 protein. Three hours after the treatment, peritoneal cavity was washed by 10 mL PBS. At the same time, whole blood was collected by cardiac puncture and serum was extracted. Concentrations of HMGB1 in serum and peritoneal cavity wash were determined using ELISA. P.C.W stands for peritoneal cavity wash. (A-E) * — Indicates significant differences (P < 0.05) from data obtained during the LPS-only, TLR agonists-only or HMGB1-only challenge. Data represent the mean ± S.E.M. of three independent experiments.