Figure 2. LA1 induces intermediate-affinity conformation in CD11bA and reduces TLR-stimulated proinflammatory cytokines.

(A) IL-1β, IL-6, TNF-α, and MCP-1 levels in primary mouse macrophage supernatants treated with vehicle DMSO (C), LA1 (20 μM), LPS (50 ng/ml), or LPS+LA1 for 8 hours (MCP-1) or 12 hours (IL-1β, IL-6, and TNF-α). Bars show mean ± SEM (n = 3) from 1 of at least 3 independent experiments. (B) IL-6 and TNF-α levels in supernatants of human macrophages treated with vehicle DMSO (C), LPS (50 ng/ml), or LPS+LA1 for 12 hours. Bars show mean ± SEM (n = 3) from 1 of at least 2 independent experiments. (C) IL-6 and TNF-α levels in supernatants of human macrophages treated with vehicle DMSO (C), R848, or R848+LA1 for 12 hours. Bars show mean ± SEM (n = 3) from 1 of 2 independent experiments. (D) IFN-β levels in the sera of WT or CD11b^–/– mice treated with DMSO (C), LPS (100 ng/ml), or LPS+LA1 for 4 hours. Bars show mean ± SEM (n = 3). (E) IFN-β levels in supernatants of primary WT or CD11b^–/– macrophages treated with DMSO (C), LA1 (20 μM), LPS (100 ng/ml), or LPS+LA1 for 12 hours. Bars show mean ± SEM (n = 3) from a representative experiment. ^#Not detectable. (F) Survival of C57BL/6 mice (n = 12) subjected to CLP and treated with vehicle (1% DMSO in saline) or LA1 (2 mg/kg/d). (G) IL-1β, IL-6, and TNF-α in plasma obtained 24 hours after C57BL/6 mice were subjected to either sham surgery (C) or CLP and treated with either vehicle (CLP+Veh) or LA1 (CLP+LA1). Bars show mean ± SEM. (H) Body weight loss (percentage of the initial weight) over 14 days in C57BL/6 mice (n = 20 per group) infected with WT H1N1 virus and treated daily with either vehicle (H1N1 + Vehicle) or LA1 (H1N1 + LA1). Weight loss in noninfected animals (control) and noninfected animals treated with LA1 (Control + LA1) is also shown. Each data point represents mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; A–E, 1-way ANOVA, Tukey’s test; F–H, Student’s t test).