Preserved TLR4-mediated MyD88-independent pathway in HIV⁺ human macrophages. (A) Human macrophage IL-10 release is mediated by TLR4. U937 and U1 cells were incubated with lipid A (10 μg/mL) in the presence or absence of neutralizing anti-TLR4 antibody (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 by ELISA. Data shown are mean ± SEM of 4 independent experiments done in triplicate. *P < .01 compared with U937 with lipid A alone; **P < .05 compared with U1 with lipid A alone. (B-C) Lipid A–mediated release of IL-10 and RANTES is preserved in HIV⁺ macrophages. U937 and U1 cells were incubated with or without lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 (B) or RANTES (C) by ELISA. Data shown are mean ± SEM of 4 independent experiments done in triplicate. (D-E) Functional silencing of human MyD88 did not affect TLR4-mediated release of RANTES (D) and IL-10 (E). Cells were challenged with lipid A and incubated for 24 hours. Cell-free supernatant was assayed for RANTES and IL-10 by ELISA. Results are representative of 3 independent experiments done in triplicate. (F-H) Healthy and HIV⁺ AMs show similar levels of IL-10 release (F), similar levels of the TLR4 adaptor molecule MD2 (J), and surface expression of TLR4 coreceptor CD14 (H). (F) Healthy AMs and HIV⁺ AMs were incubated with or without lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 by ELISA. Data shown are mean ± SEM from 4 independent experiments in triplicate (n = 4 subjects for each group). (G) Total cell lysates from healthy AMs and HIV⁺ AMs were probed with specific anti-MD2 antibody by Western blot, with β-actin probed for protein loading. (H) Healthy AMs and HIV⁺ AMs were incubated with PE-conjugated anti-CD14 antibody or isotype control antibody, and surface expression was determined by flow cytometry. Representative blot and profiles were similar of 3 independent experiments (n = 3 subjects for each group).