Figure 1
Figure 1. Reduced TLR4-mediated TNFα release associated with impaired MyD88-dependent signaling pathway in HIV+ human macrophages. (A) Human macrophage TNFα release is mediated by TLR4. Human macrophage 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 TNFα 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) Reduced TLR4-mediated MyD88-IRAK interaction and IRAK phosphorylation in HIV+ alveolar macrophages (AMs). Healthy AM (B left) and HIV+ AM (B right) were incubated with lipid A (10 μg/mL) up to 60 minutes, detergent soluble extracts were immunoprecipitated with anti-MyD88 antibody and immunoblotted with anti–p-IRAK antibody. Densitometric analysis for each p-IRAK band is displayed beneath Western blot. Western blot is a representative of 3 independent experiments with similar results (n = 3). *P < .01 unstimulated control; **P < .01 compared with healthy in the presence of lipid A with time. (C) Reduced TLR4-mediated NF-κB nuclear translocation in HIV+ macrophages. U937 and U1 cells were incubated with lipid A (10 μg/mL) up to 120 minutes, nuclear and cytoplasmic extracts were isolated and probed with anti-p65 antibody. Densitometric analysis of p65 bands for each lane is displayed beneath Western blot. Western blot is a representative of 4 independent experiments with similar results. *P < .01 compared with unstimulated control; **P < .01 compared with U937 cells in the presence of lipid A with time. (D) Functional silencing of human MyD88 leads to marked diminution of TLR4-mediated TNFα release in U937 cells. Western blot analysis of human MyD88 after gene silencing with the use of MyD88 shRNA and scrambled shRNA. β-Actin was used to monitor protein loading after stripping the membrane. A representative blot shows results from 1 experiment with similar results of 3 independent experiments (left). U937 cells were pretreated with shRNA MyD88 and scrambled shRNA. Cells were differentiated with phorbol ester, challenged with lipid A and incubated for 24 hours. Cell-free supernatant was assayed for TNFα by ELISA. Results are representative of 3 independent experiments in triplicate.

Reduced TLR4-mediated TNFα release associated with impaired MyD88-dependent signaling pathway in HIV+ human macrophages. (A) Human macrophage TNFα release is mediated by TLR4. Human macrophage 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 TNFα 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) Reduced TLR4-mediated MyD88-IRAK interaction and IRAK phosphorylation in HIV+ alveolar macrophages (AMs). Healthy AM (B left) and HIV+ AM (B right) were incubated with lipid A (10 μg/mL) up to 60 minutes, detergent soluble extracts were immunoprecipitated with anti-MyD88 antibody and immunoblotted with anti–p-IRAK antibody. Densitometric analysis for each p-IRAK band is displayed beneath Western blot. Western blot is a representative of 3 independent experiments with similar results (n = 3). *P < .01 unstimulated control; **P < .01 compared with healthy in the presence of lipid A with time. (C) Reduced TLR4-mediated NF-κB nuclear translocation in HIV+ macrophages. U937 and U1 cells were incubated with lipid A (10 μg/mL) up to 120 minutes, nuclear and cytoplasmic extracts were isolated and probed with anti-p65 antibody. Densitometric analysis of p65 bands for each lane is displayed beneath Western blot. Western blot is a representative of 4 independent experiments with similar results. *P < .01 compared with unstimulated control; **P < .01 compared with U937 cells in the presence of lipid A with time. (D) Functional silencing of human MyD88 leads to marked diminution of TLR4-mediated TNFα release in U937 cells. Western blot analysis of human MyD88 after gene silencing with the use of MyD88 shRNA and scrambled shRNA. β-Actin was used to monitor protein loading after stripping the membrane. A representative blot shows results from 1 experiment with similar results of 3 independent experiments (left). U937 cells were pretreated with shRNA MyD88 and scrambled shRNA. Cells were differentiated with phorbol ester, challenged with lipid A and incubated for 24 hours. Cell-free supernatant was assayed for TNFα by ELISA. Results are representative of 3 independent experiments in triplicate.

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