Figure 3. Inflammasome activation requires viral entry by clathrin-mediated-endocytosis.

(A) The HIV entry receptor CD4 was blocked on monocytes with mAb prior to addition of HIV_BaL culture supernatant (HIV_BaL,). IL-18 production at multiple anti-CD4 mAb concentrations is shown relative to isotype control-treated monocytes. Symbols represent the mean ± S.D. of n = 4 experiments, (black triangles represents the mAb concentration previously determined to prevent HIV infection of CD4+ T-cells. CD4 blockade did not alter IL-18 production from monocytes at concentrations >100 fold that required to block infection. (B) The HCV entry receptor CD81 was similarly neutralized using anti-CD81 mAb. CD81 blockade did not alter IL-18 production from monocytes cultured with plasma from HCV_{Subject 180} (HCV_{Subject 180}). Symbols represent the mean ± S.D. of n = 4 experiments. (C) Receptor-mediated HIV entry or fusion was inhibited by adding Maraviroc (MVC) or T20, respectively to cultures of HIV_BaL and monocytes. IL-18 production was measured after 24 h with no inhibition seen. Bars represent the mean ± S.D. of n = 8 replicates. Clathrin-mediated or clathrin-independent endocytosis was inhibited by pre-incubating monocytes with MβCD, Genistein or Dynasore then adding (D) HIV_BaL or (E) HCV_{Subject 180}. IL-18 production was significantly reduced by Dynasore and MβCD, suggesting the clathrin-mediated endocytosis of the virus is required for inflammasome activation by HCV and HIV. (F) LPS was added to treated cells and IL-18 or TNF-α production measured after 24 h to assess cellular function independent of inflammasome activation. Bars represent the mean ± S.D. of n = 8 replicates, (*) denotes comparisons with p≤0.05 compared to the untreated cells.