Fig. 1. Both chemical inhibition and genetic loss of TLR4 results in an increase in the phagocytosis of C. neoformans in both murine and human macrophages.

a J774A.1 macrophages or (c) human monocyte derived macrophages (HMDMs) were treated with DMSO (control) or 0.2 μM TAK-242, a TLR4 specific inhibitor, for 1 h before infection with non-opsonised C. neoformans. b Immortalised bone marrow derived macrophages (iBMDM) from wildtype and Tlr4^−/− macrophages were infected with non-opsonised C. neoformans. Phagocytosis was quantified as the number of individual internalised cryptococci within 100 macrophages. Figures are representative of at least three independent experiments. HMDM data represents two independent experiments with separate donors. d Representative image showing the phagocytosis of GFP-labelled C. neoformans (Cn-GFP) by wildtype and Tlr4^−/− iBMDM. Calcofluor White (CFW) was used to stain extracellular fungi. White arrows show phagocytosed fungi. Scale bar = 10 μm. The intracellular proliferation (IPR) of C. neoformans was measured in (e) J774A.1 macrophages and (f) wildtype and Tlr4^−/− iBMDMs using timelapse imaging. Images were captured every 5 mins for 18 h. The number of internalised fungi per 100 macrophages at the ‘first frame’ (T0) and ‘last frame’ (T10) was quantified and IPR was determined using the equation: IPR = T10/T0. Data is representative of two independent experiments. All data shown as mean ± SEM; n = 3 per condition; statistical significance was evaluated using an unpaired two-sided t-test; P-values are shown above each graph. Source data are provided as a Source Data file.