Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition
Fig 8
The mar1Δ mutant induces increased TNF-α production from macrophages and dendritic cells.
(A) Macrophage activation by mar1Δ cells is not dependent on cell viability. WT, mar1Δ, and mar1Δ + MAR1 cells were incubated for 16–18 hours at 37°C in TC medium. Bone marrow-derived macrophages (BMMs) were co-incubated with live or heat-killed (HK, 1–2 hours at 65°C) C. neoformans strains at a multiplicity of infection (MOI) of 10:1, C. neoformans:BMMs. TNF-α levels (pg/ml) were assayed from the supernatant by ELISA. Data represent means from 3 replicates per strain per condition from 3 independent experiments (n = 9). *, p < 0.05; ***, p < 0.001 mar1Δ vs. WT as determined by two-way ANOVA with Tukey’s multiple comparisons test. (B) mar1Δ induces increased TNF-α production by dendritic cells. C. neoformans cells were incubated as described above and co-incubated with bone marrow derived dendritic cells (BMDCs) at an MOI of 10:1, C. neoformans:BMDCs. TNF-α levels were assayed from the supernatant by ELISA. Data represent means from 3 replicates per strain from 2 independent experiments (n = 6). **, p < 0.01 mar1Δ vs. WT as determined by one-way ANOVA with Tukey’s multiple comparisons test. (C) Macrophage activation by mar1Δ cells is dependent on pre-culturing in TC medium. Cells were incubated for 16–18 hours in YPD (30°C) or TC (37°C) prior to co-incubation with BMMs and TNF-α quantification by ELISA as above. Data represent means from 3 replicates per strain (n = 3). ****, p < 0.0001 mar1Δ/TC vs. WT/ TC as determined by two-way ANOVA with Tukey’s multiple comparisons test. mar1Δ/YPD vs. WT/YPD, not significant. (D) Cell wall material isolated from mar1Δ induces increased macrophage activation. WT, mar1Δ, and mar1Δ + MAR1 reconstituted cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. 10 μg/ml of cell wall material was co-cultured with BMMs and TNF-α was quantified from the supernatant by ELISA. Data represent means of 3 independent cell wall isolations in 3 independent experiments (n = 9 for each strain). *, p < 0.05 mar1Δ vs. WT as determined by one-way ANOVA with Tukey’s multiple comparisons test.