PMC

Wild-type and TLR9-deficient macrophages exhibit comparable phagocytosis of C. albicans and S. cerevisiae and similar phagosomal maturation. WT and TLR9KO macrophages were incubated with fluorescent C. albicans (A) or S. cerevisiae (B) at an MOI of 5:1 for 2 h. Cells were washed with PBS to remove extracellular yeast. Cells were then analyzed by flow cytometry. WT (C) and TLR9KO (D) macrophages expressing CD63-mRFP1 were exposed to C. albicans at an MOI of 1:1 and imaged immediately using spinning disk confocal microscopy. WT (E) and TLR9KO (F) macrophages expressing CD82-mRFP1 were exposed to C. albicans at an MOI of 1:1 and imaged immediately using spinning disk confocal microscopy.

TLR9 is responsible for modulation of TNF-α production in WT macrophages. (A) Functional complementation of TLR9KO macrophages with TLR9-GFP restores the WT phenotype in response to CpG. TLR9KO macrophages were transduced with either a GFP-tagged version of wild-type TLR9 (TLR9-GFP) or a GFP-tagged deletion mutant lacking N-terminal TLR9 proteolytic cleavage residues 441 to 470 (ΔTLR9-GFP). (B and C) Functional complementation of TLR9 decreases the TNF-α level to the WT level in response to both C. albicans (B) and S. cerevisiae (C) yeast cells. Macrophages were exposed to live or autoclaved yeast cells at the indicated MOI or target-to-effector-cell ratio, respectively, and incubated for 6 h. Supernatants were analyzed for TNF-α by ELISA. Data represent means and SD of cytokine concentrations and are representative of three independent experiments.

The fungal component responsible for triggering TLR9 recruitment is sustained after loss of fungal cell wall structural integrity. (A) The cell wall ultrastructure of live C. albicans and S. cerevisiae is disrupted by autoclave treatment. Transmission electron microscopy images show live and autoclaved C. albicans (left) and S. cerevisiae (right). Bar, 500 nm. (B) TLR9 recruitment to C. albicans and S. cerevisiae phagosomes is retained after autoclave treatment. Confocal microscopy images of RAW macrophages expressing TLR9-GFP (green) are shown with their DIC counterparts. RAW cells were incubated with live or autoclaved C. albicans or S. cerevisiae yeast cells for 1 h. Representative images are shown in all panels. One focal plane is shown. Bar, 5 μm.

TLR9 deficiency leads to increased macrophage TNF-α production in response to C. albicans and S. cerevisiae. (A) Immortalized bone marrow-derived TLR9KO macrophages are unresponsive to CpG but retain responsiveness to other TLR agonists. WT and TLR9KO cells were incubated with 10 ng/ml Pam3CSK4, 1 ng/ml LPS, or 1 μM CpG for 4 h, and supernatants were analyzed for TNF-α by enzyme-linked immunosorbent assay (ELISA). (B and C) TLR9KO macrophages produce significantly more TNF-α than WT macrophages in response to C. albicans (B) and S. cerevisiae (C). WT and TLR9KO macrophages were exposed to live or heat-killed yeast cells at the indicated MOI or target-to-effector-cell ratio, respectively, and incubated for 6 h. Supernatants were analyzed for TNF-α by ELISA. Data represent means and SD and are representative of at least four independent experiments. P values were <0.04 (B) or <0.01 (C) for comparing cytokine secretion by WT and TLR9KO macrophages at all indicated ratios.

Fungi from different taxonomic groups trigger phagosomal recruitment of TLR9. (A to F) Confocal microscopy of RAW macrophages expressing TLR9-GFP (green). One focal plane is shown. Bar, 5 μm. (A) TLR9 is robustly recruited to C. albicans and A. fumigatus phagosomes. RAW cells were incubated with A. fumigatus resting conidia and C. albicans yeast cells for 1 h. A representative macrophage that has phagocytosed one C. albicans yeast cell (right) and one A. fumigatus resting conidium (left) is shown, demonstrating that both phagosomes have acquired TLR9. The DIC image demonstrates the presence of the two fungal organisms within the cell. (B) TLR9 accumulation on C. albicans phagosomes is retained during hypha formation. RAW cells were exposed to C. albicans yeast cells and incubated for 4 h. (C and D) TLR9 is recruited to S. cerevisiae (C)- and M. furfur (D)-containing phagosomes. RAW cells were incubated with yeast cells for 1 h. (E) Zymosan recruits TLR9 to the phagosome. The white arrow indicates the phagocytosed zymosan particle. RAW cells were incubated with zymosan for 30 min. (F) C. neoformans recruits TLR9 to the fungal phagosome. Representative images are shown in all panels.

TLR9 deficiency leads to increased macrophage activation and microbicidal activity against C. albicans and S. cerevisiae. (A) TLR9 deficiency results in increased nitric oxide production in response to C. albicans and S. cerevisiae. WT and TLR9KO macrophages were pretreated with 10 ng/ml IFN-γ overnight and then exposed to heat-killed C. albicans or S. cerevisiae yeast cells at the indicated target-to-effector-cell ratio or to 0.1 μM CpG or 1 ng/ml LPS for 36 h. Supernatants were analyzed for nitrite by Griess assay as a measure of macrophage activation. Data represent means and SD of cytokine concentrations and are representative of three independent experiments. In comparing WT and TLR9KO macrophages, P values were <0.0001 for C. albicans and <0.03 for S. cerevisiae at all indicated target-to-effector-cell ratios. (B) TLR9 deficiency results in increased fungal cell damage of C. albicans and S. cerevisiae. WT and TLR9KO macrophages were incubated with C. albicans yeast cells for 2 h or with S. cerevisiae for 6 h. Fungal cell survival was then measured by XTT assay. Data represent means and SD for at least four independent experiments, each tested in triplicate. In comparing WT and TLR9KO macrophages, P values were <0.02 for C. albicans and <0.003 for S. cerevisiae at all indicated target-to-effector-cell ratios. (C) TLR9KO macrophages show more potent fungicidal activity against S. cerevisiae. WT and TLR9KO macrophages were incubated overnight and then assessed for fungal survival by measuring CFU. Data represent means and SD for at least four independent experiments, each tested in triplicate. P values were <0.02 for comparing WT and TLR9KO macrophages at all indicated target-to-effector-cell ratios.