Figure 6.

IRF7 and TLR3 are recruited to L. donovani phagosomes and IFN-β can revert macrophage leishmanicidal activity. (A–F) Thioglycolate-recruited macrophages from C57BL/6 mice were cultivated on glass coverslips overnight in RPMI-FCS and then washed and infected with late-stage promastigotes of L. donovani at a 10:1 parasite:macrophage ratio for 3 h at 37°C in RPMI-BSA. After, the monolayers were extensively washed with HBSS to remove extracellular parasites, fixed with paraformaldehyde or cultivated up to 72 h (as indicated in the images) in RPMI-FCS at 37°C, before fixation. Cells were labeled with (A–C) anti-IRF7 antibodies or (D–F) anti-TLR3 antibodies, followed by the appropriate secondary antibody conjugated to Cy3. Arrows point to TLR3 co-localized with parasites and arrowheads point to parasites not co-localized with TLR3. (G) Macrophages of the 14M1.4 cell line were treated with mitomycin C, washed, and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio in DMEM-BSA. After 3 h, the monolayers were washed with HBSS to remove extracellular parasites, fixed with paraformaldehyde and Giemsa-stained, or further incubated for 24 h, 48 h, or 72 h in DMEM-FCS at 37°C before fixation and staining. Where indicated, 200 ng/ml IFN-β was added to cultures that had been previously infected for 3 h and washed for removal of extracellular parasites, and cultivated for 48 h or 72 h in DMEM-FCS. The number of intracellular parasites was determined under a light microscope. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. *p < 0.05, ***p < 0.001.