Figure 7.

TLR3 is required for the induction of type I IFN, IL10, and OASL2 and for parasite growth in macrophages. Thioglycolate-recruited peritoneal macrophages were infected with stationary-phase promastigotes of L. donovani at a 10:1 parasite:macrophage ratio for 2 h, cultures were washed, and RNA was extracted or cultures were incubated in RPMI-BSA until 6 h before extraction. cDNA was made and used as templates in qPCR for the determination of the relative mRNA levels for (A) IFN-β, (B) IFN-α, (C) IL10, and (D) OASL2. Poly I:C was used at 25 µg/ml. The experiment was repeated 2 independent times. The graphs show representative experiments, with the means ± SD of the triplicate technical replicates. Statistical significance was assessed using one-way ANOVA with Bonferroni posttest. (E) Peritoneal thioglycolate-recruited macrophages from C57BL/6 and tlr3 ^-/- mice were cultivated on glass coverslips overnight in RPMI-FCS and then washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI-BSA. After 3 h, the monolayers were washed with HBSS to remove extracellular parasites, fixed with methanol and Giemsa-stained, or further incubated for 24 h, 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under a light microscope. The experiment was performed 2 independent times. The graphs show a representative experiment of the means ± SD of technical replicates in triplicate. Statistical significance was assessed using two-way ANOVA with Tukey’s and Sidak’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001.