Role of MyD88 signaling in DCs for survival of mice after T. gondii infection. (A and B) Female mice 7–9 wk old were infected i.p. with 40 (A) or 200 (B) cysts of the avirulent Me49 strain, and their survival was monitored daily up to 8 wk after infection. (A) Results of WT Myd88^fl/− mice (n = 17), DC-Myd88^−/− mice (n = 14), MN-Myd88^−/− mice (n = 8), and Myd88^−/− mice (n = 6) from two separate experiments were pooled. (B) Results of WT mice (n = 9), DC-Myd88^−/− mice (n = 9), and Myd88^−/− mice (n = 4) from two separate experiments were pooled. Significant differences were found between WT mice and the DC-Myd88^−/− mice in both low and high infecting doses (log-rank test; P < 0.001), and between DC-Myd88^−/− mice and Myd88^−/− mice at low infection dose (P < 0.05). (C) The amount of T. gondii genomic DNA was measured by quantitative PCR after infection with 40 cysts of the Me49 strain. *P < 0.05; **P < 0.01. (D) The amount of T. gondii in the lavaged peritoneal cells of Tcrα^−/− mice, Rag1^−/− mice, and WT mice at day 5 after infection. Data are expressed as mean plus SD of three or four mice and are representative of two to four separate experiments. Statistical comparison is between the DC-Myd88^−/− mice and the WT control mice.

Role of MyD88 in DCs for the recruitment of inflammatory cells in acute T. gondii infection. (A–D) Peritoneal cells were enumerated and analyzed by flow cytometry after infection with 40 cysts of the Me49 strain. The numbers of (A) total infiltrating cells (peritoneal exudate cells, PECs), (B) DCs (CD11c⁺, MHC II⁺), (C) inflammatory monocytes (Mo) (F4/80^int, Ly6C⁺, Ly6G⁻), and (D) NK cells (NK1.1⁺, TCRβ⁻, MHCII⁻, F4/80⁻) are shown. Data are expressed as mean plus SD of three or four mice and are representative of two or three separate experiments. (E) Numbers of infected peritoneal cells (mean plus SD of four mice) on day 5 after infection with 100 tachyzoites of the GFP-expressing PruΔhtp strain. (F) Median fluorescence intensity (MFI) of surface MHC II on inflammatory monocytes. Statistical comparison is between the DC-Myd88^−/− mice and the WT control mice. **P < 0.001. (G) Intracellular iNOS expression in gated inflammatory monocytes (Ly6G⁻, Ly6C⁺, F4/80⁺) on day 3 after infection as analyzed by flow cytometry.

Induction of IFN-γ in the T. gondii-infected DC-Myd88^−/− mice. (A) Induction of IFN-γ mRNA in total peritoneal cells as measured by quantitative RT-PCR. Data are expressed as mean plus SD of the normalized threshold cycle number (−ΔCt) of three or four mice and are representative of two or three separate experiments. (B–D) Intracellular IFN-γ as analyzed by flow cytometry. (B) Numbers of IFN-γ⁺ NK cells (mean plus SD of three or four mice) in the peritoneum after infection. (C) Numbers of IFN-γ⁺ T cells in the peritoneum after infection. Statistical comparison is between the DC-Myd88^−/− mice and the WT control mice. *P < 0.05; **P < 0.01. (D) Representative FACS plots of intracellular IFN-γ staining on gated NK cells (NK1.1⁺, TCRβ⁻) on day 2 and day 3 after infection.

Induction of IL-12 in the T. gondii-infected DC-Myd88^−/− mice. (A–D) Induction of IL-12 in peritoneal cells was analyzed after infection with 40 cysts of the Me49 strain. (A) Total amount of IL-12p40 (mean plus SD of three or four mice) in the supernatant of peritoneal lavage measured by ELISA. (B) Representative flow cytometry plots of intracellular IL-12p40 staining of gated DCs (CD11c⁺, MHC II⁺) at day 3 after infection. (C) Correlation analysis of the amount of IL-12p40 and the induction of IFN-γ mRNA in the peritoneum at day 2 and day 3 after infection. The amount of IL-12p40 was determined as in A, and the level of IFN-γ mRNA was determined as in Fig. 3A. ◇, WT mice; ◆, DC-Myd88^−/− mice. (D–F) The effects of IL-12 treatment of mice infected with 40 cysts of the Me49 strain. (D) Representative flow cytometry plots of intracellular IFN-γ in gated NK cells 1 d after supplementation with IL-12 or with PBS control on day 1 after infection. (E) Representative flow cytometry plots of intracellular iNOS in gated inflammatory monocytes on day 3 after infection. Mice in this experiment received supplementation with IL-12 or with PBS control on day 1 and 2 after infection. (F) Total amount of T. gondii in the peritoneal cells at day 7 after infection. Four mice of each strain were treated twice with PBS or 150 ng IL-12 daily on day 1 and day 2. Statistical comparison is between the DC-Myd88^−/− mice treated with or without IL-12. **P < 0.01.

Activation of T cells in the DC-Myd88^−/− mice. Mice were infected with 40 cysts (A–C) or 100 cysts (D) of the Me49 strain. (A and B) Splenocytes were analyzed for intracellular IFN-γ and TNF 8 d later after in vitro stimulation with anti-CD3 for 4 h in the presence of brefeldin A. (A) The total number of IFN-γ⁺ CD4⁺ and CD8⁺ T cells in the spleens (mean plus SD of four or five mice). (B) Representative flow cytometry plots of intracellular IFN-γ and TNF staining of gated CD4⁺ and CD8⁺ T cells. (C and D) Mice were treated with either PBS or 150 ng IL-12 daily from day 1–3. T. gondii tissue cysts were enumerated in brain homogenates of surviving mice at day 28 after infection. Statistical comparison is between WT mice and the DC-Myd88^−/− mice or the DC-Myd88^−/− mice treated with or without IL-12. *P < 0.05. **P < 0.01. (D) The survival 7- to 8-wk-old female mice was monitored daily up to 6 wk after infection. Results of PBS-treated WT mice (n = 12), DC-Myd88^−/− mice (n = 10), IL-12-treated WT mice (n = 5), DC-Myd88^−/− mice (n = 5), and Myd88^−/− mice (n = 4) are shown. A significant difference was found between DC-Myd88^−/− mice treated with or without IL-12 (log-rank test; P < 0.01).