T-bet-dependent ILC1- and NK cell-derived IFN-γ mediates cDC1-dependent host resistance against Toxoplasma gondii

Abstract

Host resistance against intracellular pathogens requires a rapid IFN-γ mediated immune response. We reveal that T-bet-dependent production of IFN-γ is essential for the maintenance of inflammatory DCs at the site of infection with a common protozoan parasite, Toxoplasma gondii. A detailed analysis of the cellular sources for T-bet-dependent IFN-γ identified that ILC1s and to a lesser degree NK, but not TH1 cells, were involved in the regulation of inflammatory DCs via IFN-γ. Mechanistically, we established that T-bet dependent innate IFN-γ is critical for the induction of IRF8, an essential transcription factor for cDC1s. Failure to upregulate IRF8 in DCs resulted in acute susceptibility to T. gondii infection. Our data identifies that T-bet dependent production of IFN-γ by ILC1 and NK cells is indispensable for host resistance against intracellular infection via maintaining IRF8+ inflammatory DCs at the site of infection.

Fig 1. T-bet is critical for inflammatory DCs and host resistance during acute T. gondii infection.

(A) Survival of WT (●), Tbx21^-/- (▼), and Rag2^-/- (♦) mice infected i.p. with 20 cysts of the ME49 strain of T. gondii. WT and Tbx21^-/- mice were i.p. infected and PECs were harvested on days 0, 5, and 8 (B, C, D) and Lin^-CD11c⁺MHCII⁺ DCs were analyzed by flow cytometry. Average frequencies of Lin^-CD11c⁺MHCII⁺ DCs (B, C, D) in the PECs were analyzed on days 0, 5, and 8 following infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using Log-rank (Mantel Cox) test or unpaired t-test analysis of individual groups, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, standard error mean.

Fig 2. T-bet is essential for sustaining IRF8+ DCs.

(A-F) WT and Tbx21^-/- mice were infected i.p. with 20 cysts of T. gondii. (A, C, E) Representative contour plots and (B, D, F) average frequencies of Lin^-CD11c⁺MHCII⁺IRF8⁺ DCs in the PECs as analyzed on days 0, 5, and 8 of infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using unpaired t-test analysis of individual groups, ****P<0.0001. Error bars, standard error mean.

Fig 3. Early T-bet-dependent IFN-γ is critical to maintain inflammatory IRF8+ cDCs1.

(A) IFN-γ analysis by ELISA of serum in mice following T. gondii infection on days 2, 3, and 5 post-infection. (B-F) Tbx21^-/- mice were i.p. infected with T. gondii and treated with or without IFN-γ. (B) Representative contour plots and (C) average frequencies of Lin^-CD11c⁺MHCII⁺ DCs in the PECs were analyzed on day 5 following infection. (D) Representative contour plots and absolute quantification of (E) Lin^-CD11c⁺MHCII⁺IRF8⁺ DCs in the PECs were analyzed on day 5 following infection. (F) Mean fluorescent intensity (MFI) of Lin^-CD11c⁺MHCII⁺ DC IRF8 expression in the PECs was analyzed on day 5 post-infection. Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using (A, E, F) unpaired t-test analysis of individual groups or (C) one-way Anova with Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, standard error mean.

Fig 4. IFN-γ and ILC1s are required for sustaining inflammatory DCs during T. gondii infection.

(A, B) WT and Rag2^-/- mice were i.p. infected with 20 cysts of T. gondii and then treated with anti-IFN-γ, anti-NK1.1, or anti-Thy1 antibodies during infection. (C) Average frequencies of CD11c⁺MHCII⁺ DCs in the PECs were analyzed on day 8 following infection and antibody treatment. (D-G) WT and Tbx21^-/- mice were infected i.p. with 20 cysts of T. gondii. (D) Day 3 representative contour plots and (F) average frequencies of CD127-NKp46+IFN-γ+ NK cells in the PECs were analyzed on days 3 and 5 following infection. (E) Day 3 representative contour plots and (G) average frequencies of CD127⁺NKp46⁺T-bet⁺ ILC1s cells in the PECs were analyzed on days 3 and 5 following infection. (H) Sort-purified NK cells (CD45+CD3-CD19-NKp46+CD127- NKs) and ILC1s (CD45+CD3-CD19-NKp46+CD127+) from the PECs of WT mice (S6C Fig) infected with T. gondii were adoptively transferred into Tbx21^-/- mice on day 2 post infection, and the presence of DCs (Lin-CD11c+MHCII+) was analyzed by flow cytometry 3 days later (day 5 post infection). Results are representative of three-independent experiments involving at least 3 mice per group. Statistical analyses were done using unpaired t-test analysis of individual groups, or (C) one-way Anova with Tukey’s multiple comparison test, *P<0.05, ***P<0.001, ****P<0.0001.

Fig 5. cDC1s are essential for host resistance against intracellular infection.

(A, B, C) WT, DC-Irf8^-/-, and Irf8^-/- mice were infected i.p. with 20 cysts of T. gondii. (A, B) Representative contour plots, average frequencies, and absolute number of (A) Lin^-CD11c⁺MHCII⁺ and (B) Lin^-CD11c⁺MHCII⁺IRF8⁺ DCs in the PECs were analyzed on day 5 following infection. (C) WT, DC-Irf8^-/-, and Irf8^-/- mice were i.p. infected with T. gondii and pathogen burden was assessed from PECs and spleen of mice following infection by qPCR on day 5 post-infection. (D) Survival of WT (●), Tbx21^-/- (▲), and DC-Irf8^-/- (■) mice infected i.p. with 20 cysts of T. gondii. Results are representative of four independent experiments involving at least 3 mice per group. Statistical analyses were done using (D) Log-rank (Mantel Cox) test or (A-C) one-way Anova with Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars, standard error mean.