Fig. 5.

Expansion of IFN-γ–producing NK cells correlates with neonatal inflammation. (A, Left) Representative flow cytometry plots of IFN-γ–producing cells in neonatal livers of TLR9^fsWT/+β-actin-cre⁺ and TLR9^fsTM/+β-actin-cre⁺ mice. (A, Right) Quantification of IFN-γ⁺ cells in neonatal livers of mice of the indicated genotypes. Data combined from multiple litters are shown as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparisons posttest. Mouse numbers: TLR9^fsWT/+ β-actin-cre⁺, n = 5; TLR9^fsTM/+ β-actin-cre⁺, n = 19; TLR9^fsTM/+ Cd11c-cre⁺, n = 8; TLR9^fsTM/+ Lyz2-cre⁺, n = 7. (B) Expansion of IFN-γ⁺ NK1.1⁺ cells in TLR9^fsTM/+β-actin-cre⁺ neonates. Shown are representative flow cytometry plots of NK1.1 and IFN-γ staining on CD3^–CD19^–NK1.1⁺ gated cells from neonatal livers of the indicated mice. (C) Quantification of IFN-γ⁺CD3^–CD19^–NK1.1⁺ subsets of indicated genotypes. Results from multiple litters are shown as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparisons posttest. Mouse numbers: TLR9^fsWT/+ β-actin-cre⁺, n = 5; TLR9^fsTM/+ β-actin-cre⁺, n = 9; TLR9^fsTM/+ Cd11c-cre⁺, n = 8; TLR9^fsTM/+ Lyz2-cre⁺, n = 7. (D) Quantification of GFP⁺ and GFP^– CD3^–CD19^–NK1.1⁺ cells in neonatal livers. Results are combined from multiple litters and shown as mean ± SEM and analyzed using the two-tailed Student’s t test. Mouse numbers: TLR9^fsWT/+ β-actin-cre⁺, n = 5; TLR9^fsTM/+ β-actin-cre⁺, n = 15. (E) Analysis of IFN-γ secretion by GFP⁺ and GFP^– total cells by ELISpot. Quantification of IFN-γ secreting cells of indicated genotypes. Results combined from multiple litters are shown as mean ± SEM and analyzed using the two-tailed Student’s t test. Mouse numbers: TLR9^fsWT/+ β-actin-cre⁺, n = 7; TLR9^fsTM/+ β-actin-cre⁺, n = 7. (F) Neonatal liver cells from TLR9^fsTM/+ B-actin-cre⁺ mice secrete more IL-18 and TNF compared to littermate controls. Shown are cytokine levels of adherent fetal liver cells cultured from TLR9^fsTM/+ β-actin-cre⁺ and TLR9^fsTM/+ β-actin-cre^– neonates. Results are combined from two independent experiments and analyzed using the two-tailed Student’s t test. Mouse numbers: TLR9^fsTM/+ β-actin-cre^–, n = 9; TLR9^fsTM/+ β-actin-cre⁺, n = 8. (G) Neonatal macrophages from TLR9^fsTM/+ β-actin-cre⁺ and TLR9^fsTM/+ Cd11c-cre⁺ neonates secrete significantly more TNF and IL-12p40 compared with TLR9^fsTM/+ Lyz2-cre⁺ neonates. Results are combined from multiple experiments and analyzed using one-way ANOVA with Tukey’s multiple comparisons posttest. Mouse numbers: TLR9^fsTM/+ β-actin-cre^–, n = 10; TLR9^fsTM/+ β-actin-cre⁺, n = 8; TLR9^fsTM/+ Cd11c-cre^–, n = 7; TLR9^fsTM/+ Cd11c-cre⁺, n = 12; TLR9^fsTM/+ Lyz2-cre^–, n = 8; TLR9^fsTM/+ Lyz2-cre⁺, n = 6. In all panels, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.