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. 2020 Jan 24;117(6):3074–3082. doi: 10.1073/pnas.1911579117

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Fig. 3. — Expression of dysregulated TLR9 by neonatal macrophages and Ly6C^hi monocytes correlates with liver inflammation and pancreatitis. (A) Survival curve of indicated genotypes. Results are combined from multiple litters and analyzed using Log-rank (Mantel-Cox) test. (B) TLR9^fsTM/+ β-actin-cre⁺ and TLR9^fsTM/+ Cd11c-cre⁺ neonates exhibit an increase in LSK, eGMP, and Ly6C^hi monocyte cell populations in the liver compared with TLR9^fsTM/+ Lyz2-cre⁺ neonates. 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^fsTM/+ β-actin-cre^–, n = 15; TLR9^fsTM/+ β-actin-cre⁺, n = 13; TLR9^fsTM/+ Cd11c-cre^–, n = 9; TLR9^fsTM/+ Cd11c-cre⁺, n = 4 and n = 10; TLR9^fsTM/+ Lyz2-cre^–, n = 10; TLR9^fsTM/+ Lyz2-cre⁺, n = 11. (C) Histology scores of liver (Left) and pancreas (Right) sections from the indicated mice. Pathology scoring scheme for liver: 0 = hepatocytes contain diffuse cytoplasmic lipid vacuoles; 1 = hepatocytes contain many small cytoplasmic lipid vacuoles; 2 = hepatocytes contain a few small cytoplasmic lipid vacuoles; 3 = hepatocytes contain no lipid. Pathology scoring scheme for pancreas: inflammation with exocrine cell necrosis or zymogen depletion or both: 0 = unaffected; 1 = minimal diffuse to mild multifocal; 2 = mild diffuse to moderate multifocal; 3 = moderate diffuse to marked multifocal. Data are combined from multiple experiments and analyzed using Kruskal–Wallis ANOVA and Dunn’s multiple comparisons test. Mouse numbers: TLR9^fsTM/+ β-actin-cre^–, n = 6; TLR9^fsTM/+ β-actin-cre⁺, n = 8; TLR9^fsTM/+ Cd11c-cre^–, n = 7; TLR9^fsTM/+ Cd11c-cre⁺, n = 6; TLR9^fsTM/+ Lyz2-cre^–, n = 6; TLR9^fsTM/+ Lyz2-cre⁺, n = 11. (D) TLR9^fsTM/+ β-actin-cre⁺ neonates suffer from anemia. Blood hemoglobin levels from the indicated mice are shown. Results combined from multiple experiments are shown as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparisons posttest. Mouse numbers: TLR9^fsTM/+ β-actin-cre^–, n = 14, TLR9^fsTM/+ β-actin-cre⁺, n = 12; TLR9^fsTM/+ Cd11c-cre^–, n = 7; TLR9^fsTM/+ Cd11c-cre⁺, n = 7; TLR9^fsTM/+ Lyz2-cre^–, n = 8; TLR9^fsTM/+ Lyz2-cre⁺, n = 6. (E) The frequency of TLR9-expressing neonatal macrophages correlates with disease severity. Quantification of CD45⁺CD3e^–CD19^–Ly6G^–CD11b⁺F4/80⁺Mertk⁺ TLR9 (GFP⁺) macrophages from the neonatal livers of the indicated genotypes. Results combined from multiple experiments are shown as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparisons posttest. Mouse numbers: TLR9^fsTM/+ β-actin-cre⁺, n = 13; TLR9^fsTM/+ Cd11c-cre⁺, n = 12; TLR9^fsTM/+ Lyz2-cre⁺, n = 6. (F) The frequency of macrophages and Ly6C^hi monocytes expressing TLR9 (GFP⁺) and TLR7 (TdTomato⁺). Results combined from multiple experiments are shown as mean ± SEM and analyzed using the two-tailed Student’s t test. Mouse numbers: TLR9^GFP/GFP, n = 12; TLR7^TOM/TOM, n = 6. (G) Representative histograms of TLR9 and TLR7 expression in Ly6C^hi monocytes and macrophages (CD45⁺CD3e^–CD19^–Ly6G^–Ly6C^–CD11b⁺F4/80⁺) from adult bone marrow from TLR9^GFP/GFP or TLR7^TOM/TOM reporter mice compared with B6 WT controls (gray). In all panels, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.