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. 2021 Sep 7;12:700501. doi: 10.3389/fimmu.2021.700501

Figure 4.

Figure 4

Absence of IL-10 changes the kinetics of monocyte activation in the BM. B6 and B6.Il10-/- mice were infected with L.donovani amastioges in the tail vein for 14, 28 and 56 days. Data from the BM are shown (A–G). (A) Parasites burden in the bone marrow determined by limiting dilution assay at the indicated time point. Two-way ANOVA applied on log10 transformed data: P<0.0001; statistical power: 11.9%%; n≥6; post hoc test Šídák’s MCT: statistical difference reached at day 28 and day 56 (P<0.0001; ****). (B) Dot plot representative of nos2 and MHCII expression by Ly6C+CCR2+ monocytes. (C–F) Histogram (C), bar graph showing MFI (D), percentage of MHCII (E) and percentage of nos2 (F) on BM Ly6C+CCR2+ monocytes. Two-way factorial analysis applied in (E): P=0.001; statistical power: 8.2%; n≥3; post hoc test EMM: statistical difference reached at day 14 and day 56 (P<0.0001; ****); and two-way factorial analysis applied in (F): P=0.013; statistical power: 6.1%; n≥4; post hoc test EMM: statistical difference reached at day 14 (P<0.0077; **). (G, I) Production of IFN-γ and TNF by CD4+ T cells in the BM at the different time point post-infection and (H, J) percentage of IFN-γ produced by CD4+ T cells after PMA-Ionomycin-BFA stimulation for 4h (G, H) and ex vivo (BFA stimulation only, (I, J). Brown-Forsythe ANOVA applied in (H): P<0.0001; statistical power: 43.8%; n≥4; post hoc test Dunnett’s T3: statistical difference reached at day 56 (P=0.0003; ***); 95% CI: 8.16 – 30.46; and Brown-Forsythe ANOVA applied in (J): P=0.0002; statistical power: 47.5%; n≥4; post hoc test Dunnett’s T3: statistical difference reached at day 56 (P=0.0032; **); 95% CI: 4.99 – 24.47. (K–M) B6 mice infected with L. donovani were injected with an anti-IL-10 depleting antibody or the isotype control for 14 days starting 1 day prior to the infection (K) Parasite load determined by limiting dilution assay in the BM at day 14 p.i. Welch’s t test applied: P=0.0717; 95% CI: -1.86 – 0.115; statistical power: 13.9%; n=5. (L) Percentage and (M) MFI of MHCII expressed on Ly6C+CCR2+ monocytes. Brown-Forsythe ANOVA applied in (L): P<0.0001; statistical power: 100%; n≥3; post hoc test Dunnett’s T3: statistical difference reached in NA vs. aIL10R (P<0.0001;****; 95% CI: -79.79 – -53.75) and CLT vs. aIL10R (P<0.0001; ****; 95% CI: -67.77 – -49.41); and Brown-Forsythe ANOVA applied in (M): P=0.0009; statistical power: 100%; n≥3; post hoc test Dunnett’s T3: statistical difference reached in NA vs. aIL10R (P<0.0034; **; 95% CI: -4367 – -1169) and CLT vs. aIL10R (P<0.0034; **; 95% CI: -4361 – -1175). For (A–J), data are derived from analysis of 6 to 7 individual mice (2 to 4/experiment) of each strain at each time point pooled from two independent experiments and are shown as mean ± SD. Data in (B, C, G) and I are representative plots. For (K–M), data are the pool of 5 mice/group/time point. Mean ± SD is shown. Experiment was performed once. In (A, D–F) median with 95% CI is shown. For (H, J) statistical details are found in Table S1.