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. 2020 Mar 3;11:306. doi: 10.3389/fimmu.2020.00306

Figure 3.

Figure 3

Inflammation and IFNγ concentrations in heart tissue, leukometry and CD8+ T-cells in the spleen of chronically T. cruzi-infected mice. ccl3+/+ and ccl3−/− mice were infected with 100 trypomastigote forms of the Colombian T. cruzi strain and analyzed at 120 dpi. (A) Representative sections of heart tissues of infected ccl3+/+ and ccl3−/− mice were IHC for CD8 marker. Horizontal bar indicates 30 μm. (B) IHC data showing total inflammation and the presence of CD4+, CD8+, and F4/80+ (macrophages) cells in 100 microscopic field of heart tissue of T. cruzi-infected ccl3+/+ and ccl3−/− mice. (C) IHC data showing the presence of Pfn+ and IFNγ+ in 100 microscopic field of heart tissue of T. cruzi-infected ccl3+/+ and ccl3−/− mice. (D) Hearts of NI and infected mice ccl3+/+ and ccl3−/−were collected, extracts were prepared and IFNγ concentrations were estimated by ELISA. Horizontal gray bar shows cytokine levels in non-infected age-matched control mice (means). Data are expressed as ng of CCL3 per 100 mg of heart tissue. (E) Peripheral blood was collected; red blood cells were lysed with Turk's reagent and the total leukocytes counted. (F) Flow cytometric analysis reveals the percentage of total CD8+ T-cells and CCR5+ LFA-1+ among CD8+ T-cells in the spleen of T. cruzi-infected ccl3+/+ and ccl3−/− mice and respective NI controls. After selection of singlets (FSC-Lin × FSC-Area, R1), dead-cell exclusion (FSC-A × SSC-Lin, R2), TCR × CD8 dot plot (gating on TCR+ CD8+ cells, R3), LFA1 × CCR5 dot plot were analyzed. Each experimental group consisted of three non-infected mice and four to five T. cruzi-infected mice. Experiments were repeated twice. Data are presented as means ± SE. *p < 0.05, **p < 0.01 and ***p < 0.001, comparing T. cruzi-infected and NI mice; #p < 0.05 and ##p < 0.01, comparing T. cruzi-infected ccl3+/+ and ccl3−/− mice. (t-Student test, ANOVA Bonferroni posttest).