Fig 2: IL-33 supplementation fails to prevent trTreg reduction in established infection.(A) IL-33 concentration in plasma and skeletal muscle (SM) lysates obtained from Foxp3-GFP mice at different days post infection (dpi). Muscle IL-33 concentration was normalized to total protein content. Data are presented as individual replicates (circles) and mean (line). Dashed line in plasma indicates the assay’s limit of detection. (B) Representative dot plots and cumulative data (N = 4–6 replicates) showing the frequency of ST2+ KLRG-1+ Tregs (trTregs) within total Tregs isolated from the spleen of non-infected (NI) and infected (INF) (21 dpi) Foxp3-GFP mice. Left dot plots correspond to uncultured Tregs (ex vivo), while middle and right dot plots correspond to Tregs activated with anti-CD3+anti-CD28+IL-2 with or without IL-33 for 72 hours. Statistical significance was determined by Friedman test. (C) Experimental scheme illustrating the treatment of Foxp3-GFP mice with an established infection with intraperitoneal (i.p.) IL-33 or IL-33+IL-2. Created with BioRender.com. (D) Dot plots depicting the frequency of trTregs within total Tregs in the spleen of NI Foxp3-GFP mice 72 hours after receiving 3 doses of i.p. IL-33 or PBS, as described in (C). (E-H) Analysis of trTreg response and disease progression in INF mice treated with intraperitoneal IL-33 or IL-33+IL-2 as described in Fig 3C. (E) Graphs displaying trTreg frequency within total Tregs (upper row) and absolute number (bottom row) in SM, liver and spleen at 21 dpi. In all cases, values from untreated NI mice are also shown for comparison. In SM, counts are normalized to tissue weight. (F) Violin plots indicating the distribution of plasma LDH, GOT, GPT, CPK and CPK-MB activities, and glucose concentration at 21 dpi. N = 3–7 per group. (G) Percentage of total body weight reduction at 21 dpi compared to 15 dpi. (H) Survival curve in the different experimental groups. N = 6–11 per group. (E and G) Bars indicate the mean ± SEM. Squares in SM represent values obtained from pools of 2–4 mice, while circles in the remaining plots represent values from individual mice. Statistical significance was determined by Kruskal-Wallis test (A); Friedman test (B); one-way ANOVA (E); Mann-Whitney test (F and G) and Mantel-Cox test (H). P values in (A) are relative to 0 dpi: *p < 0.05; **p < 0.01; while in (E-H) are relative to PBS. Data are representative of two (A) and one (D-H) independent experiments.

Fig 3: IFN-γ limits trTreg expansion both in vitro after IL-33 stimulation and in vivo during T. cruzi infection.(A) Representative dot plots and cumulative data (N = 3) showing the frequency of ST2+ KLRG-1+ Tregs (trTregs) within total Tregs isolated from the spleen of non-infected (NI) Foxp3-GFP mice and incubated for 72 h with anti-CD3+anti-CD28 together with the addition of IL-2; IL-2+IL-33 and IL2+IL33 plus cytokines associated to Th1 signals. Statistical significance was determined by Friedman test. P values are relative to IL-2 condition: *p < 0.05. (B) Experimental scheme illustrating the treatment of Foxp3-GFP mice with anti-IFN-γ or anti-TNF at different days post infection (dpi). Created with BioRender.com. (C) Survival curve in the different experimental groups. N = 4–8 per group. (D-H) Analysis at 21 dpi of mice receiving anti-IFN-γ or isotype control. (D) Parasite counts in blood. (E) Total splenocyte counts (left) and skeletal muscle (SM) leukocyte counts (right). (F-H) Representative dot plots showing Tregs frequency within CD4+ cells in spleen (F) and SM (G), and trTreg frequency within total Tregs in spleen (H). Bars on the right indicate the frequency and absolute numbers (mean ± SEM) of the gated cell populations in the respective dot plots on the left. (E-H) Circles in bars represent individual replicates. (E and G) Cell counts in SM are normalized to tissue weight. Statistical significance was determined as follow: Mantel-Cox test (C) Mann-Whitney test for parasitemia (D and G) and unpaired t test (E, F and H). Data were collected from 1 (anti-TNF treatment) or 2 (anti-IFN-γ or isotype control treatment) experiments.