*3.2. The Presence of Peritoneal HO-1+ Cells Associates with Increased Splenic CD4+ CD25+ and CD8<sup>+</sup> CD25<sup>+</sup> Cells during Infection*

Considering that HO-1 can induce regulatory T cells [34–37], we analyzed the presence of both CD4*<sup>+</sup>* and CD8*<sup>+</sup>* cells in spleens of infected mice. Although we could not find any significant differences between the percentage of CD4*<sup>+</sup>* and CD8*<sup>+</sup>* cells during the infection, we did observe that they significantly increased in number after 15 dpi (Figure 2A and Supplementary Figure S2). We also analyzed the presence of splenic CD25*<sup>+</sup>* CD4*<sup>+</sup>* (Figure 2B and Supplementary Figure S2) or CD8<sup>+</sup> (Figure 2C and Supplementary Figure S2) T cells. Again, no significant differences were found in the percentage of these cells during the infection, while their number significantly increased after 15 dpi. Finally, we analyzed the presence of CD4*<sup>+</sup>* T cells in livers from infected animals and did not find any difference in their percentage nor their number (Figure 2D and Supplementary Figure S3). However, the number, but not the frequency, of hepatic CD25*<sup>+</sup>* CD4*<sup>+</sup>* T cells was increased in advanced infected mice (Figure 2E and Supplementary Figure S3). Further analyses demonstrated that the number of splenic CD25*<sup>+</sup>* CD4*<sup>+</sup>* and CD25*<sup>+</sup>* CD8*+*cells positively correlated with the number of peritoneal HO1*<sup>+</sup>* cells (Figure 2F).

**Figure 1.** HO-1 expression in F4/80+ peritoneal cells inversely correlates with ROS/RNS production. Mice (*n* = 4–6) were infected with 10 metacercariae and sacrificed at 1, 8, 15, and 21 dpi. Non-infected

mice were used as control (NI). (**A**) Clinical signs including hemorrhage, splenomegaly, and macroscopic liver damage were assessed to evaluate the severity of the disease [17]. ALT activity in sera was used as a marker of liver damage. (**B**) Analysis by flow cytometry of HO-1+ cells in PEC from infected and control (NI) mice. (**C**) Frequency and cell number of HO-1<sup>+</sup> cells in the peritoneal cavity of infected and control (NI) animals by flow cytometry. (**D**) Analysis by flow cytometry of F4/80<sup>+</sup> HO-1<sup>+</sup> cells in PEC from infected and control (NI) mice. (**E**) Frequency and cell number of F4/80<sup>+</sup> in the peritoneal cavity of infected and control (NI) animals by flow cytometry. (**F**) HO-1 expression in F4/80+ in the peritoneal cavity of infected and control (NI) mice. Letters on histograms correspond as follows: a: NI, b: 1 dpi, c: 15 dpi, d: 21 dpi. Median fluorescence intensity is shown (MeFI) in the plot. (**G**) ROS/RNS quantification in F4/80<sup>+</sup> in the peritoneal cavity of infected and control (NI) mice using the DCFDA probe by flow cytometry. Letters on histograms correspond as follows: a: NI, b: 1 dpi, c: 15 dpi, d: 21 dpi. Median fluorescence intensity is shown (MeFI) in the plot. (**H**) Murine RAW264.7 macrophages were cultured in the presence of 75 μg/mL of FhTE or CoPP (100 μM/mL) and SnPP (50 μM/mL) overnight at 37 ◦C. Then, cells were collected and incubated for 30 min at 37 ◦C in PBS with the DCFDA probe and analyzed by flow cytometry. The RNS/ROS levels are shown as the ratio between FhTE/medium (FhTE), CoPP + FhTE/CoPP (CoPP/FhTE), and SnPP + FhTE/SnPP (SnPP/FhTE). Representative experiments are shown. Asterisks indicate significant differences with *p* < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.
