*3.3. HO-1 Activity Decreases the Production of ROS/RNS by F4/80+ Cells and Correlates with an Increase of Splenic Regulatory CD4+ T Cells Induced by F. hepatica Infection*

In order to evaluate whether HO-1 interferes with the production of ROS/RNS, we treated *F. hepatica*-infected mice with the HO-1 inhibitor SnPP. SnPP treatment was associated with a decrease in the clinical signs of infected mice (Figure 3A). In addition, SnPP treatment of infected mice abrogated the increase of HO-1*<sup>+</sup>* cells, both in frequency and number, induced by the infection, since no significant difference was found in infected mice with respect to the control group with SnPP treatment (Figure 3B and Supplementary Figure S4). Surprisingly, a significant increase in F4/80*<sup>+</sup>* cell number, but not frequency, was found in both SnPP-treated and non-treated infected mice (Figure 3C and Supplementary Figure S4). Indeed, the F4/80+ cell number was higher in SnPP-treated infected mice. Nevertheless, F4/80*<sup>+</sup>* cells of SnPP-treated infected mice produced higher levels of ROS/RNS than control infected mice (Figure 3D), although they expressed similar levels of HO-1 (Figure 3E). Of note, the expression of ICOSL in peritoneal F4/80*<sup>+</sup>* cells of infected mice was significantly reduced with SnPP treatment (Figure 3F). Lastly, SnPP treatment during *F. hepatica* infection did not induce an increase in the number of splenic CD4+ T cells (Figure 3G) or CD4*+*/CD25*<sup>+</sup>* T cells (Figure 3H), although these cells expressed higher levels of CTLA4 in the absence of SnPP treatment (Figure 3H). Altogether, these results suggest that HO-1 activity inhibited by SnPP decreases the production of ROS/RNS during fasciolosis and correlates with an increase of splenic regulatory CD4*<sup>+</sup>* T cells in a process that might involve ICOSL in antigen-presenting cells or CTLA4 expression in Tregs.

To complement these results, we evaluated whether the inhibition of HO-1 by SnPP treatment affected the recruitment or the phenotypical characteristics of peritoneal F4/80*<sup>+</sup>* cells at the early stages of *F. hepatica* infection. To this end, we analyzed F4/80*<sup>+</sup>* cells in the peritoneal cavity of SnPP-treated mice at 1 dpi and compared them with both non-treated infected and control mice. We observed the presence of two different cell populations according to F4/80 expression (Figure 4A and Supplementary Figure S1). SnPP treatment increased both the frequency and number of F4/80int cells, while it reduced F4/80hi cells in the peritoneal cavity of infected mice at 1 dpi (Figure 4B). Nevertheless, F4/80int cells expressed similar levels of HO-1 (Figure 4C) and ROS/RNS (Figure 4D) in F4/80int cells regardless of SnPP treatment. Interestingly, peritoneal F4/80int cells expressed higher levels of CCR2 (Figure 4E), while only those from SnPP-treated infected mice expressed significantly increased levels of IL-33R (Figure 4F), which could be related to the initiation of an early immune response against the parasite. Thus, these results suggest that the presence of F4/80int IL-33R+ cells in the peritoneum is induced by SnPP treatment, which in turn protects mice from infection.

**Figure 2.** Peritoneal HO-1+ cells correlate with increased splenic CD4<sup>+</sup> CD25+ and CD8+ CD25<sup>+</sup> cells during *F. hepatica* infection. Mice (*n* = 4–8/group) were infected with 10 metacercariae and sacrificed at 1, 8, 15, and 21 dpi. Non-infected mice were used as control (NI). (**A**) Frequency and cell number of splenic CD4<sup>+</sup> or CD8<sup>+</sup> cells from infected and control (NI) mice. (**B**,**C**) Frequency and cell number of splenic CD4<sup>+</sup> CD25<sup>+</sup> (**B**) or CD8+ CD25<sup>+</sup> (**C**) cells from infected and control (NI) mice. (**D**) Frequency and cell number of hepatic CD3<sup>+</sup> CD4+ cells from infected and control (NI) mice. (**E**) Frequency and cell number of hepatic CD4<sup>+</sup> CD25<sup>+</sup> cells from infected and control (NI) mice (right). (**F**) Splenic CD4+ CD25+ or CD8+ CD25+ cell number in function of the number of peritoneal HO-1<sup>+</sup> cells in NI and infected mice. The mean of CD4+ CD25<sup>+</sup> (in Figure 2B), CD8 CD25+ (in Figure 2C), and HO-1<sup>+</sup> cells (in Figure 1C) was plotted. Gate analyses by flow cytometry are shown in Supplementary Figures S2 and S3. The results shown represent one experiment. Asterisks indicate significant differences with *p* < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

**Figure 3.** The HO-1 pharmacological inhibitor SnPP decreases the production of ROS/RNS by peritoneal F4/80<sup>+</sup> cells and correlates with an increase of splenic FoxP3+CD25+/CD4+ T cells induced by *F. hepatica* infection. The HO-1 pharmacological

inhibitor SnPP decreases the production of ROS/RNS by peritoneal F4/80+ cells and correlates with an increase of splenic FoxP3+CD25+/CD4+ T cells induced by *F. hepatica* infection. Mice were injected with SnPP (40 mg/kg) or vehicle (PBS) one day before infection and every 4 days until the end of the experimental protocol. Mice (*n* = 5/group) were infected with 10 metacercariae (day 0) and sacrificed at 20 dpi. Non-infected (NI) mice (*n* = 3/group) both treated and untreated with SnPP were used as control. (**A**) Clinical signs of infected mice were analyzed to assess disease severity [17]. (**B**) Frequency and cell number of HO-1+ cells in the peritoneal cavity of SnPP-treated or untreated infected and control (NI) animals by flow cytometry. (**C**) Frequency and cell number of F4/80+ cells in the peritoneal cavity of SnPP-treated or untreated infected and control (NI) animals by flow cytometry. (**D**) ROS/RNS quantification in F4/80+ cells of the peritoneal cavity of SnPP-treated or untreated infected and control (NI) mice using the DCFDA probe by flow cytometry. HO-1 (**E**) and ICOSL (**F**) expression in F4/80+ cells in the peritoneal cavity of SnPP-treated or untreated infected and control (NI) mice. (**G**) Splenic CD4+ T cell frequency and number from SnPP-treated or untreated infected and control (NI) mice. (**H**) Splenic CD4+/CD25+FoxpP3+ cell frequency and number from SnPP-treated or untreated infected and control (NI) mice (left). CTLA4 expression in splenic CD4+/CD25+FoxpP3+ cells from SnPP-treated or untreated infected and control (NI) mice. Gate analyses by flow cytometry are shown in Supplementary Figures S2 and S4. Results obtained for one representative experiment are shown. Asterisks indicate significant differences with *p* < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

**Figure 4.** Analyses of peritoneal F4/80+ cells at the early events of *F. hepatica* infection. Mice (*n* = 5/group) were injected with

SnPP (40 mg/kg) or vehicle (PBS) one day before infection, infected with 10 metacercariae (day 0), and sacrificed at 1 dpi. Non-infected (NI) mice (*n* = 3/group) both treated and untreated with SnPP were used as a control. (**A**) Analysis of F4/80int and F4/80hi cells in PEC from SnPP-treated or untreated infected and control (NI) mice by flow cytometry. (**B**) Frequency and cell number of F4/80int (upper plots) or F4/80hi (lower plots) cells in the peritoneal cavity of SnPP-treated or untreated infected and control (NI) animals. HO-1 (**C**), ROS/RNS (**D**), CCR2 (**E**), and IL-33R (**F**) expression in peritoneal F4/80int and F4/80hi cells by flow cytometry. "nd" means none detected, since barely any F4/80hi cells in SnPP-treated infected mice were detected. Median fluorescence intensity is shown (MeFI) in the plot. Gate analyses by flow cytometry are shown in (**A**) and Supplementary Figure S1. 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.
