*3.5. Deficiency of Functional NADPH Oxidase Partially Protects Mice from Liver Damage Induced by F. hepatica and Limits the Production of IL-10*

Considering the fact that SnPP treatment protected mice from parasite infection and that reduced levels of *gp91phox* mRNA were found in PECs from infected mice, we analyzed the infection in gp91phox knockout mice. gp91phox deficiency reduced the clinical signs and liver damage induced by *F. hepatica* infection (Figure 6A and Supplementary Figure S5). This partial protection was associated with a lower increase of HO-1*<sup>+</sup>* peritoneal cells, both in frequency and number (Figure 6B). Moreover, HO-1<sup>+</sup> cells from gp91phox knockout infected mice expressed lower levels of MHCII (Figure 6C) and CD40 (Figure 6D), but not CD80 (Figure 6E), than those from wildtype mice, indicating that NADPH oxidase may play a role both in the immune response and the pathogenesis induced by *F. hepatica* infection. Further characterization of the peritoneal cells from these mice indicated that the increase of F4/80*<sup>+</sup>* cells was abrogated in the absence of gp91phox (Figure 7A), and as expected, very low levels of ROS/RNS produced by these cells (Figure 7B). Additionally, these cells expressed lower levels of Sirpα (Figure 7C), ICOSL (Figure 7D), and IL-10 (Figure 7E). Finally, lower numbers of CD4*<sup>+</sup>* (Figure 7F and Supplementary Figure S6) and CD4*+*/CD25*+*FoxP3*<sup>+</sup>* Tregs (Figure 7G and Supplementary Figure S6) were found in gp91phox knockout infected mice with respect to wildtype mice. However, no significant differences in the production of IFNγ by splenocytes stimulated with parasite components (FhTE) between gp91phox knockout and wildtype infected mice were detected (Figure 7H).

**Figure 5.** mRNA levels of antioxidative and oxidative genes in the infection by *F. hepatica*. Mice (*n* = 5/group) were injected with SnPP (40 mg/kg) or PBS 1 day before infection, infected with 10 metacercariae (day 0), and sacrificed at 20 dpi. *nrf2* (**A**), *catalase, gpx1, gpx2, sod1, sod2* (**B**), and *p47phox* and *gp91phox* (**C**) gene expression in PECs from SnPP-treated and control infected mice evaluated by qRT-PCR. mRNA levels were analyzed by qRT-PCR with respect to *gapdh* expression in PECs from SnPP-treated and control infected mice (PBS). Results were compared to the infected (control) group of mice and represented as the ratio between gene expression in SnPP-treated and control mice. Asterisks indicate significant differences with *p* < 0.05, performed by the Student's *t*-test.

#### *3.6. IL-10 Signaling Is Crucial for HO-1 Expression in F. hepatica-Infected Mice*

Considering the fact that IL-10 induces HO-1 expression that can favor the production of IL-10, and that IL-10 is crucial for Treg differentiation [36], we analyzed whether there was a relationship between IL-10 signaling and HO-1 expression during *F. hepatica* infection. To this end, we treated infected mice with a neutralizing antibody of IL-10 receptor (IL-10R). The results demonstrate that IL-10R blocking reduced the clinical signs associated with parasite infection (Figure 8A). Although the recruitment of F4/80*<sup>+</sup>* cells in the peritoneum of infected mice was not affected by IL-10R neutralization (Figure 8B), it abrogated the elevated expression of HO-1 induced by *F. hepatica* infection (Figure 8C). Interestingly, IL-10R neutralization reduced the frequency, but not the number, of CD4*<sup>+</sup>* (Figure 8D and Supplementary Figure S7) and CD4*+*CD25*<sup>+</sup>* (Figure 8E and Supplementary Figure S7) T cells in the spleens of infected animals. Altogether, these results indicate that IL-10 signaling is essential for HO-1 expression of F4/80*<sup>+</sup>* cells during *F. hepatica* infection, likely affecting the differentiation of regulatory T cells.

**Figure 6.** Gp91phox (gp91p-) knockout (*n* = 6–8/group) and littermate control (*n* = 2/group) mice were infected with 10 metacercariae and sacrificed at 20 dpi. Non-infected mice were used as a control. (**A**) Clinical signs analyzed to assess disease severity [17]. Quantification of liver damage by ALT activity in sera. (**B**) Flow cytometry analysis of HO-1<sup>+</sup> in PECs from gp91phox knockout mice and littermate controls (upper panel). (**C**) Frequency and cell number of HO-1+ cells in the peritoneal cavity of infected and non-infected animals by flow cytometry (lower panel). MHCII (**C**), CD40 (**D**), and CD80 (**E**) expression in HO-1<sup>+</sup> cells in the peritoneal cavity of infected and control mice. Letters on histograms correspond as follows: a: infected gp91phox, b: infected wildtype littermates. Median fluorescence intensity is shown (MeFI) in the plot. Asterisks indicate significant differences with *p* < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.

**Figure 7.** Peritoneal F4/80+ cells from wildtype infected mice present a regulatory-like phenotype. Gp91phox (gp91p-) knockout

(*n* = 7–8/group) and littermate control (*n* = 2–4/group) mice were infected with 10 metacercariae and sacrificed at 20 dpi. Non-infected mice were used as a control. (**A**) Flow cytometry analysis of F4/80+ cells in PEC from gp91phox knockout mice and littermate controls (upper panel). Frequency and cell number of F4/80<sup>+</sup> cells in the peritoneal cavity of infected and non-infected animals by flow cytometry (lower plots). ROS/RNS (**B**), Sirpα (**C**), ICOSL (**D**), and IL-10 (**E**) expression in F4/80+ cells in the peritoneal cavity of infected and uninfected mice. Letters on histograms correspond as follows: a: infected gp91phox, b: infected littermates, c: uninfected gp91phox, and d: uninfected littermates. Median fluorescence intensity is shown (MeFI) in the plot. (**F**) Frequency and cell number of splenic CD4+ cells from infected and non-infected mice. (**G**) Frequency and cell number of splenic CD4+/CD25+ Foxp3+ cells from infected and non-infected mice. Gate analyses by flow cytometry are shown in (**A**) and Supplementary Figure S1. (**H**) IFNγ levels in culture supernatants of splenocyte proliferation assay cultured with FhTE for 5 days at 37 ◦C. 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 8.** IL-10 signaling is essential for HO-1 expression in *F. hepatica*-infected mice. Fifteen μg of monoclonal rat IgG2a anti-IL-10R antibody was administrated by intraperitoneal injection the day before and after infection with *F. hepatica* and every 3 days until sacrifice (*n* = 5–8/group). The control group (*n* = 4/group) received an isotype control antibody. At day 20 post-infection, animals were sacrificed and splenocytes were analyzed by flow cytometry. (**A**) Clinical signs were analyzed to assess disease severity [17]. (**B**) Analysis by flow cytometry of F4/80+ cells in PEC from infected and non-infected (NI) mice showing frequency and number of F4/80+ cells in PECs. (**C**) HO-1 expression in F4/80+ cells in the peritoneal cavity of infected and uninfected mice. (**D**) Frequency and cell number of CD4+ T cells in spleens from infected and non-infected (NI) mice. (**E**) Frequency and cell number of CD4+ CD25+ T cells in spleens from infected and non-infected (NI) mice. Gate analyses by flow cytometry are shown in (**B**) and Supplementary Figure S7. Representative results of one representative are shown. Asterisks indicate significant differences with *p* < 0.05, performed by one-way ANOVA followed by Tukey's test with multiple comparisons.
