*2.2. Parasite Protein Extract (FhTE)*

FhTE was prepared from live adult flukes obtained from infected bovines. Flukes were washed for 1 h at 37 ◦C with phosphate buffered saline (PBS), pH 7.4, sonicated, and then centrifugated at 40,000× *g* for 60 min [4,30]. Finally, protein lysates were dialyzed against PBS. FhTE protein concentration was measured using the bicinchoninic acid assay (Sigma, St. Louis, MO, USA). In order to remove endotoxin contamination, FhTE was applied to a column containing endotoxin-removing gel (detoxi-gel, Pierce Biotechnology, Waltham, MA, USA), and endotoxin levels were quantified using the Limulus Amebocyte Lysate kit Pyrochrome (Associates of Cape Cod, East Falmouth, MA, USA) and found to be lower than 0.05 EU/mL. Furthermore, at the used concentrations, FhTE did not induce the production of pro-inflammatory cytokines such as IL-12 or IL-6 [4,30]. The concentration of *F. hepatica* extracts used in the in vitro experiments did not modify cell viability, as evaluated by the MTT (2-[4,5-dimethyl-2-thiazolyl]-3,5-diphenyl-2H-tetrazolium bromide) assay [4,17,30].

#### *2.3. F. hepatica Infections*

#### 2.3.1. Parasite Infections, Animal Treatments, and Sample Obtention

The infection was achieved by orally administrating 10 *F. hepatica* metacercariae (Montevideo, Uruguay) per mouse. Mice were bled and peritoneal exudate cells (PECs), spleens, and livers were removed after 1, 8, 15, and 21 days post-infection (dpi), while non-infected animals were used as controls. Each experimental group contained at least four mice. PECs and hepatic leukocytes were obtained as already described [31]. Red cells were lysed with ammonium chloride potassium buffer.

HO-1 activity was inhibited using SnPP (40 mg/kg), and vehicle (PBS, 200 μL) was used as a control. The SnPP dose was within a range of doses used in previous works [32,33]. Mice received intraperitoneal injections of SnPP 1 day before infection, 1 day after infection, and every 4 days until the end of the experimental protocol (between 19 and 21 dpi). When gp91phox- and non-infected littermates were used (*n* = 6–8/group), infections were performed in the same conditions as previously described. In order to neutralize IL-10 receptor (IL-10R), BALB/c mice (*n* = 6–8/group) were intraperitoneally injected with 15 μg of monoclonal rat IgG2a anti-IL-10R (clone 1B1.3A from BioXcell, Lebanon, NH, USA) or an isotype-matched control antibody (clone HRPN from BioXcell, Lebanon, NH, USA), the day before and after infection with *F. hepatica* and every 3 days until animal sacrifice at 20 dpi. Blood samples were obtained, and PECs, spleens, and livers were removed. The infection severity was assessed with a defined clinical score according to the following parameters: presence or absence of peritoneal hemorrhage, presence of macroscopic liver damage and splenomegaly, and the amount of cell content in the peritoneal cavity [17], where the minimum score was 0 and the maximum was 10. The alanine aminotransferase

(ALT) activity in sera was used to quantify liver damage, determined with a commercial kit (Spinreact, Girona, Spain) according to the manufacturer's instructions.

#### 2.3.2. Proliferation Assay and Cell Culture

Splenocytes (0.5 × <sup>10</sup>6/well) from infected mice or uninfected naïve mice (control group) were cultured for 5 days at 37 ◦C and 5% CO2, in RPMI-1640 with 400 μg/mL of glutamine (Capricorn, Ebsdorfergrund, Germany) complete medium containing 10% heat-inactivated fetal bovine serum (FBS, Capricorn Scientific, Ebsdorfergrund, Germany), 50 mM of 2-mercaptoethanol, 100 U/mL of penicillin, and 0.1 mg/mL of streptomycin (Merk, Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of FhTE (75 μg/mL), as previously described [30]. An IFNγ-specific sandwich ELISA assay (Biolegend, San Diego, CA, USA) was used to quantify IFNγ levels in culture supernatants.

RAW264.7 macrophages were cultured at 0.5 × <sup>10</sup>6/mL in complete RPMI medium in the presence or absence of the HO-1 inductor (CoPP) and inhibitor (SnPP) (50 and 100 μM, respectively) or FhTE (75 μg/mL) overnight at 37 ◦C. Afterwards, the ROS/RNS production was determined as described in the following section.
