*2.5. Flow Cytometry*

For flow cytometry analysis, spleens from infected mice and controls were aseptically removed 72 h post-infection, homogenized in Hanks' Balanced Salt Solution (Sigma Aldrich, Roswell-Park, St. Louis, MO, USA) and, when necessary, red blood cells were lysed. The following monoclonal antibodies (mAb) were used (at previously determined optimal dilutions) for surface antigen staining after pre-incubation with anti-mouse CD16/CD32 for Fc γR blocking. For dead cell exclusion, all samples except single-stained controls were first incubated with allophycocyanin (APC) eFluor 780 Fixable Viability Dye (eBioscience, San Diego, CA, USA) diluted 1:1000 in PBS for 30 min at 4 ◦C. For surface staining, cells were incubated with the following monoclonal antibodies: anti-mouse GR1 Fluorescein isothiocyanate (FITC)-conjugate, anti-mouse CD80 Phycoerythrin (PE)-conjugate, anti-mouse F4/80 Peridinin-chlorophyll protein Cyanin 5.5 (PerCp Cy5.5)-conjugate, anti-mouse CD86 PE-cychrome 7 (PE-Cy7)-conjugate, anti-mouse CD11c BV421-conjugate (all from BD Biosciences, San Jose, CA, USA), anti-mouse CD11b BV510-conjugate, and anti-mouse major histocompatibility complex (MHC) class II APC conjugate (eBiosciences, San Diego, CA, USA). Data acquisition was performed in a FACSCantoTM II system (BD Biosciences, San Jose, CA, USA) using the FACSDIVATM software (BD) and compensated and analyzed in FLOWJO version 9.7.5. (Tree Star Inc., Ashland, OR, USA). A biexponential transformation was applied to improve data visualization; 10<sup>6</sup> cells were stained per sample.

#### *2.6. Histopathologic Examination and Immunohistochemistry*

Livers were fixed in buffered formalin and embedded in paraffin for hematoxylin-eosin (HE) and periodic acid–Schiff (PAS) histopathologic analysis, as previously described [51,52].
