*3.4. Immunomodulation*

Immunohistochemical analysis revealed a very distinct cytokine profile between uninfected (negative control) and infected animals (positive and experimental groups), as well as between untreated rats and those subjected to some types of therapy (Figure 3). In this regard, animals without *T. cruzi* infection exhibited the lowest levels of pro-inflammatory cytokines (IFN-γ and TNF-α), and the pro-oxidant marker, iNOS. On the other hand, as a result of infection, a significant increase of TNF-α was observed in infected and untreated rats with a concomitant reduction of the anti-inflammatory IL-10. With respect to treatments, LIMOX and BNZ stimulated a significant increase in IFN-γ immunoreactivity, compared to the negative control; with LIMOX as the only therapy able to reduce TNF-α levels in a significant manner (compared to positive control). Interestingly, the highest levels of the anti-inflammatory cytokine IL-4 were exhibited by the infected animals treated with LIMOX, at a significant difference with respect to the other groups (negative control, LIMOXBNZ, and BNZ). Correspondingly, this treatment was the only therapy able to reestablish the IL-10 levels to those found in uninfected animals. Finally, the iNOS analysis revealed a notable reduction of this oxidant marker (similar to the uninfected model) given


by the experimental treatments (LIMOX, LIMOXBNZ, and BNZ), in comparison with the infected and untreated animals (Figure 3).

(**a**)



**Figure 3.** Immunohistochemical analysis of cardiac tissue (**a**) Percentages of immunoreactivity obtained on cross sectional cuts of heart tissue by specific antibodies for Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), Interleukin (IL)-4, IL-10, and inducible Nitric Oxide Synthase

(iNOS); (**b**,**c**) Comparison of immunoreactivity percentages within groups. Negative: untreated and uninfected animals; Positive: untreated and infected animals; LIMOX: infected animals treated with an essential oil fraction of *L. alba* carvone chemotype enriched in limonene (68.9 mg/kg/day) with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX and benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). All *p* values were calculated by comparison within groups. \* *p* ≤ 0.05. Data are representative of six independent experiments and values are expressed in mean ± SEM.
