*3.1. The NO-Resistant 2853 L. braziliensis Strain Is More Infective to Macrophages In Vitro*

To verify the difference of resistance or susceptibility to NO of the two previously characterized *L. braziliensis* strains [17,20], the inhibitory concentration (IC50/4 h) of NaNO2 (NO donor) was evaluated in vitro using replicative promastigotes (logarithmic phase), collected at third day of growth (Figure S1). Confirming the previously reported phenotypes, 2853 strain was significantly more resistant to NO than 2856, exhibiting IC50/4 h of 27.6 ± 1.7 and 21.2 ± 0.9 mM, respectively (Figure 1A). The infection index of peritoneal macrophages, which corresponds to percentage of infected host cells × number of parasites per 100 cells, was significantly higher with the NO-resistant parasites (2853) than with the NO-susceptible (2856), even after the treatment of infected macrophages (24 h post-infection) with the 2 mM NaNO2. Additionally, the treatment with 2 mM L-NAME, a specific NOS inhibitor, led to a significant increase of the infection index observed in 2856-infected macrophages, in comparison to 2853-infected ones (Figure 1B).

Furthermore, analysis of peritoneal macrophages supernatants showed that both *L. braziliensis* strains triggered NO-mediated response in infected cells; however, the RNS levels were significantly higher after the infection with NO-resistant parasites, reaching an increase of 3.5-fold in comparison to NO-susceptible strain. Treatment with NaNO2 also potentiated the nitrosative stress started by parasite infection, with such increase being more pronounced in 2853-infected cells. Interestingly, treatment with the NOS

inhibitor decreased the production of RNS in both infections, abolishing its detection in the supernatant of 2856-infected cells but not in 2853-infected ones (Figure 1C).

**Figure 1.** In vitro evaluation of *L. braziliensis* NO resistance and RNS levels under nitrosative stress. (**A**) Parasites naturally resistant (2853 strain) or susceptible to NO (2856 strain) were exposed to NaNO2 (ranging 0.06–32 mM) during 4 h. Values correspond to the NaNO2 concentration that reduces 50% parasite viability at 4 h of exposure. Dot plots represent mean ± SD of four independent experiments. Statistical differences by t test (\*\* *p* = 0.002). (**B**) Peritoneal macrophages obtained from BALB/c mice were infected for 24 h with promastigotes of each strain. After that, cells were treated with 2 mM NaNO2 or 2 mM L-NAME for 48 h, completing 72 h of infection. Controls correspond to untreated cells infected for 72 h; infection index = percentage of infected host cells × number of parasites per 100 cells. (**C**) Macrophage supernatants were collected, and RNS levels were analyzed by Griess Reagent according to manufacturer's instructions. Graphs represent mean ± SD of at least three independent experiments. Significance of differences between strains were determined by t test using the Holm–Sidak method for multiple comparisons (\* *p* < 0.05; \*\*\*\* *p* < 0.0001). Significance of differences between treatments in comparison to control were determined by two-way ANOVA followed by Dunnett's multiple comparisons test (# *p* ≤ 0.01); ND = no detected.
