*3.2. NO-Susceptible Parasites Trigger a More Intense ROS-Dependent Response in Peritoneal Macrophages*

Since nitrosative and oxidative stresses work together for host cell's microbicidal mechanisms [13], we evaluate whether resistance to NO in *L. braziliensis* strains can influence the infective capacity after oxidative burst. To test this, infected peritoneal macrophages were treated with 150 μM H2O2 and several antioxidants for 48 h. Treatment with the prooxidant molecule significantly decreased the infection index observed in NO-susceptible group, amplifying the differences found between the infections caused by 2853 and 2856. On the other hand, the antioxidants presence increased the infection by NO-susceptible parasites, leading 2856 strain to reach infection index higher than those observed with the NO-resistant parasites (Figure 2A). Treatment with H2O2 also decreased by 52.2% the infection caused by the NO-susceptible parasites in comparison to control group, while

antioxidants tested increased up to 2.5-fold the infection index of 2856-infected cells. In contrast, neither H2O2 nor most of the antioxidant molecules altered the infection index by NO-resistant strain; only the incubation with SOD decreased by 48% the 2853-infection index in relation to control (Figure 2A).

**Figure 2.** Effect of ROS and RNS levels in peritoneal macrophages infected in vitro with *L. braziliensis* strains resistant and susceptible to NO. Peritoneal macrophages obtained from BALB/c mice were infected with promastigotes for 24 h. After that, cells were treated with 150 μM H2O2, 30 U/mL SOD, 40 U/mL catalase, or 1 μM mitoTEMPO for 48 h, completing 72 h of infection. Controls correspond to untreated cells infected for 72 h. (**A**) Infection index, which corresponds to percentage of infected host cells × number of parasites per 100 cells. (**B**) Host cells were incubated with 100 μM Amplex Red and 50 U/mL HRP for 30 min in respiration buffer and analyzed for detection of ROS levels. (**C**) Macrophage supernatants were collected, and the production of RNS was analyzed by Griess Reagent, according to manufacturer's instructions. Graphs represent mean ± SD of at least four independent experiments. Significance of differences between strains were determined by *t* test using the Holm-Sidak method for multiple comparisons (\* *p* ≤ 0.01). Significance of differences between treatments in comparison to control was determined by two-way ANOVA followed by Dunnett's multiple comparisons test (# *p* ≤ 0.05); ND = no detected.

Besides RNS, both *L. braziliensis* strains also trigger ROS-mediated responses in peritoneal macrophages; however, ROS levels were 1.7-fold higher in 2853-infected cells than in 2856-infected ones (Figure 2B). As expected, the treatment with H2O2 increased ROS levels in supernatants of peritoneal macrophages (up to 2-fold), and antioxidant molecules significantly decreased the oxidative burst in all analyzed conditions. Nevertheless, even in the presence of antioxidants, ROS levels were significantly higher (~3-fold) in macrophages infected with the NO-resistant strain than in those infected with the NO-susceptible. Interestingly, the treatment with catalase was able to abolish completely the H2O2 detection in both infection conditions (Figure 2B).

To test the existence of a relation between the oxidative and nitrosative metabolism during *L. braziliensis* infection, supernatants of those peritoneal macrophages were also evaluated regarding to RNS levels. Remarkably, in all conditions, after treatment with H2O2 or antioxidants, macrophages infected with the NO-resistant strain produce among ~3.3-fold to ~9-fold higher levels of RNS than those infected with the NO-susceptible strain (Figure 2C). Treatment with H2O2 significantly increased the production of RNS (up to 1.9-fold) in cells infected by both strains, in comparison to infected control group, while antioxidants modulated RNS production only in 2856-infected macrophages, decreasing nitrosative levels. Interestingly, macrophages infected with the 2853 did not suffer such modulation, maintaining high levels of RNS even in the presence of antioxidant molecules (Figure 2C).
