Fig 1.
The macrophage cells, challenged with arsenic unexposed (Ld) and exposed (LdAS) parasites of same strain, showed an infection pattern of 1.78x102±50 and 5.43x102±91 amastigotes/100 cells (100x), (Pic 1 and Pic 2) respectively.
The percentage of their infectivity is plotted in bar diagram (***P≤0.001) (A), and their survivability against different concentration of SAG compared to negative (Ld with drug) and positive (LdAS without drug) controls (B) is also calculated. The data are expressed as mean ± SEM (n = 5) of independent experiment in triplicate and significance was determined by paired t test, one-way ANOVA with Tukey’s post hoc multiple comparison tests.
Fig 2.
The arsenic exposure to Leishmania parasite enhanced the production of reduced thiol and down-regulated the mitochondrial membrane potential (Δψm) as the concentration of reduced thiol was enhanced in LdAS (1.3-fold in 1 mg/L and 1.7-fold in 1.5 mg/L) compared to Ld, was directly proportional to degree of arsenic exposure (A). The result of Δψm was evaluated using JC-1 dye, that showed 1.8-fold and 2.2-fold depolarization of Δψm in LdAS (1 and 1.5 mg/L) respectively compared to Ld. The magnitude of depolarization was also directly proportional to quantity of arsenic exposure to Leishmania parasite (B). The lactate production and glucose consumption were also increased in LdAS compared to that of Ld in a dose-dependent fashion. The level of intracellular lactate in parasite (LdAS & Ld) was estimated by parasites harvesting, homogenizing in assay buffer and analysing by spectrophotometric technique. LdAS (1 and1.5 mg/L) produced significantly 1.87-fold and 2.28-fold more lactate compare to that of Ld (C). The level of glucose consumption was estimated in LdAS and Ld, by cultivating parasites in glucose free M199 medium, following centrifugation and evaluating in supernatant by spectrophotometric technique. The glucose levels have been observed 1.79-fold and 2.5-fold less in LdAS (1 and 1.5 mg/L) respectively than Ld. The results are represented through bars (D) and the data are represented as mean ± SEM (n = 5), the significance of experiments in triplicate, was determined by one-way ANOVA with Tukey’s post hoc multiple comparison tests (*P≤0.05; **P≤0.005; ***P≤0.001).
Fig 3.
The LdAS dampened the production of reactive oxygen intermediates compared to that of Ld.
The NO production was determined in peritoneal macrophages stimulated with Ld and LdAS followed by incubation in CO2 incubator for 48 hrs, thereafter supernatant was collected, NO was measured using griess reagents (p value <0.0056) (A). The ROS was measured in peritoneal macrophages stimulated with Ld and LdAS by method as described in “Materials and methods”. The results are shown in a bar diagram (p value <0.0079) (B), where data are expressed as mean ± SEM (n = 5), and significance of experiment in triplicate was determined by one-way ANOVA with Tukey’s post hoc multiple comparison tests (*P≤0.05; **P≤0.005; ***P≤0.001).
Fig 4.
The splenocytes stimulated with LdAS significantly up-regulated intracellular anti-inflammatory and down-regulated pro-inflammatory cytokines.
The anti-inflammatory cytokines IL-4 (p value <0.0033) (A) & IL-10 (p value <0.0034) (B) and pro-inflammatory cytokines IL-2 (p value <0.0049) (C) and IFN-γ (p value <0.0004) (D) were determined by flow cytometry. The data are represented as mean ± SEM (n = 5). All experiments were performed in triplicate and significance was determined by one-way ANOVA with Tukey’s post hoc multiple comparison tests (*P≤0.05; **P≤0.005; ***P≤0.001).
Fig 5.
The extra-cellular cytokines, produced by splenocytes stimulated with LdAS and Ld were also measured by ELISA and results showed, up-regulation of anti-inflammatory cytokine IL-10 (p value <0.0013) (A) and down-regulation of pro-inflammatory cytokines IFN-γ (p value <0.0039) (B) and IL-12 (p value <0.0015) (C) produced by splenocytes stimulated with LdAS compared to Ld.
The data are shown in scatter plot, as mean ± SEM, experiments were performed in triplicate and significance was determined by ANOVA with Tukey’s post hoc multiple comparison tests (*P≤0.05; **P≤0.005; ***P≤0.001).
Fig 6.
The LdAS stimulated macrophages (CD14+ cells) significantly down-regulated the intracellular pro-inflammatory cytokines.
For this, the splenocytes derived macrophages, stimulated with LdAS & Ld, stained with surface FITC labelled CD14+ and intracellular cytokines stained with individually PE labelled IL-12 & TNF-α antibodies were analysed by flow cytometry as described in “Material and methods”. The data of experiment in triplicate represent mean ± SEM (n = 5) and significance was determined by one-way ANOVA with Tukey’s post hoc multiple comparison tests (*P≤0.05; **P≤0.005; ***P≤0.001).