PMC

(A) Gating strategy for the analysis of neutrophil phenotypes. Neutrophils were purified from healthy donors and infected with GFP-expressing L. amazonensis (5 parasites: 1 neutrophil) in RPMI 1640 plus 10% FBS. (B) 3h after infection, neutrophils were characterized by flow cytometry and data analyzed by FlowJo software (in duplicate, n = 5 experiments). Green and black bars represent, respectively, the GFP positive and negative cells. The white bars represent non-exposed group (unstimulated). The mean ± SD of parental percentage of GFP+, GFP- and non-exposed cells were compared by Friedman paired test with Dunn’s post test.

(A) Sera of leprosy patients with various clinical presentations were collected and sCD163 concentrations measured by ELISA. Household contacts without symptoms or signs of leprosy (Contacts) were used as a control group. The mean ± SD sCD163 levels in patients with indeterminate leprosy (IL) (n = 9; 114 ± 49,57 ng/mL), true tuberculoid leprosy (TT) (n = 14; 90,29 ± 44,06 ng/mL), borderline leprosy (BL) (n = 14; 97,71 ± 47,97 ng/mL) and lepromatous leprosy (LL) (n = 10; 177,6 ± 62,18 ng/mL), as well as contacts (n = 23; 90,78 ± 31,55 ng/mL) were compared by Mann-Whitney test. ROC curves comparing sCD163 concentrations from TT versus LL (B) and Contacts versus LL patients (C) were constructed and are shown.

(A) Gating strategy for analysis of the cytokine profiles of CD163+ and CD163- monocyte/macrophages. PBMC were collected from healthy donors and the adherent cells cultured for 5 days in RPMI 1640 plus 20% FBS. The cells were incubated with L. amazonensis strain (10 parasites: 1 macrophage) and L. infantum-isolate 1 (5:1), and incubated with antibodies specific for intracellular cytokines, prior to analysis by flow cytometry (n = 6 experiments, in duplicate). (B) Frequency of IL-4+ cells, (C) MFI of IL-4-PerCPCy5.5, (D) iMFI of IL-4 analysis, (E) Frequency of IL-10+ cells, (F) MFI of IL-10-APC, (G) iMFI of IL-10 analysis, (H) Frequency of IL-12+ cells, (I) MFI of IL-12-APC, (J) iMFI of IL-12 analysis, (K) Frequency of TNF-α+ cells, (L) MFI of TNF-α-PerCPCy5.5, (M) iMFI of TNF-α analysis.

(A) sCD163 levels were measured in sera of VL patients of different clinical status. The mean ± SD sCD163 levels of patients with classical VL at D0 (D0-Classic, n = 33) (152,1 ± 67,86 ng/mL), D30 (n = 19) (98,79 ± 58,58 ng/mL) and of patients of severe VL at D0 (D0-SVL) (n = 13) (241,5 ± 76,88 ng/mL) were compared by Mann-Whitney test. Sera from Leishmania-infected individuals without symptoms or signs of VL (DTH+, n = 11, 72,55 ± 25,68 ng/mL) and healthy individuals from non-endemic regions (HC, n = 8, 49,0 ± 23,71 ng/mL) were included as control groups. Spearman correlation analyses between sCD163 concentrations were performed versus (B) spleen size, (C) liver size and (D) neutrophil count. (E) Paired analysis of sCD163 levels of VL patients before and after treatment (n = 15, p = 0.0455, paired t test). ROC curves of sCD163 concentration comparing HC versus (F) D0-Classic, (G) D0-Classic versus D30 and (H) D0 versus D0-SVL group.

(A) Gating strategy for analysis of monocyte/macrophage phenotypes. PBMC were collected from healthy donors and the adherent cells cultured for 5 days in RPMI 1640 plus 20% FBS. The cells were infected with L. amazonensis-GFP (10 parasites: 1 macrophage), two isolates of L. infantum CellTracker stained (5:1) or BCG CellTracker stained (2:1), and incubated with antibodies specific for macrophage surface molecules prior to analysis by flow cytometry. (B) Surface CD163 expression was quantified after 24h in infected, GFP+ cells (in duplicate, n = 5 experiments). (C) The macrophages were analyzed by FlowJo software (in duplicate, n = 6 experiments). Green and black bars represent, respectively, the infected, GFP+ and uninfected, GFP- cells after 24h of exposure to L. amazonensis GFP-expressing strain. White bars are the non-exposed group (unstimulated). The mean ± SD parental percentage of the cell populations (B and C) were compared by Friedman paired test with Dunn’s post test. (C) MFI analysis of GFP in the different populations according to the surface phenotype, 24h after infection (n = 7 experiments in duplicate). The mean ± SD MFI of each population was compared by Friedman paired test with Dunn’s post test. (D) Spearman correlation analysis between CD163-PE and Leishmania-GFP fluorescence for each cell of the analysis (one representative of 7 experiments in duplicate).