PMC

Amastigote-induced cytokine production. a-c Thioglycollate-induced neutrophils were cocultured with amastigotes of L. amazonensis (La) or L. braziliensis (Lb) at a MOI of 5 for 24 h. The levels of IL-22, TNF-α and IL-18 in culture supernatants were assessed by multiplex. d Neutrophils were cocultured with amastigotes for 4 h followed by flow cytometric detection of IL-22 in Ly6G+ CD11b+ cells. * p < 0.05, ** p < 0.01, between indicated groups. Med = Control neutrophils; n.s. = not significant.

Neutrophil degranulation in response to Leishmania amastigotes. a Validation of a FACS-based degranulation assay. Thioglycollate-induced neutrophils were treated with cytochalasin B (CytoB, 5 mg/ml) for 10 min and then with fMLP (10 mM) for 4 h, resulting in a decrease in intracellular MPO staining. The MFI of MPO in treated samples was then compared to that of the controls (Med). b Neutrophil degranulation in response to amastigotes (MOI of 5) and/or LPS. *** p < 0.001, between indicated groups.

Amastigote-induced neutrophil activation and oxidative burst. Thioglycollate-induced neutrophils were cocultured with unlabeled (a, d, e) or CFSE-labeled amastigotes (b, c) at a MOI of 5 for 4 h, followed by flow cytometric analysis. a MFI of surface CD11b on control neutrophils (Med) and neutrophils exposed to unlabeled amastigotes of L. amazonensis (La) or L. braziliensis (Lb). b, c Imaging flow cytometry data, showing CD11b expression on control neutrophils (Med), neutrophils carrying no parasites (0), 1 parasite or ≥3 parasites, respectively. d Oxidative burst in total Ly6G+ neutrophils in medium alone or in response to amastigotes. e Oxidative burst in Ly6G+ Rho 123+ neutrophils, demonstrating that the intensity of oxidative burst was significantly greater in L. braziliensis-infected cells than in L. amazonensis-infected cells. * p < 0.05, ** p < 0.01, *** p < 0.001, between indicated groups.

Neutrophil uptake of L. amazonensis and L. braziliensis amastigotes. Thioglycollate-induced peritoneal neutrophils were cocultured with CFSE-labeled amastigotes (MOI of 5) for 4 h. Cells were then analyzed by both FACS (a) and imaging flow cytometry (b, c) to assess parasite uptake. a A histogram shows CD11b+ Ly6G+ neutrophil uptake of CFSE-labeled parasites. b Representative events from imaging flow cytometric analysis showing (left-right) an uninfected neutrophil (red) in close contact with an amastigote (green) and neutrophils infected with 1, 2 and 3 parasites, respectively. c Graphic representation of imaging flow cytometry, showing the percentage of infected cells carrying the designated number of L. amazonensis or L. braziliensis parasites. La = L. amazonensis-infected neutrophils (solid gray line); Lb = L. braziliensis-infected neutrophils (solid black line); Med = uninfected neutrophils (dotted line).

Neutrophil leishmanicidal activity against L. amazonensis (La) and L. braziliensis (Lb) amastigotes. Thioglycollate-induced neutrophils were cocultured with amastigotes at a ratio of 1 parasite/10 neutrophils (MOI of 0.1) for 6 h in the presence or absence of PMA (100 nM) and/or DNase (100 U/ml). a Reduced survival of L. braziliensis amastigotes after coculture with control neutrophils or PMA-activated neutrophils as well as improved parasite survival in PMA-activated wells via treatment with DNase. b Survival of L. amazonensis amastigotes was not significantly altered by the presence of control or PMA-activated neutrophils. c Bone marrow-derived macrophages were infected with amastigotes (MOI of 3) for 24 h followed by the addition of neutrophils (at a ratio of 3: 1 neutrophils/macrophages) for 24 h. Neutrophil-mediated clearance of amastigotes from infected macrophages was measured 3–4 days later via counting (see Materials and Methods). ** p < 0.01, *** p < 0.001, between indicated groups. n.s. = Not significant.