Leishmania amazonensis amastigotes trigger neutrophil activation but resist neutrophil microbicidal mechanisms

Abstract

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.

Fig 1

Neutrophil phagocytosis of L. amazonensis parasites. (A) Thioglycolate-elicited peritoneal neutrophils were cocultured with CFSE-labeled axenic amastigotes (AxAm) or metacyclic promastigotes (Pm) for 4 h. In some groups, neutrophils were pretreated with cytochalasin D (Cyto D) (20 μM) or parasites were opsonized in serum from infected mice. Neutrophils were identified by forward/side scatter characteristics and by Ly6G positivity. CFSE positivity in the boxes shown represents Ly6G⁺ neutrophils carrying parasites. Values are mean percentages of CFSE⁺ cells ± 1 standard deviation (SD). (B) Percentages of CFSE⁺ neutrophils (PMN) carrying amastigotes after 4 h in medium alone (Med) or in the presence of LPS (100 ng/ml) or GM-CSF (200 ng/ml). Data are pooled from 3 independent repeats and are shown as means ± standard errors. *, statistically significant differences (P < 0.05) between the groups.

Fig 2

Ultrastructural analysis of amastigote uptake by neutrophils. Neutrophils were cocultured with serum-coated amastigotes for 4 h, fixed, and prepared for analysis via electron microscopy. A characteristic neutrophil is depicted, exhibiting a multilobular nucleus (N), electron-dense granules (arrowheads), and 2 intracellular amastigotes (asterisks). A flagellar remnant is also clearly visible in the amastigote on the left (arrow). Bar, 2 μm.

Fig 3

Neutrophil activation and oxidative burst after contact with parasites. (A) Mean fluorescence intensity (MFI) of CD11b on neutrophils resting in medium (Med) or cocultured with metacyclic promastigotes (Pm), axenic amastigotes (AxAm), or lesion-derived amastigotes (Am). (B) MFI of rhodamine 123 (Rho 123) in dihydrorhodamine 123-labeled Ly6G⁺ cells after 4 h of coculture with parasites. (C) Oxidative burst in Ly6G⁺ Rho123⁺ neutrophils, indicating that the extent of burst on a per-cell basis does not differ for neutrophils cocultured with promastigotes or amastigotes. (D) ROS production in neutrophils infected with PKH26-labeled axenic amastigotes for 4 h, showing that the majority of ROS was generated in PKH26^hi (amastigote-laden) cells. All data are pooled from at least 2 independent experiments and shown as means ± standard errors. * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) indicate statistically significant differences between groups. NS, not significant.

Fig 4

Neutrophil cytokine production in response to parasites. Neutrophils were cocultured with axenic amastigotes (AxAm) or metacyclic promastigotes (Pm) for 24 h. Production of TNF-α (A) and IL-10 (B) was analyzed by ELISA. Results are pooled from 2 independent repeats and shown as means ± standard errors. * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) indicate statistically significant differences between groups. NS, not significant.

Fig 5

Accelerated neutrophil apoptosis after L. amazonensis amastigote uptake. (A) Percentages of PS⁺ PI⁻ neutrophils after 18 h of culture in medium (Med) or after coculture with axenic amastigotes (AxAm) or lesion-derived amastigotes (Am). (B) Neutrophil apoptosis in medium alone or in response to axenic amastigotes in the presence or absence of GM-CSF (20 ng/ml). All data in panels A and B are pooled from at least 2 independent repeats and shown as means ± standard errors. NS, not significant. * (P < 0.05) and *** (P < 0.001) indicate statistically significant differences between the groups. (C) Comparison of apoptosis in resting neutrophils, CFSE⁺ (parasite-carrying) neutrophils, and CFSE⁻ (bystander) neutrophils. PS surface exposure in neutrophils was measured via binding of annexin V (AnxV). The percentages of apoptotic cells were calculated by dividing the number of PS⁺ cells by the total number of cells for each group. Values are mean percentages of apoptotic cells ± 1 SD. Shown are representative results of one of three independent repeats.

Fig 6

Neutrophil killing of promastigotes and amastigotes. Luciferase-expressing metacyclic promastigotes (Pm) and axenic amastigotes (AxAm) were cocultured with neutrophils (at an MOI of 0.1) for 0, 6, or 18 h. Cells were lysed and treated with luciferin substrate to elicit photon emission. Photon emission of 0-h samples was used as a reference for 100% parasite survival, and the subsequent decay in signal at 6 h and 18 h was used to estimate the extent of parasite killing. (A) Representative graph showing photon intensity in relative light units. (B) Survival of axenic amastigotes and metacyclic promastigotes cocultured with neutrophils for 6 h and 18 h. (C) Survival of lesion-derived amastigotes (Am) and axenic amastigotes opsonized in fresh serum from infected mice (AxAm-Opso) after 6 h and 18 h of coculture with neutrophils. The data in panels B and C are pooled from at least 2 independent experiments and shown as means ± the errors.