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-␣), 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.

Interactions between Neutrophils and <b><i>Leishmania braziliensis</i></b> Amastigotes Facilitate Cell Activation and Parasite Clearance

“…To better understand amastigote interactions with other innate immune cells, we recently examined neutrophil activation and microbicidal activity against L. amazonensis amastigotes. We found that amastigotes are a poor stimulus for neutrophil activation and are highly resistant to neutrophil microbicidal activity [7]. These findings supported an earlier report from our laboratory that L. amazonensis amastigotes are not killed by purified human histones [8], which are important microbicidal components of neutrophil extracellular traps (NETs).…”

“…CFSE-labeled parasites were co-cultured with neutrophils at a multiplicity of infection (MOI) of 5 for 4 h, as described previously [7]. Cells were washed, blocked with anti-CD16/CD32, and stained with anti-Ly6G-AlexaFluor 647 (BioLegend, San Diego, CA) and anti-CD11b-PE-Cy7 (BD Bioscience, San Jose, CA).…”

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

“…Despite observations of murine neutrophils to the contrary [21,23], our data also showed activation of “bystander” human PMNs in cultures containing L. infantum infected neutrophils. Bystander activation was not observed when fresh PMNs were incubated in conditioned medium from either L. infantum or L. infantum infected neutrophils.…”

“…Our observation that neutrophil populations exhibit heterogeneous responses to parasites in culture is consistent with observations in vitro and in vivo that PMNs take up, and though they may kill some parasites early in infection [13], they are unable to control Leishmania infection [5,22,23,25]. Our data raise the curious possibility of “silent” parasite uptake by a neutrophil subset.…”

“…They are the first cell to migrate to, and take up parasites at the site of in vivo infection with either L. major [5,18–20] or with L. infantum [6] species. Indeed, there appears to be distinct differences in the way various species of Leishmania result in activation and otherwise affect neutrophils [10,21–23]. In the context of disease progression, it has been suggested that, rather than control the infection, neutrophils exacerbate disease onset either by delaying apoptosis, by impairing activation of dendritic cells, or by serving as a vehicle route for silent entry of the parasite [4,24–26].…”