Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

Abstract

Neutrophils (PMNs) are recruited in high numbers to sites of host infection by the protozoan parasites of the genus Leishmania. Although PMNs are capable of phagocytizing Leishmania parasites and are potent producers of anti-microbial compounds including reactive oxygen species (ROS), they are unable to control the establishment of infection. Prior studies document production of ROS in isolated PMNs incubated with Leishmania under conditions allowing phagocytosis, but without a measure of single cells’ responses it cannot be discerned whether PMN activation and ROS production is suppressed or ineffective in the cells that internalize the parasite. To address these interactions, we engineered a strain of fluorescent, mCherry-expressing Leishmania infantum (mCherry-Li). By infecting isolated human PMNs in vitro with mCherry-Li, we observed ready association of the parasites with PMNs in a time- and dose-dependent fashion. We also examined production of PMN ROS (using the fluorescent compound DHR123) and PMN activation (as evidence by loss of surface CD62L expression). Whereas many Li-associated (mCherry+) PMNs responded to parasite interactions and uptake with ROS production and/or activation, a proportion exhibited neither response. Furthermore, a large proportion of mCherry - “bystander” PMNs displayed both ROS production and activation. The heterogeneous response of PMNs to Leishmania exposure leads us to hypothesize, first, that some PMNs exhibit decreased activation upon phagocytosis of Leishmania, and could support their maintenance. Second, responses of bystander PMNs may contribute to a local inflammatory environment that is ineffective at parasite clearance.

The Modulation of NADPH Oxidase Activity in Human Neutrophils by Moroccan Strains of Leishmania major and Leishmania tropica Is Not Associated with p47phox Phosphorylation

“…Recently, we reported that L. major and L. tropica modulated the production by macrophages of nitric oxide (NO) in a different manner [ 15 ]. However, controversial results have been reported regarding ROS production by Leishmania -infected PMNs [ 16 , 17 ]. Early recruitment of PMNs at the site of infection and production of ROS could contribute to the resolution or aggravation of the pathology according to species and strains.…”