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.

Keywords: Flow cytometry; Leishmania; Neutrophils.

Figure 1

Generating recombinant Leishmania expressing genes encoding fluorescent markers, using the pIR1SAT expression vector. (A) Schematic showing the mCherry pIR1SAT recombinant plasmid and the gene order upon into genome of L. infantum . dashed lines indicate the expected sites of homologous recombination. (B) Integration of fluorescent marker genes in parasites was confirmed by Southern blotting. Genomic DNA from transgenic dTomato, mCherry, and wild type L. infantum were digested with SmaI, separated on an agarose gel and incubated with [³²P]–DNA probe to the streptothricin acetyltransferase (SAT) gene in pIR1SAT. (B,C) Confirmation that mCherry- expressing L. infantum express fluorescent protein after infection of neutrophils. PMNs isolated from a healthy donor were stained with cytoplasmic CFSE dye, allowed to adhere to fibrinogen-coated coverslips and then infected with mCherry-Li, pseudo colored red. Pictured are images of PMN with both internalized parasites (C) and the combination of internalized and adherent parasites (D) Scale bar is equal to 5 microns (μm).

Figure 2

mCherry-Li uptake by human neutrophils (A) Unopsonized mCherry-Li were incubated with human neutrophils at 37°C at an MOI of 5:1. After 20, 40 or 80 minute, cultures were harvested, fixed, and processed for flow cytometry. Uninfected PMNs were used to set the mCherry expression gate. (B) Increasing doses of human-serum opsonized mCherry-Li were incubated with human neutrophils for 30 minutes then fixed and analyzed by flow cytometry. Histograms represent increasing numbers of mCherry⁺ (infected) PMNs of total neutrophil population. MFI (mean fluorescence intensity) is indicated. (C) PMNs were exposed to mCherry-Li fixed, then analyzed, either by flow cytometry or by light microscopy to determine percentage of internalized parasites. Results are representative of two separate experiments. (D) Using CFSE-labeled Li parasites, the same protocol as above was performed, except that internalized and surface-bound parasites were quantified for each MOI.

Figure 3

ROS production and activation by PMNs associated with mCherry-Li and “bystander” uninfected neutrophils. (A) Flow plots from a 30 minute infection of human neutrophils with opsonized mCherry-Li. (B) shows the expression of DHR123 in infected PMNs, indicating the amounts of ROS generated. PMNs were incubated with either mCherry-Li at the indicated MOI, or buffer, or PMN supernatant for 10 minutes at 37°C. DHR123 was then added to cultures, and after an additional 20 minutes at 37°C, cells were fixed and stained for flow cytometry. PMN supernatant was collected from a previous PMN infection (MOI 5:1, 30 minutes). (C) Flow cytometry plots of surface CD62L and DHR123 in mCherry⁺ or mCherry⁻ PMNs incubated for 30 minutes with mCherry-Li at 37°C is shown. Negative and positive controls were PMNs incubated with buffer, or with opsonized zymosan (OZ), respectively.