Distinct neutrophil effector functions in response to different isolates of Leishmania aethiopica

Abstract

Background: In Ethiopia, cutaneous leishmaniasis is mainly caused by Leishmania (L.) aethiopica parasites and presents in three main clinical forms. It is still not clear if the host immune response plays a role in the development of these different presentations. Since neutrophils are likely to be one of the first immune cells present at the site of the sand fly bite, we set up an in vitro model of infection of neutrophils with L. aethiopica and assessed some of the main neutrophil effector functions: association with and internalisation of parasites, apoptosis and ROS production. We used three freshly isolated clinical isolates and one isolate that has been kept in culture for decades.

Results: Our results showed by flow cytometry that all four L. aethiopica isolates had the ability to associate with neutrophils. The three clinical isolates of L. aethiopica associated more efficiently with neutrophils than the long-term cultured L. aethiopica. At 18 h, two distinct populations of neutrophils were identified that associated with L. aethiopica, CD15^high and CD15^low neutrophils. Confocal microscopy demonstrated that all isolates can be internalised. Our results also showed that all parasites induced apoptosis in L. aethiopica-associated neutrophils. Moreover, our results showed that after 2 h, L. aethiopica-associated neutrophils upregulated their production of ROS, but to a greater extent with the long-term cultured L. aethiopica. After 18 h of incubation, CD15^lowparasite⁺ showed an impaired ability to produce ROS compared to CD15^highparasite⁺.

Conclusions: Using this in vitro model, our results show that different L. aethiopica parasite isolates, most notably long-term cultured parasites, had differential effects on neutrophil effector functions.

Keywords: Leishmania aethiopica; Apoptosis; Neutrophils; Phagocytosis; ROS.

Fig. 1

Association of neutrophils with different isolates of Leishmania aethiopica; 1 × 10⁵ cells/ml neutrophils were co-cultured with 1 × 10⁶ cells/ml FR labelled L. aethiopica isolates for 2 h (A, B) and 18 h (C–F). The percentages of neutrophils associated with L. aethiopica were determined by flow cytometry. A Dot plot showing neutrophils unassociated (gate R4) and associated (gate R5) with L. aethiopica. B % of neutrophils associated with the four different isolates of L. aethiopica. C Dot plot showing the three different population of neutrophils: CD15^{intermediate (int)}parasite⁻ (gate R3), CD15^highparasite⁺ (gate R4) and CD15^lowparasite⁺ (gate R5). D % of CD15^highparasite⁺ associated with the four different isolates of L. aethiopica. E % of CD15^lowparasite⁺ associated with the four different isolates of L. aethiopica. F Comparison in FR MFI between CD15^highparasite⁺ and CD15^lowparasite⁺ for each L. aethiopica. Data are presented as scatter plot with bar (mean with standard deviation), with each dot representing the value for one experiment. Statistical differences were determined using Kruskal-Wallis (dotted line) and Mann-Whitney (solid line) tests

Fig. 2

Internalisation of Leishmania aethiopica by neutrophils; 1 × 10⁵ cells/ml neutrophils were co-cultured with 1 × 10⁶ cells/ml FR labelled L. aethiopica lab for 2 h (A) and 18 h (B) and cells were labelled as described in “Methods”. The red arrows point to the parasite (FR), the green arrows to the CD15 (Alexa Fluor 555) and the blue arrow to the nucleus (DAPI). These are representative images of at least three independent experiments

Fig. 3

% change in apoptotic neutrophils between the different parasite isolates; 1 × 10⁵ cells/ml neutrophils were co-cultured with 1 × 10⁶ cells/ml FR labelled L. aethiopica isolates for 2 h (A) and 18 h (B, C). The % change was measured by deducting the % apoptotic (as defined by Annexin V⁺7-AAD⁻) CD15^high neutrophils co-cultured with the parasites from the % of apoptotic neutrophils cultured in the absence of parasites. Data are presented as scatter plot with bar (mean with standard deviation), with each dot representing the value for one experiment. Statistical differences were determined using Kruskal-Wallis (dotted line) and Mann-Whitney (solid line) tests

Fig. 4

% change in ROS MFI in neutrophils between the different parasite isolates; 1 × 10⁵ cells/ml neutrophils were co-cultured with 1 × 10⁶ cells/ml FR labelled L. aethiopica isolates for 2 h (A) and 18 h (B, C). The % change was measured by deducting the ROS MFI in the neutrophils co-cultured with the parasites from the ROS MFI in the neutrophils cultured in the absence of parasites. Data are presented as scatter plot with bar (mean with standard deviation), with each dot representing the value for one experiment. Statistical differences were determined using Kruskal-Wallis (dotted line) and Mann-Whitney (solid line) tests