Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria

Abstract

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.

Fig. 1. IP-opsonized merozoites stimulate ROS generation by neutrophils and monocytes.

a Median DCF fluorescence intensity (MFI) of neutrophils (blue) and monocytes (red) after incubation with merozoites opsonized with immune plasma (IP), IP and cytochalasin D (IP + CytD), or non-immune plasma (NP); and b phagocytes with engulfed parasite (OP+, detected by EtBr signal) of IP-opsonized merozoites show higher levels of DCF signals compared to those without detectable engulfed parasites (OP−, no EtBr signal). Left y-axes show the MFI of neutrophils, whereas the right y-axes show the MFI of monocytes for the DCF signal. Boxes indicate the median and interquartile range. c Kinetics of isoluminol chemiluminescence for peripheral blood leukocytes (PBLs) incubated with merozoites opsonized with IP (red) and NP (blue). Median values are in bold. d Area under curve (AUC) of isoluminol chemiluminescence for PBLs incubated with merozoites opsonized with IP, NP, and IP plus the ROS scavengers (catalase and superoxide dismutase) (IP + Scavenger). Values shown are from a single independent experiment (n = 12) (i.e., single IP and single NP sample were tested using the same batch of merozoites with PBLs from 12 donors in a single assay). P values for panel b were determined by Wilcoxon signed-rank test, whereas for panels (a, d) were determined by a Friedman test and Dunn’s multiple comparisons test.

Fig. 2. ROS generation after incubation with IP-opsonized merozoites depends on FcγRIII (CD16) in neutrophils and FcγRII(CD32) in monocytes.

PBLs treated with 10 μg/ml of antibodies against CD16 (FcγRIII), CD32 (FcγRII), or CD64 (FcγRI) for 30 min were then incubated with IP-opsonized merozoites. Graphs show the relative DCF of neutrophils (a) and monocytes (b) or relative area under curve (AUC) of isoluminol signal of PBLs (c) of specific FcγR blocker using the untreated cells (IP) as reference. Horizontal lines represent median values. P values were determined by the Friedman test and Dunn’s multiple comparisons test. The values shown are from a single independent experiment (n = 12).

Fig. 3. ROS generation by IP-opsonized merozoites in blood phagocytes depends on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2).

PBLs were incubated with immune plasma (IP) or non-immune plasma (NP) plus 2.5 µM diphenyleneiodonium (DPI, an inhibitor of NOX2) and/or 500 µM 4-aminobenzoic acid hydrazide (ABAH, an inhibitor of myeloperoxidase (MPO)). Graphs show the relative DCF or isoluminol signal of neutrophils (a), monocytes (b), and PBLs (c) using the untreated cells (IP) as reference. Horizontal lines represent median values. P values were determined by the Friedman test and Dunn’s multiple comparisons test. The values shown are from a single independent experiment (n = 12).

Fig. 4. IP-opsonized ROS generation in blood phagocytes depends on the phosphoinositide 3-kinase signaling pathway.

PBLs were incubated with immune plasma (IP) or non-immune plasma (NP) plus 250 nM Gö6983 (an inhibitor of protein kinase C) and/or 100 nM wortmannin (an inhibitor of phosphoinositide 3-kinase). Graphs show the relative DCF or relative area under curve (AUC) of isoluminol chemiluminescence signal of neutrophils (a), monocytes (b), and PBLs (c) using the untreated cells (IP) as reference. Horizontal lines represent median values. P values were determined by the Friedman test and Dunn’s multiple comparisons test. The values shown are from a single independent experiment (n = 12).

Fig. 5. ROS generated in blood phagocytes by IP-opsonized merozoites is associated with protection from febrile malaria.

PBLs incubated with merozoites opsonized with plasma samples (108 Ghanaian and 121 Indian cohorts) were analysed for DCF signal and isoluminol signal quantification. Study participants were classified into susceptible and protected individuals (Ghanaian: n = 63 and 45, respectively and Indian: n = 48 and 73, respectively) based on their febrile malaria status. The DCF signal of susceptible and protected Ghanaian and Indian cohorts (a, c, respectively) were compared in neutrophils (blue) and monocytes (red). Moreover, isoluminol signal (AUC) in PBLs was compared between susceptible and protected cohorts (a, c, black). Left y-axis (a, c) shows the median fluorescence intensity (MFI) of neutrophils and monocytes DCF signal. Right y-axis (a, c) shows the area under the curve (AUC) of the PBLs isoluminol signal. Ghanaian and Indian cohorts (b, d, respectively) were categorized into two equal groups based on the median DCF signal, and to calculate the risk of suffering from febrile malaria during the follow-up period, the Cox-regression model was used to compare the high group with the low group (reference group). Values represent age-adjusted (circles), age-plus monocytes DCF (mono)-adjusted (square), and age-plus neutrophils DCF (neu)-adjusted (triangles) hazard ratios at 95% confidence intervals. P values were determined by Mann–Whitney tests.

Fig. 6. Correlation between opsonic phagocytosis of IP-opsonized merozoites and ROS generation in blood phagocytes.

Scatterplots with linear regression lines show the relationship between intracellular ROS production and opsonic phagocytosis in neutrophils (a) and monocytes (b). The production of extracellular ROS and opsonic phagocytosis in neutrophils and monocytes are depicted in panels c, d, respectively. Pearson’s correlation coefficient (r) and corresponding P values are shown in each plot (n = 108).