ARTICLE
https://doi.org/10.1038/s42003-023-05118-0 OPEN
Human blood neutrophils generate ROS through
FcγR-signaling to mediate protection against febrile
P. falciparum malaria
Ebenezer Addo Ofori 1,2,7, Asier Garcia-Senosiain1,2,7, Mohammad Naghizadeh1,2, Ikhlaq Hussain Kana 1,2,
Morten Hanefeld Dziegiel 3,4, Bright Adu 5, Subhash Singh 6✉ & Michael Theisen 1,2✉
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.
1 Department for Congenital Disorders, Statens Serum Institut, Copenhagen, Denmark. 2 Centre for Medical Parasitology at Department of Immunology and
Microbiology, University of Copenhagen, Copenhagen, Denmark. 3 Blood Bank KI 2034, Department of Clinical Immunology, Copenhagen University
Hospital, Copenhagen, Denmark. 4 Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. 5 Noguchi Memorial Institute for
Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana. 6 ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha,
India. 7These authors contributed equally: Ebenezer Addo Ofori, Asier Garcia-Senosiain. ✉email: subhash0974@gmail.com; mth@ssi.dk
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P lasmodium falciparum malaria remains one of the greatest the DCF signal in neutrophils (median MFI= 59,913) was higherpublic health challenges, with an estimated 247 million new as compared to that in monocytes (median MFI= 26,769).cases and 619,000 malaria-related deaths worldwide in Neutrophils and monocytes stimulated with merozoites opso-
20211. The parasite is relatively hidden from the immune system nized with non-immune plasma (NP) displayed 14.7 (95% CI,
during its asexual stage development, which is associated with 8.10–17.50) and 1.6 (95% CI 1.40–1.70) -fold lower DCF signal
malaria symptoms and mortality. On the other hand, merozoites compared to that of IP-opsonized merozoites respectively,
released upon completion of the intraerythrocytic development, are demonstrating that DCF fluorescence induced by ROS is malaria-
highly vulnerable when they transit from one host cell to another specific with better signal-to-noise ratio observed for neutrophils
and thus become potential targets of host defense mechanisms as compared to the monocytes. The DCF signal could be abol-
(reviewed in refs. 2–4). Immunoglobulin (Ig) G antibodies constitute ished by pretreatment of cells with cytochalasin D (Cyt D),
one of the main defenses against blood-stage malaria parasites5,6. suggesting that intracellular ROS generation is related to
These antibodies can exert their anti-parasite effects in cooperation merozoite-phagocytosis (Fig. 1a). Additionally, we found that
with blood leukocytes (e.g., monocytes and neutrophils) through within the phagocytes exposed to IP-opsonized merozoites, those
antibody-dependent cellular mechanisms7–11. with detectable phagocytosed merozoites (indicated by EtBr sig-
Both neutrophils and monocytes constitute the dominant nal) contained higher levels of intracellular ROS confirming that
phagocyte population (about 55 to 75% of all blood leukocytes), the DCF signal is linked to opsonic phagocytosis (Fig. 1b).
and being the first line of innate defense and effectors of adaptive Next, we used time-lapse confocal microscopy to investigate
immunity, both share many features, but possess distinct mor- the relationship between phagocytosis and ROS production. PBLs
phological and functional properties12,13. For instance, the were incubated with EtBr-stained IP-opsonized merozoites and
metabolic burst activity of monocytes is less strong, but their ROS production was monitored with DCFH2-DA. Following
capacity to kill microorganisms is more diverse compared to phagocytosis, there was a rapid increase in the DCF signal,
neutrophils14. Several studies have suggested that both neu- confirming that these processes are connected (Supplementary
trophils and monocytes may eliminate malaria parasites through Movie 1).
phagocytosis9,10,15–17 and through the production of soluble We used membrane-impermeable isoluminol for the detection
factors with anti-parasite effects16,18–20. Each of these effector of extracellular ROS. PBLs produced much higher isoluminol-
functions requires the engagement of surface exposed FcγR by enhanced ROS signal when exposed to merozoites opsonized with
anti-parasite IgG antibodies10,15,16. In continuation with our IP compared to NP, demonstrating that extracellular ROS is
efforts to dissect the contribution of different IgG-mediated cel- malaria-specific (Fig. 1c). Addition of superoxide dismutase
lular responses in protection against malaria, we studied the role (SOD) and catalase (CAT) reduced the isoluminol-enhanced ROS
of FcγR-triggered generation of ROS in protection against signal by 8.1 (95% CI 5.40–8.30)-fold (Fig. 1d). Since SOD and
malaria. CAT do not penetrate the cell membrane, these scavengers
Cross-linking of FcγRs on neutrophils and monocytes can selectively deplete the extracellular ROS.
trigger the generation of ROS through activation of the Src family
kinases, Syk recruitment to the signaling complex, and PI3K
activation, which plays an important role in nicotinamide adenine IP-opsonized merozoite-induced ROS generation by blood
dinucleotide phosphate (NADPH) oxidase (NOX2) phagocytes depends on FcγRs. We used specific monoclonal
activation21,22. This leads to the production of large amounts of antibodies (mAbs) to block individual FcγRs, to investigate the
superoxide anion, leading to the generation of hypochlorous acid role of FcγR-signaling in the generation of ROS. Pre-incubation
(HOCl) in a reaction catalyzed by myeloperoxidase (MPO)23. As with anti-CD16 (FcγRIII) antibody led to an 82.7% reduction of
NOX2 is expressed on both the phagosomal and the plasma intracellular oxidation in neutrophils as detected by the DCF
membrane, phagocytes can release ROS both intracellularly into probe (Friedman test, P= 0.0024, Fig. 2a). Addition of anti-CD32
the phagosome and into the extracellular space24–26. (FcγRII) antibody further reduced the remaining DCF signal by
Here, we used peripheral blood leukocytes (PBLs) to study ROS another 9.9% compared to anti-CD16 antibody alone (Friedman
production intracellularly and extracellularly using freshly pur- test, P= 0.0286) suggesting that FcγRIIIB and FcγRIIA act
ified P. falciparum merozoites and naturally occurring antibodies synergistically in neutrophils for ROS generation. In contrast, the
from samples from well-established longitudinal cohort studies anti-CD32 antibody alone reduced the generation of ROS in
(LCS) performed in Ghana and India. monocytes by 21.4% (Friedman test, P < 0.0015, Fig. 2b),
demonstrating a significant role of FcγRIIA in ROS generation.
The addition of an anti-CD16 antibody further reduced the
Results remaining DCF signal by another 9.2% compared to the anti-
Anti-merozoite antibodies elicit ROS generation in neutrophils CD32 antibody alone (Friedman test, P= 0.0286), suggesting that
and monocytes. To investigate ROS generation in response to P. FcγRIIA and FcγRIIIA (as FcγRIIIB expression is known to be
falciparum merozoites, we used PBLs from whole blood samples absent in monocytes) act synergistically in monocytes for ROS
to ensure that the phagocytes were present in their natural phy- generation. We observed that the combined effect of the anti-
siological proportions and to maximize their cellular integrity. CD32 and CD16 antibodies reduced the monocyte DCF signal to
Each experiment used blood from malaria-naïve blood donors that of the background level obtained with NP (Fig. 2b).
(n= 12) and merozoites obtained from a single batch of opso- Generation of extracellular ROS by PBLs, as detected by
nization with single immune plasma, [IP] and single non- isoluminol, was significantly inhibited by the anti-CD16 antibody
immune plasma, [NP] samples, respectively, to minimize assay (59%; Friedman test, P= 0.0003) and anti-CD32 antibody (40%;
variability. First, we used the cell-permeable, broadly reacting Friedman test, P= 0.0393) individually (Fig. 2c), and synergis-
redox-sensitive fluorescent probe (DCFH2-DA) for real-time tically by a mixture of both antibodies (78%; Friedman test,
monitoring and quantification of the intracellular ROS activity 27. P < 0.0001) indicating that both FcγRIIIA/B and FcγRIIA are
Both neutrophils and monocytes generated high amounts of ROS involved in generation of extracellular ROS. Moreover, the anti-
when exposed to merozoites opsonized with decomplemented CD64 antibody neither inhibited the DCF nor the isoluminol
malarial IgG antibodies at a dilution of 1:100, as detected by DCF signals, indicating that ROS generated by phagocytes upon
fluorescence quantified by FACS (Fig. 1a). In the presence of IP, opsonic phagocytosis of merozoites does not involve FcγRI.
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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 30min 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).
ROS generation by IP-opsonized merozoites in blood phago- mediated inhibition of NOX2 activation by phorbol 12-myristate
cytes depends on NOX2. For understanding the mechanism of 13-acetate (PMA), a known NOX2 activator through protein
FcγR signaling induced by IP-opsonized merozoites for the gen- kinase C signaling27. PMA stimulated high amounts of ROS gen-
eration of ROS by PBLs, we used diphenyleneiodoium (DPI), a eration in neutrophils (median MFI= 395,646) and monocytes
commonly used inhibitor of NOX228, and compared it to DPI- (median MFI= 100,310), as detected by the DCF probe. As
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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).
expected, DPI abrogated the PMA-enhanced DCF signal in neu- (Supplementary Fig. 2c, d). Thus, PI3K activation is specifically
trophils and monocytes (Supplementary Fig. 2a, b). Similarly, the involved in FcγR-signaling, to stimulate NOX2 activation leading
addition of DPI reduced the DCF signal in neutrophils by 72% to both intracellular and extracellular ROS generation upon sti-
(Friedman test, P= 0.0008) and in monocytes by 36% (Friedman mulation of PBLs with IP-opsonized merozoites. Treatment with
test, P= 0.0002) after stimulation with IP-opsonized merozoites, wortmannin reduced the generation of extracellular ROS to near
demonstrating a role for NOX2 in intracellular ROS production by basal levels, whereas it resulted in lower reduction of the intra-
both phagocytes through FcγR signaling (Fig. 3a, b). The super- cellular ROS, indicating differential NOX2 activation at the
oxide anion generated by NOX2 is likely to generate the more phagosomal and plasma membranes.
reactive HOCl by MPO in combination with H2O2 and chloride29. Since we found that the generation of intracellular ROS in
Pre-incubation of the PBLs with the MPO inhibitor response to IP-opsonised merozoites was dependent on OP
4-aminobenzoic acid hydrazide (ABAH) before adding opsonized (Fig. 1b), we sought to investigate whether the same FcγR-
merozoites reduced the DCF signal in neutrophils by 17.8% and in signaling pathway affected both the IP-mediated phagocytosis
monocytes by 13.9% (Fig. 3a, b). However, these reductions did not process as well as the resulting ROS generation. For this, we
reach statistical significance. assessed the efficiency of phagocytosis of the IP-opsonised
The addition of DPI to PBLs reduced the isoluminol signal by merozoites by PBLs in the presence of wortmannin or Gö6983,
91.5% (Friedman test, P= 0.0005). However, no reduction was as indicated by EtBr signals (used to label the merozoite DNA) in
seen after the addition of ABAH (Friedman test, P > 0.9999) and the phagocytes. Interestingly, neither of the two kinase inhibitors
combined use of both DPI and ABAH did not cause any further alone nor in combination had any significant effect on the
reduction suggesting that extracellular ROS is likely generated efficiency of phagocytosis of the IP-opsonised merozoites,
through assembly / activation of NOX2 at the plasma indicating that the FcγR-mediated phagocytosis of the opsonized
membrane30. Taken together, these data suggest that IP- merozoites per se did not depend on PI3K / PKC signaling, which
opsonised merozoites stimulate FcγR-driven assembly of the is distinct from the downstream PI3K-signaling events involved
NOX2 system either within the phagosomal or on the plasma in FcγR-mediated generation of ROS in blood phagocytes
membrane impacting ROS generation both intracellularly as well (Supplementary Fig. 3).
as in the extracellular vicinity. Though our experimental design
precludes direct assessment of the exact cell type contributing to
the generation of extracellular ROS, striking similarities in the ROS generated in blood phagocytes by IP-opsonized mer-
patterns observed between Fig. 2a, c, and between Fig. 3a, c, ozoites is associated with protection against febrile malaria.
strongly suggest neutrophils to be the dominant cell population Having demonstrated that IP-opsonized merozoites stimulate
contributing to the generation of extracellular ROS. PBLs to generate ROS, we determined whether such responses are
associated with immunity against clinical malaria using archived
plasma samples from a longitudinal cohort survey (LCS) con-
PI3K mediated signaling involved in ROS generation stimu- sisting of 108 Ghanaian children who were considered “defini-
lated by IP-opsonized merozoites. We used specific protein tively” exposed (i.e., children who had parasitemia at one or more
kinase inhibitors of PKC and phosphoinositide 3-kinase of the monthly blood slides during the study) and who had
(PI3K)27,31, both known to be involved in NOX2 activation, to completed follow-up32. ROS in neutrophils (Mann–Whitney test;
dissect the exact mechanism of ROS generation by PBLs upon P= 0.0005) and monocytes (Mann–Whitney test; P= 0.0123), as
stimulation with IP-opsonised merozoites. The addition of the well as extracellular ROS (Mann–Whitney test; P= 0.0030), were
PI3K inhibitor wortmannin before adding IP-opsonized mer- generated at significantly higher levels by plasma samples from
ozoites to PBLs significantly reduced the DCF signal in neu- “protected” compared to the non-protected children (Fig. 5a).
trophils (Friedman test, P= 0.0047) and monocytes (Friedman Children were categorized into groups with high and low DCF- or
test, P= 0.0133) as well as the isoluminol signals (Friedman test, isoluminol signals based on the median in order to examine the
P < 0.0001) indicating inhibition of ROS generation (Fig. 4a–c). In risk of febrile malaria for each group using Cox-regression
contrast, the addition of the PKC inhibitor, Gö6983, did not models to calculate Hazards ratios (HRs). After adjustment of
inhibit intracellular (Fig. 4a, b) or extracellular ROS (Fig. 4c) age, children with high levels of DCF signal (age-adjusted
generated through FcγR signaling. As expected, Gö6983 abolished [aHR]= 0.34; 95% CI= 0.19–0.59; P < 0.0001 for neutrophils and
PMA-induced oxidation, while wortmannin had no such effect aHR= 0.53; 95% CI= 0.32–0.88; P= 0.0139 for monocytes) and
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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.
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isoluminol signal ([aHR]= 0.46; 95% CI= 0.27–0.79; P= 0.0046) P < 0.0001) and monocyte OP (Pearson r= 0.3705, P < 0.0001)
had significantly higher probability of remaining free of febrile were less strong and showed frequent samples that elicited high
malaria compared to those with low-level responses (Fig. 5b). responses in one assay and low in the other (Fig. 6).
Since the DCF signal of neutrophils was highly correlated to the
isoluminol signal (Pearson r= 0.6700, P < 0.0001) (Supplemen- Effect of ROS responses on antibody-mediated protection
tary Fig. 4a) and the monocytes (Pearson r= 0.8558, P < 0.0001) against febrile malaria. To determine the contribution of specific
(Supplementary Fig. 4b), Cox-regression models were also ROS activities to malaria immunity acquired through anti-
adjusted for neutrophil ROS as a confounder. The magnitude of merozoite IgG antibodies, LRTs were performed as follows. A
protective associations of both monocyte ROS and extracellular logistic regression model that included age and merozoite-
ROS were significantly influenced by neutrophil ROS (Fig. 5b). phagocytosis (model 1) was compared to another logistic
Next, we investigated samples and clinical data from an LCS
33 regression model (model 2), which was fitted for ROS in additionconducted in India . Intracellular ROS generated by IgG from to the variables in model 1. It was observed that the ROS activity
Indian plasma samples in neutrophils ([aHR]= 0.26; 95%CI of neutrophils, but not monocytes, had a significant influence on
0.13–0.52; P= 0.0001) but not in monocytes ([aHR]= 1.01; 95% the probability of developing febrile malaria in Ghana (OR=
CI= 0.57–1.80; P= 0.965) was strongly associated with protec- 0.12, 95% CI= 0.02–0.041; P= 0.0006) and India (OR= 0.31,
tion against febrile malaria (Fig. 5c, d). Extracellular ROS was not 95% CI= 0.11–0.81; P= 0.0177) suggesting that ROS con-
associated with protection ([aHR]= 1.28; 95%CI 0.72–2.29; tributes, at least in part, to the antibody-associated protection
P= 0.3962) from clinical malaria in this Indian cohort (Fig. 5d). against febrile malaria observed here (Table 2).
Cox-regression models were adjusted for neutrophil ROS as a
confounder as neutrophils DCF signal highly correlated to
the isoluminol signal (Pearson r= 0.5115, P < 0.0001) (Supple- Discussion
mentary Fig. 4c) and moderately correlated to monocytes Cytophilc IgG against malaria antigens play a vital role in natu-
7,34,35
(Pearson r= 0.4413, P < 0.0001) (Supplementary Fig. 4d). rally acquired immunity (NAI) against malaria by mediat-
To further strengthen the contribution of neutrophils ROS to ing a variety of anti-malaria effector functions through
malaria immunity, time to the first episode in the two LCS were interactions with the FcγRs expressed on the leukocyte
15,34,36,37
analyzed by logistic regression models that included age and surface . These effector functions include stimulation of
35,38
monocyte ROS or extracellular ROS (model 1) were compared distinct leukocyte subsets to secrete soluble cytokines for
to a second logistic regression model (model 2), which was arresting the intraerythrocytic development of the malaria para-
fitted for neutrophil ROS in addition to the variables in model 1 sites or enhancing their elimination through OP by blood
10,39
(Table 1). The results confirmed that neutrophil ROS had phagocytes . Here we found that OP leads to the generation of
significantly impacted the protective associations of monocyte both intracellular and extracellular ROS predominantly in neu-
ROS and extracellular ROS in Ghana (P = 0.00296) and trophils, but also in monocytes. Moreover, the ability of theLRT
India (P < 0.0001). Thus, neutrophil ROS is the main plasma samples to stimulate neutrophil ROS generation isLRT
predictor of protection against febrile malaria across diverse strongly associated with protection against febrile malaria, indi-
geographic malaria-endemic regions in Ghana and India. cating functional specialization amongst different leukocyte
lineages. While the use of PBLs to study ROS generation is
attractive from an experimental point of view, it also possessed
Correlation between ROS-levels and opsonic phagocytosis some inherent challenges in the neutrophil and monocyte
(OP) activity. In a previously reported study, the OP activity of populations. Firstly, due to the characteristic fragility of neu-
the same IgG preparations from Ghanaian samples were used in trophils, an experiment must utilize samples collected shortly
the present study was associated with protection from febrile (2–4 h) before use. Secondly, considering the relatively lower
malaria10. Since the ROS and OP bioassays share fundamental signal-to-noise ratio in the monocyte DCF signal, it is critical to
similarities, we investigated possible relationships between these minimize inter-assay variability by adopting a design that allows
two assays. Strong correlations were observed between neutrophil testing of all samples in a single experiment. The higher basal
ROS and OP (Pearson r= 0.7344, P < 0.0001) and monocyte ROS levels of intracellular ROS might be related to the greater content
and OP (Pearson r= 0.5986, P < 0.0001). In contrast, associations of respiratory active mitochondria in monocytes compared to
between isoluminol ROS and neutrophil OP (Pearson r= 0.4070, neutrophils40. Irrespective of this physiological difference, we
Table 1 ROS responses associated with protection against malaria.
Functional activity Model 1 Model 2 LRT P value
OR (95% CI)
Ghanaian LCS
Neutrophil ROS NA 0.16 (0.04–0.55) 0.00296
Monocyte ROS 0.38 (0.16–0.83) 1.31 (0.40–5.13)
Neutrophil ROS NA 0.15 (0.03–0.53) 0.0034
Isoluminol level 0.35 (0.15–0.80) 1.51 (0.40–7.39)
Indian LCS
Neutrophil ROS NA 0.15 (0.06–0.38) <0.0001
Monocyte ROS 0.98 (0.46–2.11) 1.73 (0.73–4.38)
Neutrophil ROS NA 0.13 (0.05–0.32) <0.0001
Isoluminol level 1.47 (0.67–3.27) 2.85 (1.14–7.84)
The table shows the P value obtained from the LRT, which compares a reduced logistic regression model (model 1) with a full logistic regression model (model 2). Model 1 was fitted for age groups (1–5
or 6–12 years in Ghana; ≤10, 11–15, or ≥16 years in India) and monocyte ROS or extracellular ROS, whereas model 2 was fitted for neutrophil ROS in addition, to the variables in model 1. Significant LRT
P values are in bold font.
CI confidence interval, LRT likelihood ratio test, OR odds ratio, ROS reactive oxygen species.
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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).
Table 2 ROS responses and merozoite-phagocytosis (OP) suggests another set of differences in effector functions elicited in
associated protection against malaria. the neutrophil and monocyte cell populations. Neutrophils and
monocytes both express FcγRIIA, with a subset of non-classical
monocytes additionally expressing FcγRIIIA42, while FcγRIIIB
Model 1 Model 2
expression is exclusive to neutrophils43. The high density of
Functional OR (95% CI) OR (95% CI) LRT P value 6
activity FcγRIIIB (⁓1.4 × 10 receptors per cell) on neutrophils may
Ghanaian LCS cause extensive receptor clustering at the membrane, leading to
Neutrophil OP 0.46 (0.20–1.00) 2.04 (0.59–9.55) 0.0006 amplified activation and ROS generation41. In neutrophils,
Neutrophil ROS NA 0.12 (0.02–0.41) FcγRIIIB appears to play a dominant role, with FcγRIIA pro-
Monocyte OP 0.41 (0.18–0.90) 0.64 (0.22–1.88) 0.2082
Monocyte ROS NA 0.51 (0.17–1.46) viding a secondary and synergistic signal for OP and ROS gen-
Indian LCS eration in response to IgG opsonized merozoite. The monocytes,
Neutrophil OP 0.22 (0.09–0.49) 0.41 (0.15–1.09) 0.0177 in contrast, appear to primarily depend on FcγRIIA-mediated
Neutrophil ROS NA 0.31 (0.11–0.81) activation signaling10 with FcγRIIIA likely to play a small role (in
Monocyte OP 0.53 (0.25–1.14) 0.52 (0.23–1.13) 0.7407
Monocyte ROS NA 1.14 (0.52–2.54) ROS generation but not in OP), indicating that distinct FcγR
subsets are involved in eliciting OP and signaling for ROS gen-
The table shows the P value obtained from the LRT, which compares a logistic regression model eration in these blood phagocyte lineages. Signaling through
(model 1) that includes merozoite-phagocytosis activity categorized into two groups divided by
the median value as indicated and age groups (1–5 or 6–12 years) with a second logistic FcγRIIIB, as it lacks its own transmembrane domain, is under-
regression model (model 2) that also includes ROS-levels categorized into two groups by the stood to occur through associations with FcγRIIA44 and may
median value in addition to the variables in model 1. The ORs shown refer to the indicated
functional activities. Significant LRT P values are in bold font. mediate a rise in cytosolic Ca2+ through mechanisms distinct
CI confidence interval, LRT likelihood ratio test, OP opsonic phagocytosis, OR odds ratio. from that of FcγRIIA activation alone, thereby leading to ROS
generation upon activation with IgG opsonized merozoites20.
consistently observed around a 1.6-fold difference between IP and Results from this study demonstrate that treatment of PBLs with
NP in monocyte DCF signals between experiments suggesting a pan-PI3K inhibitor, wortmannin, reduced the generation of
that monocyte intracellular ROS signals are measured accurately extracellular ROS to basal levels and resulted in a consistent and
in the assay used here. Thus, by adopting the present procedure, significant reduction in intracellular ROS generation. Wortmannin
we have limited sample-to-sample variability by harmonizing cell treatment has been reported to block essential membrane subunits
integrity, biochemical and physiological activities, which are likely of NOX245,46 and some of these are understood to regulate dif-
to degenerate over time. ferential activation of NOX2 between the respective phagosomal
The differences in expression of the FcγR types and the var- and plasma membranes47. Whether differential activation of
iations in their binding affinity to the structural heterogeneity of NOX2 explains the lower reduction in intracellular compared to
the interacting Fc domains of the opsonizing antibodies41, the extracellular ROS generation or other signaling pathways
COMMUNICATIONS BIOLOGY |           (2023) 6:743 | https://doi.org/10.1038/s42003-023-05118-0 | www.nature.com/commsbio 7
ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-05118-0
contribute to intracellular ROS generation remains to be investi- by anti-malarial antibodies and its significance as a strong pre-
gated. Interestingly, wortmannin did not have any effect on the dictor of protection against clinical malaria has been established
phagocytic uptake of IgG opsonized merozoites, indicating that in two geographically diverse malaria-endemic regions.
mechanisms involved in FcγR dependent phagocytosis are distinct In conclusion, findings from this study have identified neu-
from the downstream PI3K-signaling events involved in the gen- trophils as the dominant cell type for ROS elicited by anti-
eration of ROS. These results agree with previous reports where the merozoite antibodies and have established that a specific FcγR
use of PI3K inhibitors (wortmannin or LY294002) also did not signaling pathway is involved in the generation of ROS pre-
affect phagocytosis38. Collectively these findings suggest that dis- dominantly in neutrophils by protective antibodies against
tinct FcγR signaling mechanisms could be involved in control of malaria. In contrast, monocyte ROS, extracellular ROS, and OP
OP and ROS generation. are not significantly associated with clinical protection in either
In neutrophils, IgG opsonized merozoites through interactions Ghana or India. The relative contribution of ROS as an effector in
with the FcγRIIA and FcγRIIIB upon phagocytosis trigger acti- mediating parasite killing amongst other antibody-triggered leu-
vation of the NOX2 enzyme complex both in the phagosomal as kocyte effector mechanisms remain to be elucidated.
well as plasma membrane, leading to the generation of O2•−,
which is readily converted into H2O2. In the neutrophil phago-
somes, H2O2 is further converted to HOCl and derivatives Methods
thereof48,49. Inhibition of NOX2 (using DPI) completely abro- Ethics statement. The Ghanaian longitudinal cohort study was approved
gated ROS generation by neutrophils and led to a lower though, (NMIMR-IRB CPN 028/07 −08) by the Institutional Review Board of NoguchiMemorial Institute for Medical Research of the University of Ghana, Accra, Ghana.
significant decrease also in monocytes demonstrating the essential For the Indian longitudinal cohort study approval (ECR/NIMR//EC/2013//93) was
involvements of this enzyme in the ROS generation by blood obtained by the Institutional Ethics Committee of the National Institute of Malaria
phagocytes. Apparently, the minimum reduction of intracellular Research, Indian Council of Medical Research, New Delhi, India, and as part of an
ROS levels in monocytes, even after NOX2 inhibition, could inter-governmental Indo-Danish research program. Written informed consent was
40 given by study participants or their parents/guardians before sample collection.result from its high basal levels . Contrary to expectations, Ethical approval for Danish blood donor samples was given by the Scientific Ethics
inhibition of MPO (using ABAH) had no effect on the DCF Committee of Copenhagen and Frederiksberg, Denmark. Samples from anon-
signal. It is unclear whether this is due to the specificity and/or ymous Danish blood donors (18–60 years of age) obtained for control purposes at
sensitivity of the DCFH2-DA dye used for ROS detection in the Copenhagen University Hospital were used. These individuals are residents of
central Copenhagen and provided written consent to have a small portion of their
assays. Regardless of the enzymes involved in generating ROS and blood stored anonymously and used for research purposes. All data were analyzed
downstream halogen species, the role of such compounds in the anonymously.
elimination of malaria parasites requires further investigation.
The role of ROS in providing naturally acquired protection
against febrile malaria was investigated using samples and data Study populations and study designs. Population characteristics are shown in
from two well-established LCS conducted in Ghana and Supplementary Table 1. Samples and clinical data were from a longitudinal cohort
33,50 study performed in Asutsuare, Damgbe, West District, Ghana
50,52. In May of 2008,
India . Results from this study identified neutrophils as the 798 children aged 12 years and younger were enrolled and venous blood was
primary blood phagocyte contributing to IgG-mediated ROS obtained. The participants were followed up for 42 weeks for active and passive
generation upon exposure to malaria merozoites. Neutrophil- detection of malaria and symptoms. By the end of the study, the cohort was divided
derived ROS accounted for 66 and 74% reductions in the prob- into three groups: (1) those susceptible, in which parasitemia was associated with
ability of developing febrile malaria in the Ghanaian and Indian febrile malaria (axillary temperature ≥37.5 °C measured or reported and at least 1other sign of malaria such as vomiting, diarrhea, or malaise); (2) those apparently
LCS, respectively, after considering the confounding effects of protected against clinical manifestation despite parasitemia; and (3) those without
age. This finding is in agreement with a previous study demon- detectable parasitemia by microscopy and no clinical manifestations of malaria. Only
strating that extracellular ROS generated by purified neutrophils children in groups 1 and 2 were included in the present study as they were definitively
was signi cantly associated with protection from clinical malaria exposed to P. falciparum infections. It should be noted that 108 samples werefi
18 available for the present study compared to the 140 samples previously reported inin two distinct areas of Senegal and underlines the importance the same cohort10. Individuals were classified either as protected (no cases of febrile
of neutrophils in NAI10,16,18. malaria during follow-up) or susceptible (one or more cases of febrile malaria).
Intracellular ROS has been linked to phagocytosis both in The Indian study was conducted in Dumargarhi in the state of Jharkhand33. A
neutrophils and monocytes; thus, we compared individual DCF total of 945 individuals aged 1 to 82 were enrolled in May 2014. Blood samples
from 386 individuals were obtained in the first cross-sectional survey and
signals obtained here with previous data on merozoite-OP from participants were followed up actively and passively for malaria detection for
the Ghanaian LCS10. Neutrophil ROS activity significantly (LRT, 13 months. In the current study, 121 participants who were slide-positive for P.
P= 0.0006) contributed to the protective immunity observed, falciparum at any of the surveys or who has suffered from a febrile malaria episode
suggesting that though linked, neutrophil-derived ROS and OP were included. These are the same samples used in a previously published study
10.
Febrile malaria was defined as any P. falciparum parasitemia confirmed by
involve distinct operational mechanisms. Extracellular ROS, as microscopy of stained thick and thin blood smears plus reported fever or axillary
detected by isoluminol, was not strongly associated with neu- temperature ≥37.5 °C at the time of the visit. Pools of hyperimmune plasma (IP)
trophil or monocyte OP activities, making the exact role of from malaria-exposed Liberian adults and from non-immune plasma (NP) from
secreted ROS elusive. Previous studies have demonstrated that Danish blood donors never exposed to malaria served as internal controls53.
neutrophils stimulated with NOX2-activators produced com-
pounds that inhibited the asexual intraerythrocytic P. falciparum
19,51 Parasite culture and merozoite isolation. P. falciparum strain NF54 were cul-growth in vitro . However, scavengers such as CAT and SOD tured as previously described in ref. 11. Parasites were cultured in O+ erythrocytes
did not reverse the parasite growth inhibition implying that ROS at 4% hematocrit with parasite growth medium (RPMI-1640 supplemented with
was not the main mediator of this parasite killing. It may be 25 mM HEPES, 5 g/l AlbuMAX, 4 mM L-glutamine, 0.02 g/l hypoxanthine, and
speculated that ROS acts in an indirect manner through the 25 μg/ml gentamicin). The culture was kept at 37 °C in an atmosphere containing
activation of secretory granule releasing of toxic mediators with 5% O2, 5% CO2, and 90% N2. Parasitemia and developmental stages were mon-itored with thin blood smears observed by light microscopy. Smears were fixed
anti-parasite activity. The conditions that favor these different with methanol and stained with 10% Giemsa for 10 min. Parasites were synchro-
immune mechanisms remain to be understood; however, their nized by treating with 5% sorbitol for 10 min. After reaching the mature tropho-
interconnectedness seems plausible, reinforcing the perception zoite/early schizont stage, parasites were purified with a magnetic separation
that NAI against malaria is a composite of multiple mechanisms. column and cultured further in the parasite growth medium. Mature schizontswere then filtered through a 1.2 μm pore filter to obtain merozoites. Different
To our knowledge, this is the first time that PBLs have been batches of merozoites were produced for each experiment performed in a day and
used to monitor ROS generation through FcγR signaling elicited used at a leukocyte-to-merozoites ratio of 1:4.
8 COMMUNICATIONS BIOLOGY |           (2023) 6:743 | https://doi.org/10.1038/s42003-023-05118-0 | www.nature.com/commsbio
COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-05118-0 ARTICLE
Leukocytes purification. PBLs were isolated from blood samples of healthy Cytochalasin D treatment (CytD). PBLs in 100 μl of cell medium per well were
Danish donors (n= 12). All blood samples were collected within 2–4 h before use. pretreated for 30 min with 20 µg/ml cytochalasin (CytoD), an inhibitor of actin
The PBL-containing layer was obtained by centrifugation at 830 × g for 25 min at polymerization, and the experiments continued as described above for the
room temperature. Red blood cells remnant were lysed by mixing with 10 parts of OP assay.
lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA; pH 7.4) for
10 min. Cells were then centrifuged at 400 × g for 5 min and resuspended in cell Enzyme inhibition. Before the addition of opsonized merozoites, cells were incu-
medium (RPMI-1640 supplemented with 25 mM HEPES, 10% fetal bovine serum, bated for 1 h at 37 °C with 500 μM of MPO inhibitor, 4-aminobenzoic hydrazide
4 mM L-glutamine, and 25 μg/ml gentamicin) for intracellular ROS quantification (ABAH, Merck A41909); 2.5 μM of NOX2 inhibitor, diphenyleneiodonium
or Krebs–Ringer phosphate buffer (119 mM NaCl, 4.75 mM KCl, 0.420 mM CaCl2, chloride (DPI, Merck D2926); 250 nM of PKC inhibitor, Gö 6983 (Merck G1918);
1.19 mM MgSO4, 16.6 mM sodium phosphate buffer, and 5.56 mM glucose; pH 100 nM of P13K inhibitor, Wortmannin (Merck W1628) or a combination of
7.4) for extracellular ROS quantification. Cells were counted using a hemocyt-
5 these. After the incubation period, merozoites were added and the assays continuedometer and adjusted to 5 × 10 cells/ml and 5 × 106 cells/ml for intracellular and as previously described.
extracellular ROS quantification, respectively.
Neutrophil purification was performed as previously described10. In brief, a
blood sample from a healthy donor was layered on an isotonic Percoll solution Statistics and reproducibility. Each experiment included twelve biological repli-
(1.077 g/ml) and centrifuged at 800 × g for 25 min. Neutrophils in the lower layer cates. The Mann–Whitney test was used to evaluate differences between two
were further purified using the EasySep Human Neutrophil Isolation Kit (StemCell unpaired groups. To assess differences between two groups of paired observations,
Technologies) following the manufacturer’s instructions after lysis of the Wilcoxon signed-rank test was used. The Friedman test with Dunn’s multiple
contaminating red blood cells. Purified neutrophils were adjusted to 5 × 105 cells/ comparisons test was applied to estimate differences between three or more paired
ml using a cell medium. groups. Individuals were stratified into two equal groups (low or high) based on the
median DCF or isoluminol signals. The associations between time to the first
febrile malaria episode and these categorical variables were analyzed by age-
Intracellular ROS quantification. PBLs were distributed in 96-well U-bottom adjusted Cox-regression models. Correlations were assessed with the Pearson
plates containing 5 × 104 cells in 100 μl of cell medium per well. Then, 50 μl of cell correlation coefficient. The significance of the effect of ROS production was
medium with 12 μM of 2’,7’-dichlorodihydrofluorescin diacetate (DCFH2-DA, assessed by likelihood ratio tests (LRT) comparing a logistic regression model that
3 μM final concentration) and 1:200 dilution of surface staining antibodies (1:800 included age and either monocyte ROS or extracellular ROS as predictors, as well
final dilution) were added. The staining antibodies were APC anti-human CD45 as age and merozoite-phagocytosis with a second model that also included the ROS
(clone HI30; BD Biosciences 555485), BV421 anti-human CD66b (clone G10F5; values. All analyses were performed as two-sided tests. P values less than 0.05 were
BD Biosciences 562940), and APC-AF750 anti-human CD14 (clone TuK4; Thermo considered significant. Statistical analyses were performed with GraphPad Prism 9
Fisher Scientific MHCD1427) selective for leukocytes, granulocytes, and mono- (GraphPad Software, Inc.) and R version 4.1.2 with the survival and lmtest
cytes, respectively. Immediately, 50 μl of merozoites resuspended in cell medium packages54,55.
and opsonized with immune or non-immune plasma diluted 1:100 was added to
each well. After an incubation of 30 min at 37 °C, cells were washed thrice with
FACS buffer (PBS with 0.5% BSA+ 2 mM EDTA). Samples were quantified with a Reporting summary. Further information on research design is available in the Nature
CytoFLEX S (Beckman Coulter Life Sciences) flow cytometer. DCF signal was Portfolio Reporting Summary linked to this article.
determined by measuring the fluorescence using the 525/40 nm detector. Data
analysis was performed with Kaluza Analysis Software version 2.1 (Beckman
Coulter Life Sciences). The gating strategy used is shown in Supplementary Fig. 1.
Data availability
Extracellular ROS quantification. PBLs were dispensed in 96-well white opaque The data generated in this study are provided in the Supplementary Data file. Data were
plates with 5 × 105 cells in 170 μl of Krebs–Ringer phosphate buffer per well. also available from the corresponding authors upon request and pending agreement from
Isoluminol was added to a final concentration of 0.04 mg/ml. Then, 30 μl of relevant ethics committees for clinical data.
merozoites resuspended in Krebs–Ringer phosphate buffer and opsonized with
immune and non-immune plasma diluted 1:50 were added. Chemiluminescence Received: 1 December 2022; Accepted: 7 July 2023;
kinetics were recorded with a TopCount NXT Scintillation and Luminescence
Counter or a SpectraMax i3x multi-Mode microplate reader using the SoftMax Pro
7.1 software for data acquisition and analysis.
Phagocytosis assay (OP). The process for quantification of merozoite-
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