Background
It is estimated that there are currently more than 200 million malaria infections per year, resulting in more than 600,000 deaths, mainly of children under five years old and caused by infection with
Plasmodium falciparum [
1]. In addition, infection with
P. falciparum during pregnancy causes maternal malaria which results in increased incidence of pre-term births, low infant birth weight and maternal anaemia causing significant morbidity and mortality [
2,
3].
Antibody-mediated effector mechanisms against the blood stages of the parasite’s life cycle are important in protection against clinical malaria disease: in malaria-endemic regions, acquisition of antibodies to blood-stage parasites is associated with protection against death due to severe malaria by five years of age and with protection against clinical malaria by early adulthood [
4]. Important targets of protective antibodies are antigens expressed on the surface of infected erythrocytes (IE) [
5], and the major target of these antibodies is a surface protein known as PfEMP1 [
6]. In addition, acquisition of antibodies to antigens exposed on the surface of IE that adhere and accumulate in the placenta, and express the PfEMP1 variant known as Var2CSA, occurs in a gravidity-dependent manner and is associated with protection against maternal malaria as well as negative outcomes such as anaemia and low birth weight [
7‐
11].
The effector cells most likely to mediate protective effects of antibodies against circulating blood stage parasites are monocytes, which phagocytose IE [
12]. They can also accumulate as malaria pigment-laden cells in the placentas of malaria-infected pregnant women [
13‐
15]. Monocytes phagocytose IgG-opsonised IE via Fcγ receptor-mediated mechanisms [
16,
17] and secrete both pro-inflammatory and anti-inflammatory cytokines and growth factors in response to parasite ingestion which may aid in both parasite clearance and in limiting inflammation [
18,
19]. Circulating human monocytes exist as separate subsets which are identified by their expression of CD14 (the co-receptor for Toll-like receptor 4 (TLR4) recognition of bacterial lipopolysaccharide) and CD16 (FcγRIIIa: a receptor for IgG). The current convention is to define three subsets of human monocytes: classical (CD14
hiCD16-), non-classical (CD14
loCD16+) and intermediate (CD14
hiCD16+) monocytes [
20]. The biological properties of these subsets are governed by differing expression of pattern recognition and chemokine receptors. CD14
hiCD16- classical monocytes represent the major population in blood, respond strongly to bacterial products via TLR4 and infiltrate into sites of inflammation in response to the chemokine CCL2 [
21]. CD14
loCD16+ non-classical monocytes may patrol blood vessel walls and respond to viral ligands via TLR7/8. They express high levels of fractalkine receptor (CX3CR1) but migrate in response to multiple chemokines [
21]. CD14
hiCD16+ intermediate monocytes may represent a transitional form of the maturation of classical monocytes into non-classical monocytes and respond strongly to both viral and bacterial ligands [
22]. The role of different monocyte subsets in settings of parasite infection is not known.
While it is recognised that a successful vaccine strategy must generate a robust antibody response to blood stage parasites, the desired functional activities required for protective immunity are less clear. Evidence is accumulating that the ability of antibodies to promote opsonic phagocytosis of blood stage parasites is an important component of immunity [
23‐
26]. However, the major cell subsets mediating phagocytosis and the underlying mechanisms are poorly understood. This knowledge may be crucial for the development of highly protective vaccines. Monocyte phagocytosis of blood-stage malaria parasites has been previously studied using peripheral blood mononuclear cells or purified monocytes which miss interactions between serum components, uninfected erythrocytes and the phagocyte, and have not usually considered individual monocyte subset responses. Here we use a whole blood phagocytosis assay [
27] to show for the first time that CD14
hiCD16+ intermediate monocytes have a much greater phagocytic activity towards trophozoite stage malaria parasites than other monocyte subsets. Our results uncover an essential role for Fcγ receptor IIIa (CD16a) and show that complement opsonisation is required for IgG-mediated phagocytic uptake under physiological conditions and contributes to the high activity of intermediate monocytes against IE.
Methods
Ethics approval
Blood was obtained by venepuncture with informed consent from healthy volunteers without a history of malaria infection using protocols approved by The Alfred Hospital Research and Ethics Unit. Five serum samples with high IgG reactivity to CS2 IE were pooled from samples collected from pregnant women in an endemic region of Papua New Guinea [
28]. All women gave informed written consent and ethics approval was provided by the Medical Research Advisory Committee, PNG).
Parasite culture and purification
P.
falciparum laboratory lines CS2 [
29] and E8B [
30] were grown in human erythrocytes (group O, Rh
+, Australian Red Cross Blood Service) at 37 °C with 5 % CO
2 suspended in RPMI-HEPES medium supplemented with 50 μg/ml hypoxanthine, 25 nM NaHCO
3, 20 μg/ml gentamicin, 5 % heat inactivated pooled human serum and 5 % Albumax. Gelatin enrichment of knob-expressing IE was performed weekly, and IE were synchronised weekly by resuspension of culture pellets in 5 % sorbitol in water to lyse trophozoite and schizont IE. Mature pigmented trophozoite stage IE were enriched by centrifugation over Percoll gradients to >80 % purity as assessed by counting Giemsa-stained thin blood smears by microscopy.
Infected erythrocyte labeling and opsonisation
IE were opsonised at a concentration of 5 x 10
7 trophozoites/mL for 30 minutes at room temperature with rabbit anti-human erythrocyte antibodies (Cappel, MP Biomedicals, LLC; Santa Anna, CA, USA) using a sub-agglutinating 1/800 dilution of antibody in phosphate-buffered saline (PBS). In some experiments CS2 IE were opsonised with 20 % human immune serum from a pool of sera prepared from pregnant woman with placental malaria enrolled in the VT cohort in Papua New Guinea [
31]. Opsonised cells were washed in fluorescence-activated cell sorting (FACS) wash buffer (PBS, 2 % new born calf serum) and resuspended in PBS (2 x 10
8/mL) then stained with 10 μg/mL ethidium bromide (EtBr) for 30 minutes at room temperature. Following labeling with EtBr, cells were washed three times with cold FACS wash buffer and used immediately.
Whole blood phagocytosis assay
A sample of 5 mL whole blood was collected from healthy volunteers into lithium heparin blood collection tubes by venepuncture and analysed within two hours of collection. Aliquots of 50 μL whole blood were placed in polypropylene FACS tubes, then 1 x 107 EtBr-labeled IE were added. This is a ratio of approximately 200 IE per peripheral blood mononuclear cell. Cells were incubated for 30 minutes at 37 °C, or on ice as a control. After phagocytosis, cells were lysed with 3 mL 0.2 % ammonium chloride for five minutes at 22 °C, then washed with 3 mL cold FACS wash buffer. Supernatant was removed and cells were resuspended in 100 μL PBS. Cells were stained with antibodies for 30 minutes on ice, washed, then fixed with 2 % formaldehyde and analysed immediately by flow cytometry using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Monocyte subsets were identified by staining with anti CD14 APC (M5E2, BD Biosciences) and CD16 FITC (3G8, BD Biosciences), and phagocytosis was determined by measuring EtBr fluorescence in the PE channel. Gates were set using samples incubated with unopsonised, EtBr-stained IE at 37 °C. Flow data were analysed using FlowJo (version 8, Tree Star Inc.). For blocking experiments, blood was pre-incubated with the indicated concentrations of blocking antibodies for 30 minutes at 4 °C before the addition of IgG-opsonised IE. The antibodies used were 3G8 (in house (MH): blocking antibody for CD16), Fab fragment of IV.3 (in house (MH): blocking Ab for CD32a), 10.1 (Santa Cruz Biotechnology, Dallas, TX, USA : blocking Ab for CD64), H1-111 (Biolegend, San Diego, CA, USA: blocking antibody for CD11a), Bear-1 (Abcam, Cambridge, UK: blocking antibody for CD11b) and clone 3.9 (Biolegend: blocking antibody for CD11c). The antibody used to measure C3b deposition of RBC was FITC-conjugated goat fraction to human complement C3, (MP Biomedical 0855167). To measure the effect of inhibiting complement activation on phagocytosis, compstatin (R&D Systems, Minneapolis, MN, USA) was added to whole blood, from stock solutions (2 mg/ml) prepared in PBS, to a final concentration of 0–50 μM, and incubated for 15 minutes on ice before addition of iRBC and transfer to 37 °C to measure phagocytosis.
Monocyte phenotyping
Aliquots of peripheral blood mononuclear cells (PBMC) from malaria-naive healthy individuals recruited in Melbourne were incubated with predetermined saturating concentrations of the relevant antibodies. Monocytes were gated using forward and side scatter and monocyte subsets identified with CD16 PE Cy7 (3G8, BD Biosciences) and CD14 BV510 (M5E2, Biolegend) or APC (M5E2, BD Biosciences). antibodies used were: CD16 PE Cy7 (3G8, BD Biosciences), CD32a (IV.3 biotinylated Fab fragment + streptavidin APC), CD32b (in house (MH), 63X-21 biotinylated whole IgG + streptavidin APC) [
32], CD64 PerCP 5.5 (10.1, Biolegend), CD11a Alexa-488 (HI111, Biolegend), CD11b APC (ICRF44, Biolegend), activated CD11b (CBRM1/5-FITC, Biolegend), CD11c V450 (B-ly6, BD Biosciences), CD35 FITC (E11, Biolegend).
Intracellular TNF measurements
Phagocytosis of infected red blood cells (iRBC) was performed using 100 μL aliquots of whole blood as described above except that iRBC were not labeled with EtBr. A total of 20 μg/mL brefeldin A and 10 μM monensin was added and the cells incubated for four hours at 37 °C, stained with CD14 APC and CD16 PE Cy7 (30 minutes on ice), then permeabilised (Perm/Wash Buffer 1, BD Biosciences). After 10 minutes on ice, cells were stained with αTNF phycoerythrin (PE) (Mab11, BD Biosciences) for 30 minutes, washed and fixed.
Imaging flow cytometry
IE were stained with PKH26 (Sigma-Aldrich, Castle Hill, NSW, Australia) according to the manufacturer’s instructions. Aliquots of whole blood (50 μL) were incubated with 5 x 106 PKH26-stained CS2-IE for 15 minutes, and processed as above for whole blood phagocytosis except that cells were stained with CD14 Pacific Blue (M5E2, Biolegend) and CD16 PE Cy5 (3G8, BD Biosciences). Samples were acquired using an ImageStream 100 imaging flow cytometer and analysed using IDEAS software.
Discussion
Using a whole blood phagocytosis assay we studied the properties of phagocytes under conditions resembling those
in vivo as closely as possible. We show that although the CD14
hiCD16- classical and CD14
hiCD16+ intermediate monocyte subsets efficiently phagocytose IE in PBMC preparations, only the CD14
hiCD16+ subset does so in whole blood. While the CD14
hiCD16+ subset is more efficient at phagocytosis on a per cell basis, the greater number of CD14
hiCD16- monocytes suggests that they may also phagocytose significant numbers of IE in vivo. We show for the first time that phagocytosis of IgG-opsonised IE required complement opsonisation and was strongly inhibited by inhibitors of complement activation. Antibody blocking experiments showed that in whole blood phagocytosis required expression of the Fcγ receptor CD16, but not CD32a or CD64. Thus, CD16 is necessary but not sufficient for phagocytosis since non-classical monocytes, which also express CD16, failed to phagocytose IE efficiently. This is likely due to their lower expression of CR1, 3 and 4 and lower levels of activated CR3. Complement opsonisation of IE occurred rapidly
in situ during the assay, primarily via the classical pathway as we did not detect complement deposition on IE in the absence of IgG opsonisation. In whole blood of children with malaria infection, IE have C3b and C4 deposited on their surface [
36] but it is not clear whether the limiting factor for efficient phagocytosis is the extent of complement fixation, opsonisation with IgG or both. Our findings show that evaluation of immunity in vaccine trials must also take into account the ability of the elicited antibodies to fix complement as well as to promote phagocytosis. The use of the CS2 strain in this study gives relevance to anti Var2CSA vaccines currently under development for pregnancy-associated malaria and currently funded through to clinical trials. It is essential to understand how antibodies to Var2CSA function in order to design a vaccine with maximum efficacy and to evaluate responses in such trials.
We propose that co-operation between CD16 and complement receptors are required in whole blood for phagocytosis of IgG opsonised IE. Multiple complement receptors are probably involved although a likely partner for CD16 is CR3 (CD11b/CD18) which was more activated on intermediate monocytes than on other monocyte subsets, and partial inhibition of phagocytosis occurred with the blocking antibody Bear-1. CD16 is known to interact with CR3 on monocytes, enhancing its ability to bind iC3b [
37]. The selective role of CD16 expressed on monocytes may be due to specific interaction with CR3 and/or signaling differences between it and CD32a. This latter could be caused either by differences in the ITAM motifs located on the FcRγ signaling protein associated with CD16a and that in the cytoplasmic domain of CD32a, or by signaling pathways activated following phosphorylation of the cytoplasmic domain of CD16 [
38].
Our data, primarily obtained using IE opsonised with rabbit anti-human RBC antibody, were verified using IE opsonised with human immune serum. Although we did not explore the human IgG isotypes required, studies have shown that anti-malaria antibodies promoting phagocytosis are mainly cytophilic IgG1 and IgG3 [
39,
40]. We used two separate laboratory-derived
P. falciparum lines, CS2 and E8B which were both more efficiently ingested by CD14
hiCD16+ monocytes. These parasite lines express PfEMP1 adhesion molecules on the surface of the IE that bind with different ligand specificities. Thus, CS2 binds preferentially to chondroitin sulfate A [
29] while E8B binds to both CD36 and ICAM-1 [
34]. Our observations, therefore, rule out involvement of these receptors in the increased ability of CD14
hiCD16+ monocytes in phagocytosis.
Imaging flow cytometry verified that under the conditions of our experiments ingestion of IE and not surface binding were measured. This is important since we observed that fragments of bound RBC may remain attached to monocytes following hypotonic lysis of the uningested RBC which can lead to unacceptably high backgrounds when membrane stains are used to label target cells. We found that labelling of the parasite DNA within IE using EtBr was the best approach to use for measurement of IE phagocytosis, but it has the disadvantage that analysis must be performed within 30–60 minutes to avoid loss of the EtBr stain. This lessens the utility of the whole blood assay used here in clinical settings.
Our data have uncovered an important role of CD16 expressed on monocytes in responses to IE. Monocytes express the transmembrane receptor CD16a in contrast to neutrophils, which express the GPI-linked receptor CD16b. Although CD16a and CD16b have nearly identical extracellular domains, they are encoded by separate genes [
41]. Polymorphisms of CD32a and CD16b are associated with malaria severity [
42‐
45] that may reflect the ability of splenic macrophages and neutrophils, respectively, to clear opsonised parasites or, in the case of associations with severe anemia, to ingest uninfected RBC. Several polymorphisms in CD16a affect either affinity for cytophilic IgG subclasses [
46] or monocyte expression [
47]; however, to our knowledge no studies have attempted to associate these or other CD16a polymorphisms with malaria severity or vaccine responses. Given the absolute requirement of IE phagocytosis for CD16 expressed on monocytes revealed herein, such studies are indicated.
The ability of monocytes to phagocytose IE in whole blood is decreased relative to that of PBMC preparations, highlighting the caution that must be employed when interpreting results using PBMC or purified monocytes in phagocytosis assays. This may reflect the presence of inhibitory factors in serum or the large number of uninfected RBC in whole blood. Uninfected red blood cells inhibited ingestion of IgG-opsonised trophozoite stage IE consistent with observations of others using unopsonised schizont-stage IE [
35]. We found similar inhibition of all three monocyte subsets, however, suggesting that this is unlikely to underlie their differing phagocytic ability. Interestingly, human RBC membranes contain an inhibitory factor (phagocytosis-inhibitory factor, PIF) which appears to affect CR3 conformation and inhibits ingestion of both C3bi and IgG opsonised latex beads [
48]. We also provide evidence that phagocytosis by classical and intermediate monocytes in whole blood is lower than that by these cells in PBMC because of an inhibitory effect of soluble plasma components. It is possible that binding of IgG present in serum to Fcγ receptors may contribute to this, since removal of IgG using protein G sepharose beads reduced inhibition by added plasma (Additional file
4: Figure S4).
Elevated numbers of CD14
hi monocytes which co-express the chemokine receptors CCR2 and CX3CR1 have been associated with lower parasitemia and increased ADCI activity in
P. falciparum-infected individuals with uncomplicated malaria [
49]. These cells may define a specific intermediate monocyte population with an important protective role against blood stages of the parasite. It would be of interest to directly compare ADCI activity of different monocyte subsets and the phenotype of monocytes with high phagocytic and high ADCI activity to determine if the same populations are involved. A selective role for CD14
hiCD16+ monocytes in the response to
P. vivax-infected reticulocytes has recently been published [
49]. This report differs from our study in that increased phagocytosis by CD14
hiCD16+ monocytes was observed using PBMC preparations incubated for long times (four hours) with purified, infected reticulocytes. In our hands, discrete monocyte subsets cannot be identified after four hours incubation of whole blood with phagocytic targets since CD16 expression on CD16+ monocytes is decreased. A second difference between the two studies is that Antonelli et al. [
50] used PBMC prepared from patients with active
P.vivax infection which were shown to be in an activated state, whereas we have studied responses of individuals with no history of malaria infection and who therefore represent individuals at risk of primary infection. The differences between the two studies may also be due to the different phagocytic targets (
P. falciparum-infected erythrocytes versus
P. vivax-infected reticulocytes) used in the two studies. Nevertheless, both studies point to an important role for CD14
hiCD16+ monocytes in the control and response to blood-stage malaria infection.
In summary, our data show a special role for CD14hiCD16+ monocytes in the phagocytosis of trophozoite-stage P. falciparum IE. The use of whole blood assays to measure phagocytosis in place of purified PBMC or monocytes reveals that efficient phagocytosis requires both IgG opsonisation and complement components in situ. Evaluation of vaccine candidates is increasingly employing functional assays such as phagocytosis assays to determine correlates of immunity. Ideally such assays should use fresh whole blood, although this may not be feasible in some field settings. Our data suggest that care must be taken in interpreting the results of assays using purified cells and that the ability of antibodies to fix complement are likely to be as relevant as opsonic activity.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JZ performed the research with help from GF and analysed the data. YY performed research and helped analyse and interpret the image stream data. AJ designed the research and analysed the data and wrote the manuscript with help from SR. MH and JB provided vital reagents and contributed to the manuscript. All authors read and approved the final manuscript.