Malaria is a life-threatening disease transmitted by mosquitoes infected with Plasmodium parasites. Part of the parasite life cycle happens inside human red blood cells. The surface of an infected red blood cell is coated with parasite proteins, which attract the attention of white blood cells called monocytes. These immune cells circulate in the bloodstream and use a process called phagocytosis to essentially 'eat' any infected cells they encounter. However, the monocytes cannot always reach the infected cells.

Some of the proteins made by the parasites make the infected red blood cells stickier than normal. This allows the infected red blood cells to surround themselves in a protective cage of uninfected red blood cells. Known as “rosettes” because of their flower-like shape, these cages seem to protect the infected cells from attack by the immune system. Lee et al. noticed that adding white blood cells to parasite-infected red blood cells made them clump together more, but it was unclear exactly how and why this happened.

To find out, Lee et al. took fluid from around monocytes grown in the laboratory and added it to red blood cells infected with Plasmodium parasites. This made the cells clump together, suggesting that something in the fluid may potentially be alerting the parasites to impending immune attack. The fluid contained almost 700 different molecules, and Lee et al. narrowed down their investigations to the five most likely candidates. Interfering with the activities of these five proteins revealed that one – a protein IGFBP7 – not only alerted the parasites but also helped them to form the rosettes. It turns out that the parasites appear to use IGFBP7 like a bridge, linking it to two other human proteins to stick red blood cells together. Once the rosettes had formed, the monocytes were unable to eat the infected blood cells by themselves. Instead several monocytes had to work together as a team to consume the whole rosette.

Further research is now needed to shed light on this interaction between malaria parasites and human cells. Such research would be particularly relevant in the clinical setting, since some previous studies has linked the forming of rosettes to the severity of disease for malaria.

Along its intraerythrocytic development, Plasmodium spp. modifies the infected erythrocyte (IRBC) rheology (increased rigidity for P. falciparum IRBC, reduced rigidity but increased fragility for P. vivax IRBC). Such alteration increases the susceptibility of IRBC to splenic clearance (Handayani et al., 2009; Chotivanich et al., 2002); however, the parasites have developed escape strategies to avoid splenic elimination. For instance, P. falciparum expresses adhesins on the IRBC that mediate adhesion to the endothelial cells, resulting in deep microvasculature sequestration (Sherman et al., 2003). Additionally, an IRBC can bind directly to uninfected red blood cells (URBC) to form a ‘rosette’ (Russell and Cooke, 2016). This rosetting phenomenon has been described in all human malaria parasites (Angus et al., 1996); however, the functional importance of rosetting remains ambigous (Clough et al., 1998; Wahlgren et al., 1989). While the supposed role of rosetting in facilitation of merozoite invasion of URBC is unlikely; recent studies show that rosette formation may have a role in parasite immune-evasion (Clough et al., 1998; Deans and Rowe, 2006; Lee et al., 2014; Zhang et al., 2016; Moll et al., 2015; Lee et al., 2019). Theoretically, the masking of rosetting IRBC with URBC may hamper IRBC recognition and therefore their clearance by the host immune system (Moll et al., 2015). Notably, rosetting has been associated (in some but not all studies) with disease severity (Treutiger et al., 1992; Carlson et al., 1994; Rowe et al., 1995; Doumbo et al., 2009; al-Yaman et al., 1995; Ho et al., 1991). Here, we observed that the addition of leukocytes increased the rosetting rates of various P. falciparum and P. vivax isolates. We next demonstrated that IRBC stimulated monocytes to secrete products capable of stimulating rosetting, the most important being insulin growth factor binding protein 7 (IGFBP7). We further showed that IGFBP7-mediated rosetting was different from the previously described rosetting (defined here as type I rosetting), where it (we refer to this as type II rosetting) required additional serum factors to occur, in addition to the interaction between the parasite-derived ligand on IRBC surface and the receptor on the surface of URBC. Functionally, we observed that the IGFBP7-mediated type II rosetting reduced phagocytosis by monocytes, and therefore defined a new escape mechanism for the malaria parasites.

The key resources table is available as Supplementary file 1.

Effects of human leukocytes on rosetting

P. falciparum- and P. vivax-IRBC formed rosettes (Figure 1A, top panel) in the presence of 20% autologous human serum and the extent of rosetting at baseline is variable depending on the parasite isolates (Lee et al., 2014). When autologous blood leukocytes were incubated with clinical isolates, the rosetting rate increased by 10–40% depending on individual isolates for both parasite species (Figure 1B). This effect was mediated primarily by monocytes (Figure 1C). To exclude donor variability as a confounding factor, we repeated the experiment with the THP-1 cell line, which was derived from the peripheral circulation of an acute monocytic leukaemia patient (Auwerx, 1991). The rosette-stimulation mediated by THP-1 was similar to that of peripheral monocytes (Figure 1D). Addition of undifferentiated THP-1 (UT) and macrophage-like THP-1 (MT) increased rosetting rates in a dose-dependent manner. MT were more potent than UT, where the difference increased with the number of cells added (Figure 1E).

Figure 1

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Figure 1—source data 1

Raw data (rosetting rates, %) for the data set presented in the bar graph (Figure 1D).

R = biological replicate (same parasite line, but different flasks of cultures, using the same URBCs and culture media).

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Effect of THP-1 culture supernatants and supernatant fractionation

Culture supernatants (CS) of both THP-1 cell types showed similar rosette-stimulation effects (Figure 1F), with CSMT exerting a higher degree of rosette-stimulation than CSUT. Fractionation of CSMT into aqueous and lipid fractions revealed that the rosette-stimulating factors were in the aqueous fraction (Figure 2A). Subsequently, we further fractionated the aqueous fraction into high and low molecular weight sub-fractions (with a cut-off of 30 kDa). Both aqueous sub-fractions induced rosetting of P. falciparum and P. vivax (Figure 2B and C), demonstrating that the rosetting stimulation was mediated by multiple secreted hydrophilic factors, predominantly of sizes ≤ 30 kDa (particularly for P. falciparum). Heating of CSMT’s ≤ 30 kDa aqueous fraction at 56°C for 1 h did not completely abolish its rosette-stimulating effect (Figure 2D), indicating the presence of heat-stable factors. To further investigate the potential stimulating factors, mass spectrometry analysis was performed on this fraction. We identified 694 proteins (Supplementary file 2), of which complement factor D (CFD), insulin-like growth factor binding protein 7 (IGFBP7), nidogen 1 (NID1), hyaluronan-binding protein 2 (HABP2), and periostin (OSF2) were shortlisted (Supplementary file 3). Apart from having high scores in the mass spectrometry analysis (Supplementary file 2), these molecules were selected because they are secreted proteins and possess cell adhesion-related properties associated with pathological changes such as vascular injuries, inflammation, cancer development, and leukocyte recruitment (UniProt Consortium, 2015; St Croix et al., 2000; Mambetsariev et al., 2010; Ulazzi et al., 2007; Schwanekamp et al., 2016; Lindner et al., 2005; Markiewski and Lambris, 2007).

Figure 2

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Antibody neutralization assay

Antibody neutralization of IGFBP7 significantly reduced (by ~40%) the rosette-stimulation by CSMT for both parasite species (Figure 3A). Rosette-stimulation by CSMT was also reduced by anti-CFD (Figure 3B) and anti-OSF2 (Figure 3C) antibodies, albeit to a lesser extent. Antibodies against NID1 (Figure 3D) and HABP2 (Figure 3E) had no effect on CSMT-mediated rosette-stimulation. As anti-IGFBP7 had the largest inhibitory effect, further experiments focused on IGFBP7.

Figure 3

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Effect of IGFBP7 on rosetting

Addition of human recombinant IGFBP7 to leukocyte-free parasite culture stimulated rosette formation in a dose-dependent and satiable manner for P. falciparum and P. vivax isolates, reaching a plateau at 100 ng/ml (Figure 4A and B). For P. falciparum, no significant difference in rosette-stimulatory effect between the CSMT and IGFBP7 was found (Figure 4C). On the contrary, P. vivax rosette-stimulation by CSMT was significantly higher than that by recombinant IGFBP7 alone (Figure 4D). The rosette-stimulating capacity of IGFBP7 was abolished by heat denaturation at 95°C for 1 h (Figure 4E and F), thus eliminating the possibility that the effect was caused by a non-protein contaminant in the recombinant protein preparation. Furthermore, the IGFBP7 binding-inducing effect was only observed when URBC and IRBC were present in the culture. Incubation of URBC alone with IGFBP7 did not induce clustering effect on the cells (Figure 4—figure supplement 1; top panel). IGFBP7 required a minimum of 15 min to significantly stimulate rosetting. However, the rosette-stimulating effect did not significantly increase further afterwards (Figure 4G). Importantly, IGFBP7-mediated rosetting was a reversible event, where removal of the protein from the system reverted the rosetting rates to their baseline values (rosetting rates recorded prior to IGFBP7 exposure) as fast as 15 min post-protein removal (Figure 4H).

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Parasite-derived rosetting ligands essential to IGFBP7-mediated rosetting

Trypsinization of IRBC abrogated the rosette-stimulating effect of IGFBP7 (Figure 5A), suggesting the involvement of parasite-derived proteins expressed on the surface of IRBC. P. falciparum expressed different surface antigens including rosetting ligands such as the P. falciparum erythrocyte membrane proteins 1 (PfEMP1), STEVOR, and RIFIN (Chan et al., 2014). The sensitivity to low level of trypsin treatment (10 µg/ml) used here suggested that PfEMP1, rather than STEVOR or RIFIN, was likely the adhesin involved (Kyes et al., 1999; Niang et al., 2014). We further validated this hypothesis with use of the P. falciparum SBP1-KO-CS2 line, in which the skeleton binding protein 1(SBP1) gene was knocked out. SBP1 transports PfEMP1 from the parasitophorous vacuole to the Maurer’s cleft for subsequent assembly and export to the surface of IRBC. Therefore, the SBP1-KO-CS2 line is unable to express PfEMP1 on the surface of the IRBC. However, knock out of this gene does not affect the surface expression of STEVOR and RIFIN (Maier et al., 2007; Chan et al., 2016). IGFBP7 increased the rosetting rate of CS2 wild-type parasite but had no effect on SBP1-KO-CS2 rosetting (Figure 5B). Subsequently, we used two other clones of the P. falciparum NF54 line, NF54 VAR2CSA_WT clone and the mutant clone, NF54_T934D, whose PfEMP1 variant VAR2CSA is not exported onto the surface of the IRBCs (Dorin-Semblat et al., 2019). The rosetting machinery of NF54_VAR2CSA_WT responded positively to the presence of IGFBP7. On the other hand, the rosetting rates of NF54_T934D clone (lacking PfEMP1 on the surface of IRBC) were not significantly altered by IGFBP7 (Figure 5C). Of note, IRBCs with surface expression of PfEMP1 variant VAR2CSA (which include the CS2_WT and NF54_VAR2CSA_WT) did not form many rosettes, which was in parallel with earlier report (Rogerson et al., 2000). In addition, the rosettes formed by NF54_VAR2CSA_WT (Figure 5C, inset i) and NF54_T934D (Figure 5C, inset ii) were small.

Figure 5

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Figure 5—source data 1

Raw data (rosetting rates, %) for the data set presented in the bar graph (Figure 5C).

R = biological replicate (same parasite line, but different flasks of cultures using the same URBCs and culture media).

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Figure 5—source data 2

Raw data (rosetting rates, %) for the data set presented in the bar graph (Figure 5D).

R = biological replicate (same lab-adapted parasite, but conducted on three cycles of ring stages from three different flasks of cultures using the same batch of URBCs and culture media). F = flask, C = cycle.

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Expression of parasite-derived, IRBC-surface proteins such as rosetting ligands is sequential and parasite stage-specific. For example, PfEMP1 is the first rosetting ligand to be expressed on the surface of IRBC (as early as late ring stage), followed by RIFIN, and finally STEVOR (which are at the much more mature stages) (Kyes et al., 2000; Lavazec et al., 2007; Kaviratne et al., 2002; Bachmann et al., 2012; Niang et al., 2009). In other words, PfEMP1 is the only rosetting ligand available on the surface of late ring-IRBCs. We performed the IGFBP7-rosetting assessment on the late ring stages (~hour 16–26) of a laboratory-adapted clinical isolate (nine replicates across three different cycles) and found that IGFBP7 significantly increased the rates of rosette formation (Figure 5D) by the late ring stages (Figure 5E). Taken together, these results suggest strongly that PfEMP1 is essential for IGFBP7-mediated rosetting in P. falciparum. On the other hand, we could not identify which P. vivax proteins were involved in IGFBP7-mediated rosettes as the P. vivax-IRBC membrane-associated proteins have yet to be fully characterized.

Host-derived rosetting receptors essential to IGFBP7-mediated rosetting

IGFBP7 has a heparin binding domain. We hypothesized that it might be involved in the rosetting effect. When URBC treated with either heparinase I (Figure 6A) or heparinase III (Figure 6B) were mixed with purified P. falciparum- or P. vivax- IRBC, IGFBP7 did not induce rosetting. Heparinases also affected the size of rosettes (numbers of URBC in a rosette) (Figure 6C, green arrows). Notably, addition of IGFBP7 induced autoagglutination-like clustering of IRBC (which were not enzyme-treated) (Figure 6C, red arrow). IRBC-autoagglutination was absent in the control groups (untreated and enzyme-treated groups without addition of IGFBP7) (data not shown).

Figure 6

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Complement receptor 1 (CR1) expressed on URBC is a receptor for PfEMP1 in P. falciparum rosetting (Rowe et al., 1997). However, it did not play a significant role in IGFBP7-mediated rosetting for both parasite species. Anti-CR1 mAb reduced P. falciparum rosetting rates in the absence of IGFBP7, confirming that this molecule is involved in the direct interaction between IRBC and URBC (Figure 6D). However, anti-CR1 mAb had an insignificant effect on IGFBP7-induced rosetting in P. falciparum (Figure 6D). For P. vivax, anti-CR1 antibody did not inhibit rosetting, with or without addition of IGFBP7 to the culture (Figure 6E). Likewise, For P. falciparum and P. vivax (Figure 6F and G), ABO blood groups did not play significant roles in IGFBP7-mediated rosetting, as well as rosetting mediated by other CSMT-derived rosette-stimulators (Figure 6H and I).

Serum-derived co-factors in IGFBP7-mediated rosetting

All the experiments described above were performed using 20% human serum-enriched medium. However, serum filtration with a 0.45 μm filter abolished IGFBP7-induced rosetting (Figure 7A). These results indicated that other large-sized serum-derived protein aggregates or multimers might be needed for the IGFBP7-mediated rosetting effect. We hypothesized that von Willebrand factor (VWF) may be involved as VWF has been reported to absorb onto plasma-exposed surfaces (Grinnell and Phan, 1983; Mannhalter, 1993). Addition of anti-VWF antibody (25 µg/ml) did not significantly alter the baseline rosetting rates (Figure 7B). However, the presence of anti-VWF antibody prevented IGFBP7 from exerting its rosette-stimulatory effect. The specificity of rosette-inhibition by the antibodies was validated with experiments using antibody isotype controls at the same working concentration (25 µg/ml) (Figure 7—figure supplement 1A).

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When we reduced the medium’s serum enrichment to only 2%, IGFBP7 could not increase rosetting rates (Figure 7C). The protein could only exert its rosette-stimulatory effect in 2% serum-enriched medium when VWF was added. Importantly, VWF by itself did not stimulate rosetting (Figure 7C), disputing the possibility of this phenomenon as a non-specific adhesive effect of VWF and indicating that it is a co-factor of the IGFBP7-mediated rosetting.

When the media’s serum enrichment was replaced with Albumax II (a serum substitute), addition of IGFBP7 (100 ng/ml) and VWF did not significantly increase rosetting rates across the VWF concentration range tested (Figure 7D). This suggested the need for other serum-derived factors to mediate the IGFBP7 rosetting effect. We suspected that this second cofactor would either be needed in small quantity or present in high abundance in serum, so that even a 2% serum-supplied medium would be adequate to sustain IGFBP7-mediated rosetting. In addition, this co-factor should be able to interact with some of the players identified above (PfEMP1, HS, VWF) to generate IGFBP7-mediated rosettes. One candidate was thrombospondin 1(TSP-1) as it is known to bind to PfEMP1 (Cooke et al., 1994; Baruch et al., 1996) and VWF (Pimanda et al., 2004).

In serum-enriched medium, anti-TSP-1 antibody did not significantly alter baseline rosetting rates. Nevertheless, this antibody significantly blocked the IGFBP7-mediated rosette-stimulation (Figure 7E). The specificity of rosette-inhibition by this antibody was validated with experiments using antibody isotype control (Figure 7—figure supplement 1A).

When TSP-1 was added to Albumax-supplemented medium, the rosetting rates were lower than those in serum-enriched medium (Figure 7F). However, when added together (IGFBP7 + VWF + TSP-1) to the Albumax-supplemented medium, significant rosette-stimulation was observed. A high-level TSP-1 (500 ng/ml) did not induce significantly higher rosetting stimulation than the lower level TSP-1 (10 ng/ml). With the addition of VWF (2 IU/ml) and TSP-1 (10 ng/ml) to Albumax-supplemented medium, IGFBP7 stimulated rosette formation to an extent similar to that of serum-enriched medium. Importantly, without IGFBP7, the presence of TSP-1 and VWF in Albumax-supplemented medium could not increase the rosetting rates, disputing the possibility of this event as a non-specific adhesive effect and reflecting their status as co-factors in IGFBP7-mediated rosetting. Lastly, we quantitated the amount of VWF needed to facilitate IGFBP7-mediated rosetting. When supplemented with IGFBP7 and TSP-1 in Albumax-supplemented medium, VWF as low as 0.125 IU/ml was sufficient to significantly increase rosetting rates, with an optimal increment attained at 0.5 IU/ml (Figure 7G). In fact, the 20% serum-enriched media that we used for this study contained VWF higher than 0.125 IU/ml (Figure 7—figure supplement 1B).

Quantification of IGFBP7 secretion

P. falciparum IRBC-exposed peripheral monocytes secreted significantly more IGFBP7 than their unexposed counterparts or those exposed to URBC (Figure 8A). THP-1 also secreted more IGFBP7 after IRBC exposure (Figure 8B). Interestingly, parasitemia as low as 0.25% was sufficient to significantly stimulate THP-1 to secrete more IGFBP7. Further increase in parasite density (up to 16% parasitemia) did not significantly increase IGFBP7 secretion any further.

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Figure 8—source data 1

Raw data (rosetting rates, %) for the data set presented in bar graph (8F).

R = biological replicate (same parasite, but different batches of cultures grown with different batches of URBCs).

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Figure 8—source data 2

Raw data (phagocytosis rates, %) for the data set presented in bar graph (8G).

R = biological replicate (same parasite, but different batches of cultures grown with different batches of URBCs).

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Rosetting is a common characteristic of late stage-IRBC in human malaria parasites, occurring frequently in P. falciparum and P. vivax (Lee et al., 2014). It has been proposed to provide a survival advantage for the parasites (Moll et al., 2015). Earlier studies have shown that rosetting occurs between the direct interactions of the parasite-derived ligands on the IRBC (i.e. PfEMP1, RIFIN, and STEVOR proteins for P. falciparum) with various receptors on the URBC (Lee et al., 2014; Niang et al., 2014; Rowe et al., 1997; Barragan et al., 2000; Cserti and Dzik, 2007; Chen et al., 1998; Goel et al., 2015).

Here we demonstrated the existence of a different type of rosetting, which we have called ‘type II rosetting’ that does not result from the direct interaction of IRBC with URBC. It was observed in all the P. falciparum and P. vivax isolates tested. This type II rosetting differs from the classical type I rosetting as it requires bridging by soluble mediators: IGFBP7, VWF, and TSP-1 between a rosetting ligand on IRBC and HS expressed by URBC. The fast rosette-stimulating effect by the protein and its fast reversion after the protein removal from the culture suggest that IGFBP7 does not mediate rosetting via irreversible binding to neither the rosetting receptor nor ligand. Instead, it is more likely to be mediated by weaker forces. It also suggests that these soluble mediators need to be present at a minimum concentration for the rosettes to occur.

We have shown that PfEMP1 is likely the principal P. falciparum rosetting ligand in this IGFBP7-mediated type II rosetting via usage of trypsin treatments, genetically modified P. falciparum clones that cannot surface-express PfEMP1, and the late ring stages of P. falciparum, the stage of maturation that manages to surface-express only one rosetting ligand, PfEMP1. However, we cannot fully dismiss the involvement of other rosetting ligands such as STEVOR or RIFIN as these proteins are encoded by multigene families and there is a lack of tools to assess and evaluate the implication of these proteins thoroughly. To our surprise, P. vivax IRBC also interacted with IGFBP7 similarly. There are no PfEMP1 orthologues in P. vivax. Based on the rosetting trend of P. vivax post-trypsin treatments, we postulate that P. vivax has multiple rosetting ligands with different trypsin sensitivities, and the one required by IGFBP7 is highly sensitive to trypsin. IGFBP7 requires the HS moieties on URBC to exert their rosette-stimulatory effect. Interestingly, removal of HS from the surface of URBC by heparinase caused the clumping of untreated IRBC (which harboured both rosetting ligands and receptors) when supplied with IGFBP7. Importantly, without enzymatic interference, the presence of this protein does not induce non-specific binding of URBC to each other, or autoagglutination-like clumping of IRBC. We hypothesized that the IGFBP7-mediated binding occurs preferably between the URBC and IRBC under normal circumstances possibly as a result of electrostatic differences between the URBC and IRBC (Tokumasu et al., 2012).

IGFBP7 requires other serum factors, namely VWF and TSP-1 to exert its rosette-stimulating effect. For healthy individuals, the VWF levels in the serum range from 0.48 to 1.24 IU/ml (median 0.84 IU/ml), whereas individuals with underlying pathological conditions have much higher levels of serum VWF (Terpos et al., 2013; Kastritis et al., 2016). Serum TSP-1 levels in healthy individuals vary greatly (0–12060 ng/ml) (Liu et al., 2015a; Rouanne et al., 2016). We found that under serum-free conditions (Albumax- supplemented medium), concentrations of VWF as low as 0.5 IU/ml and of TSP-1 at 10 ng/ml were enough to optimally facilitate IGFBP7-mediated rosette-stimulation at IGFBP7 of 100 ng/ml (the minimum concentration needed to optimally stimulate type II rosetting). The rosette-stimulation by the presence of these three proteins was comparable to that of 20% serum-enriched medium supplied with IGFBP7, reinforcing that IGFBP7 is the limiting factor for the rosette-stimulation.

Based on the data presented here, we propose the following mechanism of interactions for type II rosetting (Figure 9). IGFBP7 binds to HS on URBC; the interaction between IGFBP7 and cell surface HS has been demonstrated and well-characterized (Sato et al., 1999). IGFBP7 has also been shown to bind to the D4-CK region of VWF (van Breevoort et al., 2012; Lenting et al., 2012; Bryckaert et al., 2015). Although heparin is found to interact with the A1 region of VWF (Bryckaert et al., 2015), it is likely that HS does not interact directly with VWF (Denis et al., 1993) as we did not observe any rosetting when VWF was added alone in the absence of IGFBP7. Therefore, the HS on URBC interacts with IGFBP7, which also interacts with VWF. In turn, VWF interacts with PfEMP1 on IRBC via TSP-1 (Cooke et al., 1994; Bryckaert et al., 2015). While the extracellular domain of PfEMP1 that binds to TSP-1 has yet to be identified, it should be noted that TSP1 has been commonly associated with PfEMP1 (Cooke et al., 1994; Janes et al., 2011). The pervasive role of TSP-1 may explain why a wide range of clinical isolates and laboratory-adapted parasite lines were capable of responding positively to IGFBP7 addition.

Figure 9

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Induction of type II rosetting in P. falciparum and P. vivax is not attributed solely to IGFBP7. Previously, it has been shown that CFD in the serum can stimulate rosetting (Luginbühl et al., 2007). This molecule was also identified in our proteomic study. Experiments with anti-CFD antibodies showed that CFD could also induce type II rosetting, but to a lesser extent. The knock down of IGFBP7 expression in THP-1 demonstrated that IGFBP7 is a major monocyte-derived rosette-stimulating factor. Culture supernatant from IGFBP7-KD_THP-1 collected after 18 h of parasite exposure could not induce rosette-stimulation. Other rosette-stimulating factors may be secreted by the cells much later. Interestingly, IGFBP7 and VWF are components in Weibel-Palade bodies, the storage granules of endothelial cells (van Breevoort et al., 2012). Future work should characterize the effect of IRBC on the secretion of IGFBP7 by endothelial cells. Of note, the reported physiologic and pathological serum concentrations of IGFBP7 vary greatly, where most of the reported normal serum IGFBP7 concentrations fall below 50 ng/ml, but the serum IGFBP7 levels in different pathological conditions (e.g. various vascular disorders and cancers) are higher (as high as ~1000 ng/ml) (Kutsukake et al., 2008; Shersher et al., 2011; Liu et al., 2015b; Shaver et al., 2016; Barroso et al., 2016). Therefore, the working concentration of IGFBP7 in this study (100 ng/ml) is still within the pathophysiologic range in clinical settings. It would be interesting to compare the serum/plasma IGFBP7 levels of uncomplicated and severe malaria patients from the same area to understand better the role of IGFBP7 in malaria pathogenesis.

The importance of monocytes/macrophages in eliminating Plasmodium during its course of infection has been reported (Groux and Gysin, 1990; Theander, 1992). Peripheral monocytes (Turrini et al., 1992; Muniz-Junqueira and Tosta, 2009), as well as the tissue-resident macrophages (Tosta et al., 1983; Sponaas et al., 2009), have been shown to engulf IRBC readily. To survive, parasites must counter or avoid this host’s immune responses. The ability to perceive a phagocyte’s secreted IGFBP7 as a signal of ‘approaching threats’ and to respond by rosetting may provide survival advantage to the parasites.

In conclusion, the host-derived IGFBP7 is used as an ‘incoming phagocyte signal’ by the IRBC, and the IRBC in turn use this protein, along with two serum factors, VWF and TSP-1, to stimulate rosette formation, which acts as an immune-evasion strategy by hampering phagocytosis of the IRBC. It is hoped that future clinical studies will investigate associations between IGFBP7 and malaria pathogenesis and immunity.

Experiments were performed with isolates of late-stage P. falciparum and P. vivax late stages unless stated otherwise (materials used in the experiments and the work flow are available in Supplementary files 1, 4–6). All incubations were conducted for 1 h under in vitro cultivation conditions unless stated otherwise. Experiments with only one cell line or one parasite line were performed with biological replicates, where the experiments were conducted with multiple sets/batches of the cell/parasite cultures under the same cultivation conditions.

All sample/data information in this study are included in the manuscript and supporting files (Supplementary files, Source data files). Of note, data represented as bar graphs are provided as source data tables (5 sets): Figure 1—source data 1; Figure 5—source data 1; Figure 5—source data 2; Figure 8—source data 1; Figure 8—source data 2.

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Author details

1. Wenn-Chyau Lee

   Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7324-5792

2. Bruce Russell

   Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

   Contribution

   Formal analysis, Supervision, Validation, Methodology

   Competing interests

   No competing interests declared

3. Radoslaw Mikolaj Sobota

   1. Systems Structural Biology Group, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
   2. Institute of Medical Biology (IMB) Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Resources, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

4. Khairunnisa Ghaffar

   Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Validation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Shanshan W Howland

   Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Zi Xin Wong

   Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

7. Alexander G Maier

   Biomedical Sciences and Biochemistry, Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Resources, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

8. Dominique Dorin-Semblat

   1. Université de Paris, Biologie Intégrée du Globule Rouge, UMR_S1134, BIGR, INSERM, Paris, France
   2. Institut National de la Transfusion Sanguine, Paris, France

   Contribution

   Resources, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

9. Subhra Biswas

   Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

   Contribution

   Resources, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

10. Benoit Gamain

    1. Université de Paris, Biologie Intégrée du Globule Rouge, UMR_S1134, BIGR, INSERM, Paris, France
    2. Institut National de la Transfusion Sanguine, Paris, France

    Contribution

    Resources, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

11. Yee-Ling Lau

    Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

    Contribution

    Resources, Project administration, Writing - review and editing

    Competing interests

    No competing interests declared

12. Benoit Malleret

    1. Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
    2. Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Contribution

    Resources, Investigation, Methodology, Writing - review and editing

    Competing interests

    No competing interests declared

13. Cindy Chu

    1. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand
    2. Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

    Contribution

    Resources, Project administration, Writing - review and editing

    Competing interests

    No competing interests declared

14. François Nosten

    1. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand
    2. Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

    Contribution

    Resources, Project administration, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7951-0745

15. Laurent Renia

    Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Project administration, Writing - review and editing

    For correspondence

    renia_laurent@immunol.a-star.edu.sg

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0349-1557

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