Introduction
Malaria is a mosquito-borne infectious disease that is responsible for more than 200 million clinical cases and 400 000 deaths annually (WHO, 2016). Although there are five species of Plasmodium that can infect humans, most cases of severe malaria are caused by Plasmodium falciparum. Cerebral malaria (CM) is one of the most severe complications of P. falciparum infection, with a case fatality rate of approximately 20% even with appropriate medical treatment (Murphy and Breman, Reference Murphy and Breman2001). Blood-stage malaria parasites express proteins on the surface of the infected red blood cell, which allows them to bind to vascular endothelial cells and avoid clearance in the spleen. This process, known as sequestration, induces obstructions in the blood flow, resulting in hypoxia and haemorrhages (Miller et al., Reference Miller, Baruch, Marsh and Doumbo2002), associated with the induction of organ-specific disease syndromes (Walter et al., Reference Walter, Garin and Blot1982; Turner et al., Reference Turner, Morrison, Jones, Davis, Looareesuwan, Buley, Gatter, Newbold, Pukritayakamee, Nagachinta, White and Berendt1994; Grau et al., Reference Grau, Mackenzie, Carr, Redard, Pizzolato, Allasia, Cataldo, Taylor and Molyneux2003; Taylor et al., Reference Taylor, Fu, Carr, Whitten, Mueller, Fosiko, Lewallen, Liomba and Molyneux2004). A large body of work indicates that inflammatory responses also contribute to severe disease (Molyneux et al., Reference Molyneux, Engelmann, Taylor, Wirima, Aderka, Wallach and Grau1993; van der Heyde et al., Reference van der Heyde, Nolan, Combes, Gramaglia and Grau2006). In addition to parasitized red blood cells (pRBCs), histopathological analysis of the brain of fatal CM cases revealed the presence of leucocytes in the brain microvasculature (Porta et al., Reference Porta, Carota, Pizzolato, Wildi, Widmer, Margairaz and Grau1993; Patnaik et al., Reference Patnaik, Das, Mishra, Mohanty, Satpathy and Mohanty1994; Armah et al., Reference Armah, Dodoo, Wiredu, Stiles, Adjei, Gyasi and Tettey2005), suggesting that recruitment of these cells to sites of parasite sequestration might result in local inflammation and contribute to disease induction.
Leucocyte trafficking is regulated by a family of chemotactic cytokines called chemokines. The observation that host cells could be found with pRBC in inflamed organs such as brain or placenta attracted interest in investigating trafficking pathways by which inflammatory cells are recruited to target organs in severe malaria. Increased levels of circulating chemokines including CCL4, CXCL4, CXCL8 and CXCL10 have been found to be associated with CM (John et al., Reference John, Opika-Opoka, Byarugaba, Idro and Boivin2006, Reference John, Park, Sam-Agudu, Opoka and Bolvin2008; Armah et al., Reference Armah, Wilson, Sarfo, Powell, Bond, Anderson, Adjei, Gyasi, Tettey, Wiredu, Tongren, Udhayakumar and Stiles2007; Jain et al., Reference Jain, Armah, Tongren, Ned, Wilson, Crawford, Joel, Singh, Nagpal, Dash, Udhayakumar, Singh and Stiles2008; Wilson et al., Reference Wilson, Jain, Roberts, Lucchi, Joel, Singh, Nagpal, Dash, Udhayakumar, Singh and Stiles2011). Amongst these factors, the CXCR3 binding chemokine CXCL10 has been identified as the most accurate independent predictor of CM mortality in children (Armah et al., Reference Armah, Wilson, Sarfo, Powell, Bond, Anderson, Adjei, Gyasi, Tettey, Wiredu, Tongren, Udhayakumar and Stiles2007; Wilson et al., Reference Wilson, Jain, Roberts, Lucchi, Joel, Singh, Nagpal, Dash, Udhayakumar, Singh and Stiles2011). In mice, CXCL10 blockade has been shown to protect P. berghei ANKA-infected mice from developing CM by reducing recruitment of CXCR3+ leucocytes to the brain (Campanella et al., Reference Campanella, Tager, El Khoury, Thomas, Abrazinski, Manice, Colvin and Lustert2008; Nie et al., Reference Nie, Bernard, Norman, Amante, Lundie, Crabb, Heath, Engwerda, Hickey, Schofield and Hansen2009). Furthermore, CXCL10 blockade was also shown to improve control of parasitaemia by favouring the accumulation of CXCR3+CD4+ T follicular helper cells in the spleen, which enhanced antibody responses to infection (Ioannidis et al., Reference Ioannidis, Nie, Ly, Ryg-Cornejo, Chiu and Hansen2016).
Consistent with its association with cerebral disease, most malaria studies investigating cellular sources of CXCL10 have focused on brain tissue. High levels of CXCL10 have been detected in the cerebrospinal fluid of children that succumbed to CM (Armah et al., Reference Armah, Wilson, Sarfo, Powell, Bond, Anderson, Adjei, Gyasi, Tettey, Wiredu, Tongren, Udhayakumar and Stiles2007). In mice, CXCL10 expression has been observed in brain endothelial cells (Campanella et al., Reference Campanella, Tager, El Khoury, Thomas, Abrazinski, Manice, Colvin and Lustert2008; Miu et al., Reference Miu, Mitchell, Muller, Carter, Manders, McQuillan, Saunders, Ball, Lu, Campbell and Hunt2008; Sorensen et al., Reference Sorensen, Lian, Ozga, Miyabe, Ji, Bromley, Mempel and Luster2018), neurons (Campanella et al., Reference Campanella, Tager, El Khoury, Thomas, Abrazinski, Manice, Colvin and Lustert2008), astrocytes (Hanum et al., Reference Hanum, Hayano and Kojima2003; Miu et al., Reference Miu, Mitchell, Muller, Carter, Manders, McQuillan, Saunders, Ball, Lu, Campbell and Hunt2008) and microglia (Ioannidis et al., Reference Ioannidis, Nie, Ly, Ryg-Cornejo, Chiu and Hansen2016). In addition, neutrophils and monocytes that are recruited to the brain during rodent malaria infection (Ioannidis et al., Reference Ioannidis, Nie, Ly, Ryg-Cornejo, Chiu and Hansen2016; Sorensen et al., Reference Sorensen, Lian, Ozga, Miyabe, Ji, Bromley, Mempel and Luster2018) have been found to be sources of CXCL10 that control the recruitment of CXCR3+ leucocytes involved in the development of CM, suggesting an important role for leucocyte derived CXCL10 in the induction of severe malaria disease symptoms. Consistent with this concept, peripheral blood mononuclear cell (PBMC)-derived CXCL10 has been shown to be associated with P. falciparum-mediated severe malaria in children (Stanisic et al., Reference Stanisic, Cutts, Eriksson, Fowkes, Rosanas-Urgell, Siba, Laman, Davis, Manning, Mueller and Schofield2014). The specific cellular sources of CXCL10 in response to P. falciparum are still unknown. In this study, we sought to identify the leucocyte populations in the blood responsible for the induction of CXCL10 in response to P. falciparum. Our results show that CD14+ monocytes are the main cellular source of CXCL10 in response to P. falciparum. CXCL10 production in response to pRBCs was also found to be stimulated via an IFN-γ-dependent mechanism.
Materials and methods
Plasmodium falciparum culture
The 3D7 strain of P. falciparum was cultured as previously described (Stanisic et al., Reference Stanisic, Cutts, Eriksson, Fowkes, Rosanas-Urgell, Siba, Laman, Davis, Manning, Mueller and Schofield2014). Parasites were synchronized by treatment with 5% sorbitol and routinely subjected to gelatin flotation to select for knob-expressing parasites. Trophozoite/schizont stage pRBCs were purified using MACS CS separation columns with a VarioMACS separator (Miltenyi Biotech).
PBMC stimulation
Peripheral blood was collected from healthy volunteers at the Volunteer Blood Donor Registry. PBMCs were then isolated by density gradient centrifugation using Lymphoprep (StemCell Technologies) and cryopreserved in liquid nitrogen until use. Cryopreserved PBMCs were thawed in complete medium (RPMI 1640 with 25 mm HEPES, 10% heat-inactivated foetal bovine serum (HI-FBS; Sigma), 100 U mL−1 streptomycin and 100 μg mL−1 penicillin), counted and then seeded into U-bottom 96-well plates (2 × 105 cells per well). PBMCs were stimulated with either uninfected RBCs (uRBCs; 2 × 105 RBCs per well), pRBCs (2 × 105 RBCs per well) or recombinant human IFN-γ (10 ng mL−1; PeproTech) for 24 h at 37°C with 5% CO2. In some experiments, PBMCs were stimulated in the presence of 20 μg mL−1 anti-IFN-γ (clone NIB42; BD Biosciences) or isotype control antibodies.
Detection of CXCL10 by enzyme-linked immunosorbent assay (ELISA)
The amount of CXCL10 in PBMC culture supernatants was determined by capture ELISA. Briefly, U-bottom 96-well plates were coated with a capture antibody (clone MAB266; R&D systems) in phosphate buffer pH 9.6 by overnight incubation at 4°C. After washing, plates were blocked with 1% BSA (Sigma) in PBS for 1 h at 37°C before incubation with culture supernatants for 2 h at room temperature (RT). Plates were then incubated with biotinylated anti-CXCL10 (clone BAF266; R&D systems) for 2 h at RT, followed by streptavidin-conjugated HRP (Pierce) for 20 min at RT. After washing, bound complexes were detected by reaction with tetramethylbenzidine and H2O2 (KPL). Absorbance was measured at 450 nm. Chemokine concentrations were calculated using recombinant CXCL10 (R&D Systems) for the preparation of standard curves.
Flow cytometry
PBMCs were stimulated with uRBCs, pRBCs or IFN-γ for 24 h. Brefeldin A (10 μg mL−1; Sigma) and monensin (2 μ m; BD Biosciences) were added to cells for the last 8 h of stimulation. After stimulation, cells were incubated for 10 min on ice with PBS containing 10 mm glucose and 3 mm ethylenediaminetetraacetic acid (EDTA) to detach adherent cells. The cells were then blocked with human IgG (10 μg mL−1; Sigma) for 10 min on ice, washed and then stained with the following antibodies for 30 min on ice: FITC-conjugated anti-human γδTCR (clone 11F2), PerCP-Cy5.5-conjugated anti-human HLA-DR (clone L234), APC-conjugated anti-human CD123 (clone 6H6), AF700-conjugated anti-human CD19 (HIB19; Biolegend), APC-Cy7-conjugated anti-human CD14 (clone MϕP9), Pacific Blue-conjugated anti-human CD11c (clone 3.9; Biolegend), Qdot 605-conjugated anti-human CD8 (clone 3B5; Invitrogen), Brilliant Violet 650-conjugated anti-human CD4 (clone OKT4; Biolegend), BV711-conjugated anti-human CD16 (clone 3G8) PE Texas Red-conjugated anti-human CD3 (clone UCHT1; Beckman Coulter) and PE-Cy7 conjugated anti-human CD56 (clone B159). All antibodies were purchased from BD Biosciences unless otherwise stated. Aqua amine reactive dye (Life Technologies) was also included for dead cell exclusion. All washes and antibody dilutions were made in FACS buffer [PBS with 0.5% BSA (Sigma) and 2 mm EDTA]. Cells were then fixed in 2% paraformaldehyde and permeabilized using Perm Buffer 2 (BD Biosciences) before intracellular staining with PE-conjugated anti-human CXCL10 (6D4/D6/G2) or an isotype control for 1 h on ice. After staining, cells were washed, re-suspended in FACS buffer and analysed on a BD Fortessa X-1 flow cytometer. Data analysis was performed using FlowJo software (TreeStar, Ashland, OR, USA). A positive response in pRBC-stimulated PBMCs was defined as a frequency of CXCL10+ cells ⩾0.1% after subtraction of the background from uRBC-stimulated cells.
Statistical analysis
Statistical analysis was performed in Prism version 8 (GraphPad Software Inc.). Wilcoxon matched-pairs signed rank tests were used to analyse matched data.
Results
CD14+ monocytes are the main cellular source of CXCL10 in response to P. falciparum
The 3D7 strain of P. falciparum has previously been shown to induce CXCL10 production in PBMCs from severe malaria cases in an endemic area of Papua New Guinea (Stanisic et al., Reference Stanisic, Cutts, Eriksson, Fowkes, Rosanas-Urgell, Siba, Laman, Davis, Manning, Mueller and Schofield2014). To determine whether this parasite was also able to induce CXCL10 production in PBMCs from malaria-unexposed individuals, PBMCs from healthy donors were stimulated with pRBCs for 24 h and CXCL10 output was determined by ELISA. PBMCs were also stimulated with autologous uRBCs as a negative control, while IFN-γ was included as a positive control. PBMCs from all donors produced CXCL10 in response to pRBC stimulation (Fig. 1A). On average, pRBC-stimulated PBMCs showed a >4-fold increase in CXCL10 levels compared to either media- or uRBC-stimulated cells. Robust CXCL10 responses to IFN-γ were also observed in each donor (Fig. 1B).
Fig. 1. CD14+ monocytes are the main cellular source of CXCL10 in response to P. falciparum. PBMCs were stimulated with uRBCs, pRBCs (A) or IFN-γ (B) for 24 h. Cell culture supernatants were then collected and CXCL10 levels were determined by ELISA. Each bar represents the mean of duplicate wells. Letters indicate individual donors. (C) PBMC were stained with fluorescent antibodies for identification of CD14+ monocytes (CD14+; P1), γδ T cells (CD3+γδ TCR+; P2), CD8+ T cells (CD3+CD8+CD4−; P3), CD4+ T cells (CD3+CD8−CD4+; P4), CD19+ B cells (CD3−CD19+; P5), CD56+ NK cells (CD3−CD19−CD16+/−CD56+; P6), pDCs (CD3−HLA-DR+CD14−CD19−CD11c−CD123+; P7) and mDCs (CD3−HLA-DR+CD14−CD19−CD11c+CD127+; P8) by flow cytometry. CXCL10 intracellular staining was evaluated within each gated population as shown in the representative contours (D). The proportion of CXCL10 responders was determined for each cell population (E). A positive response in pRBC-stimulated PBMCs was defined as a frequency of CXCL10+ cells ⩾0.1% after subtraction of the background from uRBC-stimulated cells. (F) Representative histograms showing the level of CXCL10 expression in response to pRBC by CD14+ monocytes, pDCs and mDCs. uRBC-stimulated lymphocytes are included as a background control. Numbers represent the average geometric mean fluorescence intensity (GMFI) for each cell population. The average GMFI of CXCL10 was also determined among all responder donors for each population (G). Bars represent the mean ± s.d., **P < 0.01 unpaired t-test.
To identify the specific cellular sources of CXCL10 in response to P. falciparum, PBMCs were stimulated with pRBCs or uRBCs for 24 h and then stained with a panel fluorescently labelled antibodies to assess CXCL10 production in monocytes, dendritic cells, NK cells B cells and various populations of T cells by flow cytometry. The gating strategy used to identify different leucocyte populations is shown in Fig. 1C and D. CD14+ monocytes were identified as a cellular source of CXCL10 in the majority of donors tested. In addition to CD14+ monocytes, CXCL10-producing plasmacytoid DCs (pDCs) were also detected in a small proportion of donors, while CXCL10 production was observed in monocyte-derived DCs (mDCs) from one donor (Fig. 1E). CXCL10 expression was not detected in PBMCs from three donors. The median fluorescence intensity of CXCL10 was also determined in each cell population to assess the level of CXCL10 expression (Fig. 1F and 1G). This showed that CXCL10 expression was significantly higher in CD14+ monocytes than pDCs.
PBMC CXCL10 responses to P. falciparum are IFN-γ-dependent
Although CXCL10 was initially identified as an IFN-γ-inducible factor, it has been shown that bacterial and viral agonists stimulate its production (Asensio et al., Reference Asensio, Maier, Milner, Boztug, Kincaid, Moulard, Phillipson, Lindsley, Krucker, Fox and Campbell2001; Park et al., Reference Park, Lee, Cho, Lee, Kim, Choi, Oh, Park, Kim and Lee2006; Bandow et al., Reference Bandow, Kusuyama, Shamoto, Kakimoto, Ohnishi and Matsuguchi2012; Brownell et al., Reference Brownell, Bruckner, Wagoner, Thomas, Loo, Gale, Liang and Polyak2014), suggesting that additional pathways may also contribute to the induction of this chemokine. Consistent with this, both viral infection and the TLR3 agonist polyI:C have been shown to induce CXCL10 production via activation of the NF-κB pathway (Spurrell et al., Reference Spurrell, Wiehler, Zaheer, Sanders and Proud2005; Brownell et al., Reference Brownell, Bruckner, Wagoner, Thomas, Loo, Gale, Liang and Polyak2014). Furthermore, IFN-γ-independent production of CXCL10 has also been reported (Medoff et al., Reference Medoff, Sauty, Tager, Maclean, Smith, Mathew, Dufour and Luster2002; Cheeran et al., Reference Cheeran, Hu, Sheng, Peterson and Lokensgard2003). To determine whether IFN-γ is required for CXCL10 production in PBMCs in response to P. falciparum, PBMCs were stimulated with pRBCs in the presence of anti-IFN-γ or isotype control antibodies and CXCL10 output was assessed by using ELISA. Preliminary experiments showed that anti-IFN-γ antibodies inhibit CXCL10 responses in response to IFN-γ in a dose-dependent manner, with >80% neutralization observed at an antibody concentration of 20 μg mL−1 (Supplementary Fig. 1). Based on these data, an antibody concentration of 20 μg mL−1 was used for subsequent experiments. As expected, CXCL10 production in response to IFN-γ was significantly reduced in the presence of anti-IFN-γ antibodies (Fig. 2A). Notably, IFN-γ neutralization completely abrogated CXCL10 responses to pRBCs (Fig. 2B and C). Thus, P. falciparum induces early/innate CXCL10 production in PBMCs from malaria-unexposed donors in an IFN-γ-dependent manner.
Fig. 2. CXCL10 production in response to P. falciparum is IFN-γ-dependent. PBMCs were stimulated with IFN-γ (A), pRBCs (B) or uRBCs (C), or for 24 h in the presence of 20 μg mL−1 anti-IFN-γ or isotype control antibodies. Cell culture supernatants were then collected and CXCL10 levels were determined by ELISA. Lines connect the response of individuals to each stimulus in the presence of anti-IFN-γ or isotype control antibodies. Dotted line represents the detection limit of the ELISA. *P < 0.05 Wilcoxon matched-pairs signed rank tests.
Discussion
The CXCR3 chemokine CXCL10 has been shown to be a strong independent predictor of P. falciparum-mediated CM mortality in children (Armah et al., Reference Armah, Wilson, Sarfo, Powell, Bond, Anderson, Adjei, Gyasi, Tettey, Wiredu, Tongren, Udhayakumar and Stiles2007; Wilson et al., Reference Wilson, Jain, Roberts, Lucchi, Joel, Singh, Nagpal, Dash, Udhayakumar, Singh and Stiles2011) and to promote the development of CM in mice (Campanella et al., Reference Campanella, Tager, El Khoury, Thomas, Abrazinski, Manice, Colvin and Lustert2008; Nie et al., Reference Nie, Bernard, Norman, Amante, Lundie, Crabb, Heath, Engwerda, Hickey, Schofield and Hansen2009). The identity of the cell types responsible for the production of CXCL10 in response to P. falciparum in human blood is still unknown. Here we have shown that CD14+ monocytes are the main leucocytic source of CXCL10 in the blood in response to the parasite. As CD16 expression on CD14+ monocytes was not assessed, it remains to be determined which specific monocyte population (i.e. classical, intermediate or non-classical) is responsible for the production of CXCL10 in response to P. falciparum (Ziegler-Heitbrock et al., Reference Ziegler-Heitbrock, Ancuta, Crowe, Dalod, Grau, Hart, Leenen, Liu, MacPherson, Randolph, Scherberich, Schmitz, Shortman, Sozzani, Strobl, Zembala, Austyn and Lutz2010).
Although CXCL10 was initially identified as an IFN-γ-inducible chemokine, it has also been reported that bacterial and viral agonists can stimulate its production from astrocytes (Asensio et al., Reference Asensio, Maier, Milner, Boztug, Kincaid, Moulard, Phillipson, Lindsley, Krucker, Fox and Campbell2001) and macrophages (Kopydlowski et al., Reference Kopydlowski, Salkowski, Cody, van Rooijen, Major, Hamilton and Vogel1999). Our results show that CXCL10 production by PBMCs in response to P. falciparum is IFN-γ-dependent. This finding is consistent with previous studies showing that CXCL10 production in human brain endothelial cell-PBMC co-cultures is largely IFN-γ-dependent (Khaw et al., Reference Khaw, Ball, Golenser, Combes, Grau, Wheway, Mitchell and Hunt2013). Previous studies have identified γδT cells, NK cells and conventional αβT cells as sources of IFN-γ in P. falciparum-stimulated PBMCs (D'Ombrain et al., Reference D'Ombrain, Hansen, Simpson and Schofield2007a, Reference D'Ombrain, Voss, Maier, Pearce, Hansen, Cowman and Schofield2007b), suggesting that IFN-γ production from these cells facilitates down-stream CXCL10 production from CD14+ monocytes in the blood. These studies also found that the IFN-γ response to P. falciparum was highly heterogeneous among individuals. Thus, the inability of some donors to produce CXCL10 (Fig. 1E) may reflect poor IFN-γ responsiveness to P. falciparum in these individuals.
IFN-γ has been shown to have a dual role in malaria, contributing to both the control of infection and the development of severe disease (King and Lamb, Reference King and Lamb2015). During the early stages of infection, IFN-γ can help to activate macrophages and enhance their phagocytic activity thereby promoting parasite clearance. IFN-γ also enhances monocyte-mediated antibody-dependent cellular cytotoxicity (Bouharoun-Tayoun et al., Reference Bouharoun-Tayoun, Oeuvray, Lunel and Druilhe1995). Systemic production of IFN-γ during infection has also been shown to up-regulate ICAM-1 expression on vascular endothelial cells, which promotes parasite sequestration and the development of severe disease (Amani et al., Reference Amani, Vigario, Belnoue, Marussig, Fonseca, Mazier and Renia2000). Here we found that P. falciparum-induced IFN-γ results in the production of CXC10, described to be one of the strongest biomarkers of severe malaria, suggesting that in addition to its direct effects triggered by IFN-γ-receptor mediated signalling, down-stream effects activated via IFN-γ-mediated chemokine cascades, are also important in the induction of symptomatic malaria. In addition to its well-defined role in leucocyte recruitment, CXCL10 has also been shown to exert anti-angiogenic function (Bodnar et al., Reference Bodnar, Yates and Wells2006; Yates-Binder et al., Reference Yates-Binder, Rodgers, Jaynes, Wells, Bodnar and Turner2012) and inhibit endothelial cell proliferation (Campanella et al., Reference Campanella, Colvin and Luster2010). Thus, IFN-γ-mediated production of CXCL10 during infection may also inhibit vascular growth and repair required to maintain oxygen and nutrient supply to hypoxic tissues.
Consistent with our findings here, monocytes have also been identified as a major leucocytic source of CXCL10 in CM-susceptible infected mice (Ioannidis et al., Reference Ioannidis, Nie, Ly, Ryg-Cornejo, Chiu and Hansen2016; Sorensen et al., Reference Sorensen, Lian, Ozga, Miyabe, Ji, Bromley, Mempel and Luster2018). Microglial cells and brain endothelial cells have also been identified as cellular sources of CXCL10 and stable interactions between brain endothelial cells and CD8+ T cells and are thought to be required for the induction of CM in mice (Ioannidis et al., Reference Ioannidis, Nie and Hansen2014; Sorensen et al., Reference Sorensen, Lian, Ozga, Miyabe, Ji, Bromley, Mempel and Luster2018). CXCL10 production by brain-recruited monocytes induces trafficking of CXCR3+ T cells to this site during infection, thereby promoting the development of CM (Ioannidis et al., Reference Ioannidis, Nie, Ly, Ryg-Cornejo, Chiu and Hansen2016). Interestingly, monocyte accumulation has been observed in the brain microvasculature of fatal CM cases (Hochman et al., Reference Hochman, Madaline, Wassmer, Mbale, Choi, Seydel, Whitten, Varughese, Grau, Kamiza, Molyneux, Taylor, Lee, Milner and Kim2015), raising the possibility that a similar process might take place in human CM. Consistent with this hypothesis, high levels of CXCL10 in cerebrospinal fluid have been associated with CM mortality in children (Armah et al., Reference Armah, Wilson, Sarfo, Powell, Bond, Anderson, Adjei, Gyasi, Tettey, Wiredu, Tongren, Udhayakumar and Stiles2007).
This study identified CXCL10 production from unexposed donors suggesting an important role for innate responses in the activation of this pathway. However, it remains to be determined whether additional parasite-specific adaptive responses also contribute to the CXCL10 response to P. falciparum in malaria-exposed individuals. Further work with cells from P. falciparum-infected individuals will be required to establish if CXCL10 production and its association with the development of severe malaria is further enhanced after activation of other cell populations that have been previously exposed to malaria parasites.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182019001744.
Financial support
This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS and Project Grants 1031212 and 1058665.
Conflict of interest
The authors declare no conflicting interests.
Ethical standards
All experiments were conducted in accordance with the requirements of the Walter and Eliza Hall Institute Human Research Ethics Committee, the National Health and Medical Research Council's National Statement on Ethical Conduct in Human Research and the Helsinki Declaration of 1975, as revised in 2008.
