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Macrophage migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of pro-inflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during asexual bloodstages of the parasite lifecycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question whether it affects monocyte function in a similar manner to its human counterpart.
Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localisation of the protein in bloodstage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and controls.
PfMIF protein was expressed in asexual bloodstage parasites, localised to the Maurer’s cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of toll like receptor (TLR) 2, TLR4 and CD86.
These results indicate that PfMIF is released during bloodstage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.
Over 40% of the world’s population is currently at risk of exposure to malaria with an estimated 1.5 to 3 million deaths per year attributed to Plasmodium infection [1, 2]. A majority of malaria cases occur in sub-Saharan Africa with most deaths occurring in children under 5 years of age. Plasmodium falciparum infection, especially severe malaria, is known to be associated with an acute inflammatory response. This is characterised by raised levels of pro-inflammatory cytokines such as tumour necrosis factor α (TNF-α), interferon γ (IFN-γ) and interleukin (IL)-6 . This inflammatory response may lead to an increase in the cytoadherence of parasitised erythrocytes (iRBC) to vascular endothelium resulting in more severe malarial disease . A putative protein identified during the sequencing of the P. falciparum genome showed sequence homology to the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) . Microarray studies suggested that P. falciparum MIF (PfMIF) mRNA is transcribed in late ring and early trophozoite stages of the asexual blood cycle of the parasite . The identification of a MIF homologue in P. falciparum suggested a potential mechanism contributing to the pro-inflammatory cytokine profile observed during infection.
Human MIF was one of the first cytokines identified and has a wide range of biological activities including the induction of TNF-α, nitric oxide, IL-6 and IL-8 secretion, upregulation of toll-like receptor (TLR) 4 and intercellular adhesion molecule (ICAM)-1 expression and suppression of the effects of glucocorticoids [7, 8]. MIF has been directly implicated in a wide range of infectious and immune-mediated diseases including sepsis, rheumatoid arthritis and diabetes . Interestingly, homologues of MIF have been identified in filarial nematodes and one tick species. They show remarkable similarity to mammalian MIF in both crystal structure and in vitro biological activity [9, 10]. These homologues are thought to play an important role in parasite immune evasion strategies.
The role of PfMIF during the course of P. falciparum infection has not been determined. Therefore, we generated recombinant PfMIF expressed in bacteria and investigated the expression patterns and localisation of PfMIF during the asexual blood stage cycle of the parasite. We also examined the ability of recombinant PfMIF to modulate monocyte function.
Blood samples were collected from children living in the Ngerenya area of Kilifi District, who were under active surveillance for malaria as detailed previously . We analyzed plasma from 117 children that were collected during the cross-sectional survey conducted during low transmission season in October 2003. All children were examined clinically, and venous blood samples were collected for whole blood counts and to determine the presence of malaria parasites. Children who were negative for P. falciparum bloodstage parasites by microscopy were included in the study. In August 2004 and January 2005, blood samples were collected from children attending the outpatient clinic at Kilifi District Hospital with mild, uncomplicated malaria (fever > 37.5°C, associated with a blood film positive for P. falciparum parasites and with no alternative explanation on careful clinical examination), and from children admitted to the wards with severe malaria. All 80 subjects included in this study were invited to donate a convalescence blood sample 14 days after discharge from hospital, 35 convalescent samples were collected. The study was approved by the Kenya Medical Research Institute / National Ethical Review Committee and the Oxford Tropical Research Ethical Committee. Written informed consent was obtained from the parents or guardians of the participating children.
Intraerythrocytic stage Plasmodium falciparum parasites derived from the ITG/A4 clone were cultured in vitro following the protocol described previously . Parasites cultures were synchronised using sorbitol lysis method . Tightly synchronised parasites were sampled throughout the asexual blood stages in order to perform Western blotting and immunofluorescence microscopy.
PfMIF sequence was amplified by RT-PCR from P. falciparum total RNA using oligo (dT) primers and SuperScript II reverse transcriptase (Invitrogen, US). The two terminal primers used were: 5′-GAATTCCATATGCCTTGCTGTGAAGTAATAACAAACG-3′ and 5′-CGCCCTAGGCTAGCCGAAAAGAGAACCAC-3′. The amplified DNA fragment was sub-cloned into T7/NT-TOPO expression vector that contained an in-frame N-terminal histidine-tag (Invitrogen, US). The construct containing the complete PfMIF sequence in correct orientation was transformed into Escherichia coli BL21(DE3) pLysS strain. The transformed cells were cultured for 3 hours using an overnight saturated culture as an inoculum, and then induced at 0.6 OD600 with 0.5 mM isopropyl β-D-thiogalactopyranoside for another 4 hours. The pelleted cells were lysed using Bugbuster reagent (Novagen, USA) and the crude bacterial extract was purified through Ni-NTA column (Invitrogen, USA). The peak fractions, as determined by SDS-PAGE, were subjected to ion exchange chromatography using DEAE Sepharose (Amersham Biosciences, UK). The PfMIF protein eluted between 150mM and 250mM NaCl. The purified protein was then denatured in 6M Urea containing 10mM β-mercaptoethanol for 20 minutes and subsequently the protein was dialysed against a buffer consisting of 20mM Tris-HCl, 50mM NaCl, pH 7.5 with decreasing concentrations of urea until final dialysis with no urea. Lipopolysaccharide (LPS) was removed using EndoTrap (Profos, Germany) and removal was confirmed using Limulus Ameobocyte Lysate assay (Cambrex, USA). LPS concentration in the recombinant protein was routinely found to be <2pg per μg protein.
Polyclonal anti-PfMIF serum was generated by immunization of New Zealand white rabbits with 4 subcutaneous injections of 50μg recombinant PfMIF over 35 days. The initial injection was emulsified with Freund’s Complete Adjuvant with the following 3 injection emulsified with Freund’s Incomplete Adjuvant. Serum was harvested at day 49 after the initial injection. An anti-peptide antibody was also generated in rabbits against a PfMIF specific peptide sequence (NRSNNSALADQITKC) (Sigma-Genosys, US). Polyclonal anti-serum cross-reacted with human MIF at a dilution of up 1:200 but was specific for PfMIF at higher dilutions. The anti-peptide antibody did not cross-react with human MIF (Supplementary Figure). Rabbit IgG was purified from anti-PfMIF peptide sera using Protein G Sepharose (Amersham, UK) following the standard protocol. Purified anti-PfMIF peptide IgG was used in subsequent experiments as indicated.
Parasites were sorbitol synchronized, cultures were at 3-10% parasitaemia and 200-500 μl of packed iRBCs were processed for each RNA sample. Cells were spun directly from culture and packed cells resuspended in the appropriate volume of TRIzol (Invitrogen, USA). Samples were then processed as previously described .
Tightly synchronised parasites were sampled during ring and trophozoite stages of the life cycle. A small aliquot was removed for immunofluorescence microscopy and the remaining iRBCs were lysed with 0.01% saponin (Sigma, USA) and thoroughly washed to remove haemoglobin and other RBC proteins . Purified parasite lysates were run on 12% SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher and Schuell, Germany) for immunoblotting with anti-PfMIF rabbit sera or purified IgG as indicated followed by alkaline phosphatase-conjugated swine anti-rabbit IgG secondary antibody (Dako, Denmark). Blots were developed using BCIP/NBT substrate solution (Invitrogen, USA). For the detection of PfMIF in the culture supernatant, a 4% hematocrit culture of 20% trophozoites was grown overnight to allow schizont development and rupture. Culture supernatant was then collected and spun to remove any remaining RBC and used in Western blotting as above.
Fixed and permeabilised iRBCs were stained with purified rabbit anti-PfMIF peptide IgG and anti-P. falciparum skeleton binding protein 1 (PfSBP1) (kindly provided by Prof. Catherine Braun-Breton, Dynamique Moléculaire des Interactions Membranaires, University Montpellier 2, France). PfSBP1 has been shown to localise to the Maurer’s cleft . 4′-6-Diamidino-2-phenylindole (DAPI) was used to stain the parasite nuclei (Sigma, USA). FITC conjugated anti-rabbit IgG (Dako, Denmark) and Alexa-Fluor® 546 conjugated anti-mouse IgG (Molecular Probes, USA) were used as secondary antibodies.
Monocytes were isolated from buffy coats (National Blood Service, UK) using anti-CD14 magnetic beads (Miltenyi Biotec, Germany). Monocytes were used for migration assays or cultured in 24 well plates at a concentration of 5×105 cells in 0.5ml RPMI 1640 medium (Sigma, USA) supplemented with 2mM glutamine, 50μM kanamycin and 2% pooled human serum with or without exogenous PfMIF. In some experiments, monocytes were incubated with or without PfMIF for 12 hours before addition of lipopolysaccharide (LPS) from E. coli 0111 (Sigma, UK) or Peptidoglycan (PGN) from Staphylococcus aureus (Sigma, UK) for 24 hours. After incubation at 37°C, 5% CO2 supernatant was collected for ELISA. The cells were stained for surface molecules, analysed by flow cytometry (FACScalibur, USA) and the data analysed using FlowJo (Treestar Inc., USA). The following primary antibodies were used: anti-CD54 (ICAM-1; Dako, Denmark), anti-CD40 and anti CD86 (Serotec, UK), anti-TLR2 and anti-TLR4 (eBioscience, USA), and anti-HLA DR (Dako, Denmark). The secondary antibody used was FITC conjugated anti-mouse IgG (Dako, Denmark).
Migration assays were performed using 24 well, 6.5mm Transwell membranes with 5.0μm pore size (Corning, USA). Briefly, 5×104 purified monocytes in 100μl medium were added to the upper chamber with or without 100ng/ml recombinant PfMIF or 100pg/ml LPS and 600μl of media with or without 100 ng/ml Monocyte Chemotactic Protein 1 (MCP-1) was added to the lower chamber (R&D Systems, UK). Plates were incubated immediately at 37°C (5% CO2) for 2 hours. After incubation, the base of the membrane was rinsed with 200μl fresh medium twice and cells that had passed into the lower chamber were counted using a FACScalibur flow cytometer (BD, USA).
ELISA for IL-8, IL-12 and TNF-α were performed according to manufacturer’s instructions (Pharmingen, USA). We also developed an ELISA method to determine antibodies to PfMIF in patient sera. Briefly, microtitre wells were coated with 15μg/ml recombinant PfMIF overnight, the wells were then blocked with 1% bovine serum albumin (BSA) for 1 hour, followed by the addition of patient sera diluted 1:100 in block buffer. Anti-PfMIF peptide IgG was detected using horseradish peroxidase-conjugated rabbit anti-human secondary antibody (Dako, Denmark). The colour was developed using o-phenylenediamine dihydrochloride (OPD) substrate (Sigma, UK) and optical density (OD) read at 490nm. Patients’ sera were assayed in duplicate and 7 sera from nonimmune European adults were used to control for non-specific binding. Sera that showed binding two standard deviations above the average of the European controls were considered positive.
All data were analysed using SPSS (SPSS Inc., USA). Comparisons were done using Pearson’s Chi-squared, Mann-Whitney U tests and paired t-tests.
PfMIF was initially described during the malaria genome project as a hypothetical MIF homologue. Subsequent microarray studies showed that PfMIF mRNA was transcribed during ring and trophozoite stages of the P. falciparum lifecycle . We confirmed gene expression of PfMIF in ring and trophozoite stages by Northern blot (Figure 1a). In addition, we readily detected PfMIF protein expression in both ring and trophozoites stages of the parasite asexual bloodstage cycle by Western blotting (Figure 1b). PfMIF was also detected in culture supernatant after schizont rupture (Figure 1b). However, PfMIF was not detected in saponin lysis supernatant of iRBC (containing RBC cytosol but not parasites) or in the culture supernatant before schizont rupture (data not shown). These results indicated that the protein might be released after schizont rupture into the circulation during bloodstage infection. We subsequently used indirect immunofluorescence microscopy to determine the localisation of PfMIF in iRBC during the intraerythrocytic development of the parasite (Figure 2). In ring stage parasites, PfMIF was almost exclusively located within the parasite. However, as the parasites developed into trophozoites, PfMIF was clearly detected in both the parasite and the iRBC cytosol. Here, PfMIF in iRBC appeared to be associated with distinct vesicles and co-localised with PfSBP1 suggesting an association with the Maurer’s cleft (Figure 2). This showed that PfMIF moved from the parasite into the iRBC and that the protein was associated with distinct vesicles in the cytosol.
We analysed migration, surface marker expression and cytokine secretion in monocytes, following treatment with different doses of recombinant PfMIF. We observed that random monocyte migration was significantly inhibited by treatment with 100ng/ml PfMIF (Figure 3). In addition, PfMIF significantly reduced chemotactic migration of monocytes in response to MCP-1. However, MCP-1 induced migration was still above baseline level even in the presence of 500 ng/ml PfMIF (Figure 3). The effect of PfMIF on random migration on monocytes was instable and lost approximately 10 days after purification. We therefore confirmed that PfMIF had maintained its effect on monocyte migration before each subsequent experiment.
Monocytes treated with PfMIF did not release significantly different levels of IL-8, TNF-α or IL-12 within 24 hours compared to controls (Figure 4). There was an increase of IL-8 secretion with PfMIF treatment but this was low in comparison to IL-8 secretion induced by 100pg/ml LPS and it did not reach significance (Figure 4). Preincubation of monocytes with PfMIF had no significant influence on subsequent cytokine release in response to LPS.
We subsequently analysed whether the expression of surface molecules HLA-DR,, CD40, CD86, TLR2, TLR4 and ICAM-1was altered upon incubation with PfMIF. Only TLR2, TLR4 and CD86 surface expression showed a significant reduction in response to PfMIF whereas all other surface markers remained unchanged (p<0.05 t-test; Figure 5A). By contrast, treatment of monocytes with the TLR4-ligand LPS resulted in significant upregulation of CD40, CD86 and ICAM-1 whereas the expression of TLR4 was significantly reduced (p<0.05 paired t-test; Figure 5B). Pre-incubation of monocytes with PfMIF before treatment with LPS for 24 hours did not change expression levels for any of the surface marker compared with LPS alone although TLR2 showed a trend towards reduced expression level (p=0.09 paired t-test for PfMIF 500 ng/ml). Treatment of monocytes with the TLR2-ligand PGN increased expression of CD86 only (p<0.05 paired t-test; Figure 5C). Pre-incubation with PfMIF before addition of PGN for 24 hours had no effect on the surface expression of any of the markers we analysed compared with PGN alone. Together these data would indicate that PfMIF alters monocyte function but has no effect on LPS- or PGN-mediated activation of monocytes.
Antibody responses to PfMIF were examined in samples taken from Kenyan children with acute malaria, the same patients during convalescence and healthy Kenyan children during the low transmission season. There was a significant difference in median age between acute and low transmission season subjects with the latter group older than the acute malaria patients (acute malaria: median 29 months, range 4-138; convalescent: median 26 months, range 6-70; healthy children: median 57 months, range 12-107). However, within each group there was no correlation between age and PfMIF IgG levels (Spearman’s correlation coefficient, acute malaria: r=0.076, p=0.547; convalescent: r=0.173, p=0.328; healthy children: r=0.16, p=0.221). In the healthy control group 62 children (53%) showed antibody responses to PfMIF. However, in the responder group PfMIF antibodies were not associated with age and therefore exposure. By contrast, in the acute malaria and convalescent groups 65 (81%) and 35 (100%) children made antibodies against PfMIF respectively. Therefore, there was a larger than expected proportion of positive antibody responses in acute and convalescent samples than in healthy controls (Pearson’s Chi-square, p<0.001). When only responding children from each group were taken into account, during the low transmission season the children had significantly lower levels of circulating PfMIF antibodies than seen during and immediately subsequent to P. falciparum infection (acute: OD=0.7215, range 0.1-2.10; convalescent: OD=1.0649, range 0.4-2.20; healthy: OD=0.1610, range 0.01-0.86; p<0.001 Mann Whitney test; Figure 6). This result is consistent with the antibody profiles seen in response to other malaria antigens where a rapid decrease in antibody concentration has been observed after acute infection .
We have shown here that PfMIF protein is expressed in asexual blood stages of the P. falciparum lifecycle and that the protein moved from the parasite into the iRBC cytosol and was associated with the Maurer’s cleft. Recombinant PfMIF inhibited the random migration of monocytes and reduced the chemotactic response of monocytes to MCP-1. In addition, PfMIF altered the activation of monocytes, demonstrated by surface marker expression. Finally, in plasma of children with acute malaria, antibody responses to PfMIF were readily detected. However, antibody levels were much lower during the low transmission season suggesting that PfMIF-specific antibodies are short-lived and follow a similar pattern of responses to other parasite antigens previously studied.
The localisation of PfMIF in the cytosol of iRBC is interesting in view of recent findings that the Maurer’s cleft has an important role in trafficking of parasite proteins, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), to the surface of the erythrocytes and that mammalian MIF has chaperone-like properties in vitro [18-20]. Maurer’s clefts are parasite-derived vesicular structures that appear in the RBC cytosol during the early trophozoite stage. Cooke and colleagues recently demonstrated that PfSBP1, which is associated with the Maurer’s cleft, is responsible for the final translocation step of PfEMP1 to the iRBC plasma membrane. Recent studies examining the peptide binding properties of mammalian MIF in vitro and its role in heat-induced protein aggregation have highlighted its potential role as a chaperone-like protein. In addition to its effect on monocyte function, PfMIF may play a role in protein trafficking within in the iRBC due to its apparent localisation to the Maurer’s cleft and the chaperone-like properties of MIF proteins from other species. Studies on protein trafficking in iRBC using PfMIF knockout parasites could address this question.
During the present study we observed that TLR2 and TLR4 expression on monocytes was moderately but significantly reduced in response to PfMIF. MIF has previously been shown to be required for the expression of TLR4 in mouse macrophages . However, this study only addressed endogenous MIF and the effect of the addition of exogenous MIF on TLR4 expression on macrophages, or any other cell type, has not been examined. It has long been recognised that TLR tolerance can be induced in that the treatment of cells with LPS rendering them unresponsive to further stimulation not only through TLR4 but also through other TLRs . We therefore tested whether pre-incubation with PfMIF for 12 hours would alter monocyte activation by the TLR2-ligand PGN or the TLR4-ligand LPS. Whereas LPS alone increased the surface expression of CD40, CD86, ICAM-1 but reduced the surface expression of TLR4, PGN alone increased the surface expression of CD86 only. Pre-incubation with PfMIF at either concentration had no effect on the TLR2 – or TLR4-mediated activation of monocytes suggesting that PfMIF does not play an active part in TLR tolerance that has been previously described during Plasmodium yoelii infection in mice . A reduction in TLR expression on myeloid cells has also been demonstrated in patients with filarial infections [24, 25]. T and B cells and monocytes from filarial-infected individuals were shown to exhibit significantly less TLR1, TLR2 and TLR4 expression compared to uninfected controls. The mechanism for reduced expression has not been elucidated but one hypothesis is that the MIF homologue produced and secreted by filarial nematodes may contribute to this phenomenon.
Analysis of the pathophysiological role of host-derived MIF during Plasmodium infection has so far been limited to its role in the development of malarial anemia . This study indicated that MIF knockout mice infected with P. chabaudi developed less severe anemia, had better erythroid development and improved survival compared to controls. Following from this observation it has been shown that human MIF (huMIF) levels in plasma were significantly increased during acute malarial disease in patients from Zambia . This finding is in direct contrast to a study by Awandare and colleagues that showed a significant decrease in circulating huMIF in acute malaria patients from Gabon . Neither of these studies in patients addressed the likely involvement PfMIF during acute malaria. Our study highlights the important issue that any future study should take into account the presence of parasite MIF in circulation, and hence any potential cross-reactivity between PfMIF and huMIF.
In summary, our study suggests that PfMIF is capable of influencing the host immune system. This is most clearly demonstrated by the ability of recombinant PfMIF to modulate the function of monocytes, specifically by inhibiting migration and decreasing CD86 and TLR2 and TLR4 expression in vitro. The localisation of PfMIF within the iRBC indicates that the protein is likely to be released upon schizont rupture allowing direct interactions with the host immune system.
We thank the children and their parents or guardians for participating in the study. We also greatly appreciate the help of clinical teams at the KEMRI ward and the Outpatients Clinic and the field workers of the malaria study. We would also like to thank Prof. Catherine Braun-Breton for the gift of PfSBP1 anti-sera and Dr David Muhia for his technical assistance.
This study is published with the permission of the director of KEMRI. B.C.U. holds a Wellcome Trust Senior Research Fellowship and T.N.W. holds a Wellcome Trust Senior Clinical Fellowship. S.K. and K.M. are funded by the Wellcome Trust. U.K. acknowledges support from European Commission, Alexander von Humboldt Foundation and Brunel University. This study was supported in part by the European Union BioMalPar network of excellence.
None of the authors reported any conflicting interests.
Some of the data presented in this manuscript have been presented at the Molecular Approaches to Malaria meeting, Lorne, Australia, February 2004.