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Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity

Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Open Forum Infectious Diseases MAJOR ARTICLE Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity 1,2 3 3 3 1 1 3 3 Anna E. van Beek, Isatou Sarr, Simon Correa, Davis Nwakanma, Mieke C. Brouwer, Diana Wouters, Fatou Secka, Suzanne T. B. Anderson, 4 3 5 2,6 5 David J. Conway, Michael Walther, Michael Levin, Taco W. Kuijpers, and Aubrey J. Cunnington 1 2 Department of Immunopathology, Sanquin Research and Landsteiner Laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands; Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital, Academic Medical Centre, Amsterdam, the Netherlands; Medical Research Council Unit, The Gambia at London 4 5 School of Hygiene and Tropical Medicine; Department of Pathogen and Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom; Section of Paediatrics, Imperial College London, London, United Kingdom; Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands Background. Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity. Methods. We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P.  falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity. Results. FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not cor- relate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load. Conclusions. Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by P. falciparum might be useful for malaria prevention or treatment. Keywords. complement Factor H; malaria; severity; susceptibility. Malaria accounts for a huge global burden of disease. Plasmodium complement-mediated defenses when parasites exit host cells falciparum infections account for most of this burden, and most to invade new erythrocytes or undergo sexual reproduction attributable deaths occur in young children in sub-Saharan [2]. Rapid activation of complement opsonizes invading para- Africa. Three clinical syndromes of severe malaria, occurring sites for phagocytosis or lyses them directly and stimulates the alone or in combination, predict the majority of deaths in chil- adaptive immune response. Although complement activation dren: cerebral malaria (characterized by coma), severe anemia, is likely to be protective, excessive activation would be detri- and respiratory distress (which usually indicates metabolic mental to the host, contributing to dysregulated inflammation acidosis and hyperlactatemia) [1]. Why some children develop and destruction of uninfected erythrocytes [2]. To prevent severe malaria and others do not is poorly understood. bystander damage, the complement system is tightly regu- e Th complement system is activated as part of the innate lated by various fluid phase and membrane-bound regulators. immune response to malaria [2]. P. falciparum is vulnerable to One important regulator protecting host endothelial cells and erythrocytes is the alternative pathway inhibitor factor H (FH), which normally circulates in the blood at ~300 µg/mL [3]. Received 23 March 2018; editorial decision 9 July 2018; accepted 10 July 2018. FH plays an essential role in regulating complement activ- Correspondence: A. J. Cunnington, BMBCh, PhD, DTM&H, Section of Paediatrics, Room 231, ity, and its genetic deficiency results in atypical hemolytic ure- Imperial College London, St. Mary’s Campus, Norfolk Place, London, W2 1PG, UK (a.cunning- ton@imperial.ac.uk). mic syndrome [3]. However, many microbes have evolved to Open Forum Infectious Diseases hijack FH to minimize complement activation on their outer © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases membranes. A  well-characterized example is Neisseria menin- Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits gitidis, for which high FH plasma levels increase susceptibility unrestricted reuse, distribution, and reproduction in any medium, provided the original work to invasive disease [4, 5]. P. falciparum can also recruit FH to its is properly cited. surface, protecting parasites from complement and raising the DOI: 10.1093/ofid/ofy166 Factor H Levels in P. falciparum Malaria • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Factor H and P. falciparum Histidine Rich Protein 2 (PfHRP2) Emzyme- possibility that variation in plasma levels of FH may also influ- Linked Immunosorbent Assay ence outcomes in malaria [6–9]. Enzyme-linked immunosorbent assays (ELISAs) to meas- Although in vitro studies on the mechanisms of FH binding ure Factor H and PfHRP2 were performed as previously to the malaria parasite demonstrate a plausible mechanism of described [14, 15]. evading host defense, clinical studies are needed to determine the relevance to disease outcomes. As it has previously been Statistics shown that complement activation levels return to normal Analysis was performed with GraphPad Prism v7.02 (GraphPad within a month aer a m ft alaria episode [10], we determined Software, La Jolla, CA, USA). Differences in mean values convalescent FH plasma levels of children who required hos- between groups were assessed by unpaired t test or 1-way pital treatment for P.  falciparum malaria as a proxy for the analysis of variance (ANOVA) with Tukey’s multiple compar- steady-state situation preceding infection. We compared levels isons test. Correlations were assessed using a nonparamet- with healthy community controls and between those who had ric Spearman’s correlation test. Logistic and linear regression suffered uncomplicated vs severe malaria. models were developed using the GLM function in R. The best model was assessed by the Akaike Information Criterion. METHODS RESULTS Patients and Samples Subjects were Gambian children (<16  years old) recruited To determine whether FH levels may associate with malaria from the Greater Banjul region, where malaria transmission susceptibility and severity, we measured FH in convalescent is seasonal and relatively low [11]. Convalescent heparinized samples from children with uncomplicated (n = 67) and severe plasma was obtained 28  days after presentation from chil- (n = 82) malaria and in healthy community controls (n = 173). dren (median age [interquartile range {IQR}], 5 [3–8] years) Because severe malaria is more common in younger children who had received hospital treatment for P. falciparum malaria we first tested whether FH levels were associated with age and with a parasite density >5000/µL [12–14]. Severe malaria was found that they were not significantly correlated (n  =  322, classified by the presence of 1 or more of the following: severe r  = –.034, P = .54), as was expected based on a previous study anemia (SA; hemoglobin < 5 g/dL), hyperlactatemia (LA; lac- in healthy children [16]. tate > 5 mM), cerebral malaria (CM; Blantyre Coma Scale < 3), We then tested whether FH levels were different between or prostration (SP) [14]. Samples were collected from healthy healthy controls and malaria cases. FH levels were lowest in community control children of similar age (median age [IQR], healthy controls, intermediate in convalescent uncomplicated 3.9 [2.4–5.5] years), recruited from the same region between malaria cases, and highest in convalescent severe malaria cases 2013 and 2014. Control children were not tested for asymp- (mean [95% confidence interval {CI}], 257 [250 to  264], 288 tomatic parasitemia. The study was approved by the Gambia [268 to 309], and 328 [313 to 344] µg/mL, respectively; ANOVA Government/UK Medical Research Council Joint Ethics P  <  .0001) (Figure  1A). This indicates that both the need for Committee and performed in accordance with the Declaration hospital treatment for malaria and severity of malaria associate of Helsinki. with FH. A B <.0001 .0050 .0016 .0011 600 600 400 400 200 200 0 0 HC UM SM UM SP LA SA CM Figure 1. Factor H (FH) plasma levels associate with malaria susceptibility and severity. A, FH was measured by in-house enzyme-linked immunosorbent assay in samples from healthy community control children (HC; n = 173) and in samples obtained 28 days after hospital presentation from children with uncomplicated malaria (UM; n = 67) and severe malaria (SM; n = 82). P values indicate Tukey’s multiple comparisons test performed after 1-way analysis of variance. B, Severe malaria was categorized based on major criteria of severity: severe prostration (SP; n = 69), hyperlactatemia (LA; n = 48), severe anemia (SA; n = 14), and cerebral malaria (CM; n = 29). Due to overlapping clinical features, depicted groups are not mutually exclusive. Unpaired t tests compare the mean of each group with uncomplicated malaria. Bars indicate mean ± SD. 2 • OFID • van Beek et al FH, µg/mL FH, µg/mL Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Severe malaria can manifest in several overlapping syn- To further investigate the association between FH and sever- dromes; therefore, we examined whether the higher levels of ity, we assessed relationships between convalescent FH and FH in those with severe malaria were driven by any particular several host- and parasite-derived markers measured on admis- clinical phenotype. FH levels were similar in all major severe sion. Lactate concentrations correlated significantly with FH malaria phenotypes and were all significantly higher than in levels, but platelets and hemoglobin did not (Figure  2A–C). uncomplicated malaria, with the exception of the small group Markers of circulating parasite load, parasitemia assessed from with SA (mean difference [95% CI] compared with uncom- blood film and parasites/µL, showed significant positive corre- plicated malaria: SP,  43 [18 to  69] µg/mL; LA,  43 [13 to  73] lations with FH levels (Figure 2D, E). µg/mL; SA,  41 [–8 to  90] µg/mL; CM,  37 [2 to  71] µg/mL) Measures of circulating parasite load do not quantify add- (Figure 1B). itional cyto-adherent parasites sequestered in the tissue A B r = .2816 r = –.1273 s s 20 500 P = .0009 P = .1521 0 0 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL C D r = –.1466 r = .2308 s s 20 60 P = .0805 P = .0054 0 0 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL E F G r = .2422 r = .101 r = –.2182 s s s 10 000 7 P = .0067 P = .3004 P = .0292 10 4 10 2 10 0 10 –2 0 200 400 600 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL FH, µg/mL Figure 2. Factor H (FH) plasma levels associate with severity markers. A–D, Correlations of FH plasma levels with severity markers at the time of presentation to hospital: (A) lactate, (B) platelets, (C) hemoglobin (Hb), (D) parasitemia (% of infected erythrocytes in blood film), (E) parasite density, (F) PfHRP2, (G) sequestration index [log (PfHRP2/ parasitemia)]. Spearman’s correlations. Factor H Levels in P. falciparum Malaria • OFID • 3 Parasite density, parasites/μL Hb, g/dL Lactate, mM PfHRP2, ng/mL Platelets, ×10 /L Parasitemia, % Log (PfHRP2/parasitemia) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Table 1. Logistic Regression Models to Predict Severity Model Variable No. Log Odds SE P AIC -8 Age Age 149 –0.37 0.069 5.3 × 10 165 -5 Log PfHRP2 Log PfHRP2 104 1.06 0.25 2.2 × 10 127 FH FH 149 0.0066 0.0023 .0047 205 -5 Age + log PfHRP2 + FH Age 104 –0.45 0.11 2.68 × 10 87 Log PfHRP2 0.91 0.27 .00098 FH 0.01 0.0045 .00133 Age, PfHRP2, and FH were assessed individually or combined in a multivariate model to predict severity. Abbreviations: AIC, Akaike Information Criterion; FH, Factor H. microvasculature. Total body parasite load can be assessed treatment with >5000 parasites/µL in our study, we ensured using the plasma concentration of the soluble parasite pro- high specificity of the malaria diagnosis. tein PfHRP2, which reflects both sequestered and circulating By comparing healthy children with those who had suffered a parasite numbers and is more predictive of clinical outcome definite malaria episode, we expected that even relatively small of infection [14, 17]. Surprisingly, there was no correlation differences in FH between the groups would indicate an asso- between PfHRP2 concentration and FH (Figure  2F). As an ciation between FH and susceptibility to symptomatic malaria: index of sequestration, we calculated the ratio of PfHRP2 to the outcome of a new infection in the controls could be either parasitemia (higher values would be expected to indicate rel- asymptomatic or symptomatic, whereas our malaria cases are atively more sequestration) and found that this was negatively all symptomatic. Thus our results indicate that higher levels of correlated with FH (Figure 2G). FH are associated with susceptibility to symptomatic malaria. In logistic regression models, age, PfHRP2, and FH all pre- Among subjects with malaria, the associations of FH with clin- dicted severity, and a multivariate model incorporating all 3 ical features of severe disease, lactate concentration and cir- indicated that that they have independent associations with culating parasite load, are consistent with the hypothesis that outcome (Table  1). Linear regression models indicated that higher levels of FH also predispose to severe malaria. Indeed, age, PfHRP2, and FH were also independently associated with multivariate models suggested that the association of FH with blood lactate concentration (Table 2). severity was independent of both age and parasite load. Although high FH levels may allow parasites to evade DISCUSSION complement-mediated clearance, high levels may also limit endothelial activation and expression of adhesion molecules Despite the huge evolutionary pressure malaria has exerted, involved in sequestration of parasitized erythrocytes [2]. If high surprisingly few host factors have been convincingly shown FH levels favor parasite multiplication but restrict sequestra- to influence susceptibility or severity. Of the host factors with tion, one might expect a stronger correlation with circulating the strongest evidence, almost all involve the erythrocyte [18]. parasite load than with PfHRP2 concentration (which reflects Our results suggest that natural variation in FH levels is another both circulating and sequestered parasites). This would also be determinant of malaria susceptibility and severity. consistent with the negative correlation observed between FH All children in malaria-endemic countries are potentially and the sequestration index (PfHRP2:parasitemia). susceptible to P. falciparum infection, but not all of these infec- Although total body parasite load is a strong predictor of tions will result in symptomatic malaria. The likelihood of an severity, it is not the only determinant [20]. e Th rapidity of par - infection causing symptomatic disease is dependent on para- asite growth in the circulation may also contribute to severity if site density [19]. As we only included children seeking hospital Table 2. Linear Regression Model to Predict Blood Lactate Concentration Model Variable No. Coefficient SE P AIC -11 Age Age 138 –0.11 0.015 3.02 × 10 263 -9 Log PfHRP2 Log PfHRP2 100 0.23 0.036 6.7 × 10 166 FH FH 138 0.002 0.00077 .0094 301 -8 Age + log PfHRP2 + FH Age 100 –0.073 0.012 7.4 × 10 131 -8 Log PfHRP2 0.18 0.030 3.5 × 10 FH 0.0021 0.00063 .0011 Age, PfHRP2, and FH were assessed individually or combined in a multivariate model to predict ln (lactate). Abbreviations: AIC, Akaike Information Criterion; FH, Factor H. 4 • OFID • van Beek et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 3. Parente R, Clark SJ, Inforzato A, et al. Complement Factor H in host defense and the consequent pro-inflammatory responses occur faster than immune evasion. Cell Mol Life Sci 2017; 74:1605–24. regulatory and protective responses, which are necessary to 4. Haralambous E, Dolly SO, Hibberd ML, et  al. Factor H, a regulator of comple- ment activity, is a major determinant of meningococcal disease susceptibility in limit immunopathology. Our study design precluded analysis UK Caucasian patients. Scand J Infect Dis 2006; 38:764–71. of nonsurvivors, so we cannot exclude the possibility that high 5. Józsi M. Factor H family proteins in complement evasion of microorganisms. FH levels modulated the risk of death. Front Immunol 2017; 8:1–8. 6. Simon N, Lasonder E, Scheuermayer M, et  al. Malaria parasites co-opt human e m Th ain limitation of our study is that we used convales- Factor H to prevent complement-mediated lysis in the mosquito midgut. Cell cent plasma samples, which we believe are representative of the Host Microbe 2013; 13:29–41. 7. Kennedy AT, Schmidt CQ, Thompson JK, et  al. Recruitment of Factor H as a pre-infection status of our subjects. We cannot exclude that dif- novel complement evasion strategy for blood-stage Plasmodium falciparum infec- ferences in FH levels could represent a residual effect of infec- tion. J Immunol 2015; 1501581. 8. Rosa TF, Flammersfeld A, Ngwa CJ, et  al. The Plasmodium falciparum blood tion, although we think this is unlikely because of the lack of stages acquire Factor H family proteins to evade destruction by human comple- association with PfHRP2 concentrations. A  large prospective ment. Cell Microbiol 2016; 18:573–90. 9. Simon N, Friedrich O, Kappes B. Quantification of human complement Factor study would be required to confirm that natural variation in H binding to asexual malaria blood stages by an enzyme-linked immunosorbent FH levels definitely does predict susceptibility, severity, and assay. Vaccine 2018; 1–3. mortality from malaria. Further studies will be needed to clar- 10. Roestenberg M, McCall M, Mollnes TE, et  al. Complement activation in experimental human malaria infection. Trans R Soc Trop Med Hyg 2007; ify the relative roles of complement activation and regulation 101:643–9. in malaria, but interference with parasite FH binding might be 11. Ceesay SJ, Casals-Pascual C, Erskine J, et  al. Changes in malaria indices between 1999 and 2007 in the Gambia: a retrospective analysis. Lancet 2008; considered as an adjunctive approach to limit parasite growth, 372:1545–54. enhance parasite clearance, and tackle drug resistance. 12. Walther M, Jeffries D, Finney OC, et al. Distinct roles for FOXP3 and FOXP3 CD4 T cells in regulating cellular immunity to uncomplicated and severe Plasmodium Acknowledgments falciparum malaria. PLoS Pathog 2009; 5:e1000364. 13. Walther M, de Caul A, Aka P, et al. HMOX1 gene promoter alleles and high HO-1 e a Th uthors would like to thank Ilse Jongerius (Sanquin Research) for levels are associated with severe malaria in Gambian children. PLoS Pathog 2012; critically reading the manuscript. 8:e1002579. Financial support. This work was supported by the European Union’s 14. Cunnington AJ, Bretscher MT, Nogaro SI, et al. Comparison of parasite seques- seventh Framework Program (EC-GA No. 279185; EUCLIDS, www. tration in uncomplicated and severe childhood Plasmodium falciparum malaria. J euclids-project.eu); the UK Medical Research Council (MRC) and the UK Infect 2013; 67:220–30. Department for International Development (DFID) under the MRC/DFID 15. Pouw RB, Brouwer MC, Geissler J, et al. Complement Factor H-related protein 3 Concordat agreement, which is also part of the EDCTP2 programme sup- serum levels are low compared to Factor H and mainly determined by gene copy ported by the European Union (MR/L006529/1 to A.J.C.); and core fund- number variation in CFHR3. PLoS One 2016; 11:e0152164. ing from the UK MRC to the MRC Unit The Gambia at London School of 16. de Paula PF, Barbosa JE, Junior PR, et  al. Ontogeny of complement regulatory proteins—concentrations of Factor H, factor I, c4b-binding protein, proper- Hygiene and Tropical Medicine. din and vitronectin in healthy children of different ages and in adults. Scand J Potential conifl cts of interest. All authors: no reported conflicts of Immunol 2003; 58:572–7. interest. All authors have submitted the ICMJE Form for Disclosure of 17. Hendriksen ICE, Mwanga-Amumpaire J, von Seidlein L, et al. Diagnosing severe Potential Conflicts of Interest. Conflicts that the editors consider relevant to falciparum malaria in parasitaemic African children: a prospective evaluation of the content of the manuscript have been disclosed. plasma PfHRP2 measurement. PLoS Med 2012; 9:e1001297. 18. Mangano VD, Modiano D. Host genetics and parasitic infections. Clin Microbiol References Infect 2014; 20:1265–75. 1. Marsh K, Forster D, Waruiru C, et  al. Indicators of life-threatening malaria in 19. Mwangi TW, Ross A, Snow RW, et al. Case definitions of clinical malaria under African children. N Engl J Med 1995; 332:1399–404. different transmission conditions in Kilifi District, Kenya. J Infect Dis 2005; 2. Silver KL, Higgins SJ, McDonald CR, Kain KC. Complement driven innate 191:1932–9. immune response to malaria: fuelling severe malarial diseases. Cell Microbiol 20. Cunnington AJ, Walther M, Riley EM. Piecing together the puzzle of severe 2010; 12:1036–45. malaria. Sci Transl Med 2013; 5:211ps18. Factor H Levels in P. falciparum Malaria • OFID • 5 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity

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Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Open Forum Infectious Diseases MAJOR ARTICLE Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity 1,2 3 3 3 1 1 3 3 Anna E. van Beek, Isatou Sarr, Simon Correa, Davis Nwakanma, Mieke C. Brouwer, Diana Wouters, Fatou Secka, Suzanne T. B. Anderson, 4 3 5 2,6 5 David J. Conway, Michael Walther, Michael Levin, Taco W. Kuijpers, and Aubrey J. Cunnington 1 2 Department of Immunopathology, Sanquin Research and Landsteiner Laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands; Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital, Academic Medical Centre, Amsterdam, the Netherlands; Medical Research Council Unit, The Gambia at London 4 5 School of Hygiene and Tropical Medicine; Department of Pathogen and Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom; Section of Paediatrics, Imperial College London, London, United Kingdom; Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands Background. Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity. Methods. We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P.  falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity. Results. FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not cor- relate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load. Conclusions. Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by P. falciparum might be useful for malaria prevention or treatment. Keywords. complement Factor H; malaria; severity; susceptibility. Malaria accounts for a huge global burden of disease. Plasmodium complement-mediated defenses when parasites exit host cells falciparum infections account for most of this burden, and most to invade new erythrocytes or undergo sexual reproduction attributable deaths occur in young children in sub-Saharan [2]. Rapid activation of complement opsonizes invading para- Africa. Three clinical syndromes of severe malaria, occurring sites for phagocytosis or lyses them directly and stimulates the alone or in combination, predict the majority of deaths in chil- adaptive immune response. Although complement activation dren: cerebral malaria (characterized by coma), severe anemia, is likely to be protective, excessive activation would be detri- and respiratory distress (which usually indicates metabolic mental to the host, contributing to dysregulated inflammation acidosis and hyperlactatemia) [1]. Why some children develop and destruction of uninfected erythrocytes [2]. To prevent severe malaria and others do not is poorly understood. bystander damage, the complement system is tightly regu- e Th complement system is activated as part of the innate lated by various fluid phase and membrane-bound regulators. immune response to malaria [2]. P. falciparum is vulnerable to One important regulator protecting host endothelial cells and erythrocytes is the alternative pathway inhibitor factor H (FH), which normally circulates in the blood at ~300 µg/mL [3]. Received 23 March 2018; editorial decision 9 July 2018; accepted 10 July 2018. FH plays an essential role in regulating complement activ- Correspondence: A. J. Cunnington, BMBCh, PhD, DTM&H, Section of Paediatrics, Room 231, ity, and its genetic deficiency results in atypical hemolytic ure- Imperial College London, St. Mary’s Campus, Norfolk Place, London, W2 1PG, UK (a.cunning- ton@imperial.ac.uk). mic syndrome [3]. However, many microbes have evolved to Open Forum Infectious Diseases hijack FH to minimize complement activation on their outer © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases membranes. A  well-characterized example is Neisseria menin- Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits gitidis, for which high FH plasma levels increase susceptibility unrestricted reuse, distribution, and reproduction in any medium, provided the original work to invasive disease [4, 5]. P. falciparum can also recruit FH to its is properly cited. surface, protecting parasites from complement and raising the DOI: 10.1093/ofid/ofy166 Factor H Levels in P. falciparum Malaria • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Factor H and P. falciparum Histidine Rich Protein 2 (PfHRP2) Emzyme- possibility that variation in plasma levels of FH may also influ- Linked Immunosorbent Assay ence outcomes in malaria [6–9]. Enzyme-linked immunosorbent assays (ELISAs) to meas- Although in vitro studies on the mechanisms of FH binding ure Factor H and PfHRP2 were performed as previously to the malaria parasite demonstrate a plausible mechanism of described [14, 15]. evading host defense, clinical studies are needed to determine the relevance to disease outcomes. As it has previously been Statistics shown that complement activation levels return to normal Analysis was performed with GraphPad Prism v7.02 (GraphPad within a month aer a m ft alaria episode [10], we determined Software, La Jolla, CA, USA). Differences in mean values convalescent FH plasma levels of children who required hos- between groups were assessed by unpaired t test or 1-way pital treatment for P.  falciparum malaria as a proxy for the analysis of variance (ANOVA) with Tukey’s multiple compar- steady-state situation preceding infection. We compared levels isons test. Correlations were assessed using a nonparamet- with healthy community controls and between those who had ric Spearman’s correlation test. Logistic and linear regression suffered uncomplicated vs severe malaria. models were developed using the GLM function in R. The best model was assessed by the Akaike Information Criterion. METHODS RESULTS Patients and Samples Subjects were Gambian children (<16  years old) recruited To determine whether FH levels may associate with malaria from the Greater Banjul region, where malaria transmission susceptibility and severity, we measured FH in convalescent is seasonal and relatively low [11]. Convalescent heparinized samples from children with uncomplicated (n = 67) and severe plasma was obtained 28  days after presentation from chil- (n = 82) malaria and in healthy community controls (n = 173). dren (median age [interquartile range {IQR}], 5 [3–8] years) Because severe malaria is more common in younger children who had received hospital treatment for P. falciparum malaria we first tested whether FH levels were associated with age and with a parasite density >5000/µL [12–14]. Severe malaria was found that they were not significantly correlated (n  =  322, classified by the presence of 1 or more of the following: severe r  = –.034, P = .54), as was expected based on a previous study anemia (SA; hemoglobin < 5 g/dL), hyperlactatemia (LA; lac- in healthy children [16]. tate > 5 mM), cerebral malaria (CM; Blantyre Coma Scale < 3), We then tested whether FH levels were different between or prostration (SP) [14]. Samples were collected from healthy healthy controls and malaria cases. FH levels were lowest in community control children of similar age (median age [IQR], healthy controls, intermediate in convalescent uncomplicated 3.9 [2.4–5.5] years), recruited from the same region between malaria cases, and highest in convalescent severe malaria cases 2013 and 2014. Control children were not tested for asymp- (mean [95% confidence interval {CI}], 257 [250 to  264], 288 tomatic parasitemia. The study was approved by the Gambia [268 to 309], and 328 [313 to 344] µg/mL, respectively; ANOVA Government/UK Medical Research Council Joint Ethics P  <  .0001) (Figure  1A). This indicates that both the need for Committee and performed in accordance with the Declaration hospital treatment for malaria and severity of malaria associate of Helsinki. with FH. A B <.0001 .0050 .0016 .0011 600 600 400 400 200 200 0 0 HC UM SM UM SP LA SA CM Figure 1. Factor H (FH) plasma levels associate with malaria susceptibility and severity. A, FH was measured by in-house enzyme-linked immunosorbent assay in samples from healthy community control children (HC; n = 173) and in samples obtained 28 days after hospital presentation from children with uncomplicated malaria (UM; n = 67) and severe malaria (SM; n = 82). P values indicate Tukey’s multiple comparisons test performed after 1-way analysis of variance. B, Severe malaria was categorized based on major criteria of severity: severe prostration (SP; n = 69), hyperlactatemia (LA; n = 48), severe anemia (SA; n = 14), and cerebral malaria (CM; n = 29). Due to overlapping clinical features, depicted groups are not mutually exclusive. Unpaired t tests compare the mean of each group with uncomplicated malaria. Bars indicate mean ± SD. 2 • OFID • van Beek et al FH, µg/mL FH, µg/mL Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Severe malaria can manifest in several overlapping syn- To further investigate the association between FH and sever- dromes; therefore, we examined whether the higher levels of ity, we assessed relationships between convalescent FH and FH in those with severe malaria were driven by any particular several host- and parasite-derived markers measured on admis- clinical phenotype. FH levels were similar in all major severe sion. Lactate concentrations correlated significantly with FH malaria phenotypes and were all significantly higher than in levels, but platelets and hemoglobin did not (Figure  2A–C). uncomplicated malaria, with the exception of the small group Markers of circulating parasite load, parasitemia assessed from with SA (mean difference [95% CI] compared with uncom- blood film and parasites/µL, showed significant positive corre- plicated malaria: SP,  43 [18 to  69] µg/mL; LA,  43 [13 to  73] lations with FH levels (Figure 2D, E). µg/mL; SA,  41 [–8 to  90] µg/mL; CM,  37 [2 to  71] µg/mL) Measures of circulating parasite load do not quantify add- (Figure 1B). itional cyto-adherent parasites sequestered in the tissue A B r = .2816 r = –.1273 s s 20 500 P = .0009 P = .1521 0 0 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL C D r = –.1466 r = .2308 s s 20 60 P = .0805 P = .0054 0 0 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL E F G r = .2422 r = .101 r = –.2182 s s s 10 000 7 P = .0067 P = .3004 P = .0292 10 4 10 2 10 0 10 –2 0 200 400 600 0 200 400 600 0 200 400 600 FH, µg/mL FH, µg/mL FH, µg/mL Figure 2. Factor H (FH) plasma levels associate with severity markers. A–D, Correlations of FH plasma levels with severity markers at the time of presentation to hospital: (A) lactate, (B) platelets, (C) hemoglobin (Hb), (D) parasitemia (% of infected erythrocytes in blood film), (E) parasite density, (F) PfHRP2, (G) sequestration index [log (PfHRP2/ parasitemia)]. Spearman’s correlations. Factor H Levels in P. falciparum Malaria • OFID • 3 Parasite density, parasites/μL Hb, g/dL Lactate, mM PfHRP2, ng/mL Platelets, ×10 /L Parasitemia, % Log (PfHRP2/parasitemia) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 Table 1. Logistic Regression Models to Predict Severity Model Variable No. Log Odds SE P AIC -8 Age Age 149 –0.37 0.069 5.3 × 10 165 -5 Log PfHRP2 Log PfHRP2 104 1.06 0.25 2.2 × 10 127 FH FH 149 0.0066 0.0023 .0047 205 -5 Age + log PfHRP2 + FH Age 104 –0.45 0.11 2.68 × 10 87 Log PfHRP2 0.91 0.27 .00098 FH 0.01 0.0045 .00133 Age, PfHRP2, and FH were assessed individually or combined in a multivariate model to predict severity. Abbreviations: AIC, Akaike Information Criterion; FH, Factor H. microvasculature. Total body parasite load can be assessed treatment with >5000 parasites/µL in our study, we ensured using the plasma concentration of the soluble parasite pro- high specificity of the malaria diagnosis. tein PfHRP2, which reflects both sequestered and circulating By comparing healthy children with those who had suffered a parasite numbers and is more predictive of clinical outcome definite malaria episode, we expected that even relatively small of infection [14, 17]. Surprisingly, there was no correlation differences in FH between the groups would indicate an asso- between PfHRP2 concentration and FH (Figure  2F). As an ciation between FH and susceptibility to symptomatic malaria: index of sequestration, we calculated the ratio of PfHRP2 to the outcome of a new infection in the controls could be either parasitemia (higher values would be expected to indicate rel- asymptomatic or symptomatic, whereas our malaria cases are atively more sequestration) and found that this was negatively all symptomatic. Thus our results indicate that higher levels of correlated with FH (Figure 2G). FH are associated with susceptibility to symptomatic malaria. In logistic regression models, age, PfHRP2, and FH all pre- Among subjects with malaria, the associations of FH with clin- dicted severity, and a multivariate model incorporating all 3 ical features of severe disease, lactate concentration and cir- indicated that that they have independent associations with culating parasite load, are consistent with the hypothesis that outcome (Table  1). Linear regression models indicated that higher levels of FH also predispose to severe malaria. Indeed, age, PfHRP2, and FH were also independently associated with multivariate models suggested that the association of FH with blood lactate concentration (Table 2). severity was independent of both age and parasite load. Although high FH levels may allow parasites to evade DISCUSSION complement-mediated clearance, high levels may also limit endothelial activation and expression of adhesion molecules Despite the huge evolutionary pressure malaria has exerted, involved in sequestration of parasitized erythrocytes [2]. If high surprisingly few host factors have been convincingly shown FH levels favor parasite multiplication but restrict sequestra- to influence susceptibility or severity. Of the host factors with tion, one might expect a stronger correlation with circulating the strongest evidence, almost all involve the erythrocyte [18]. parasite load than with PfHRP2 concentration (which reflects Our results suggest that natural variation in FH levels is another both circulating and sequestered parasites). This would also be determinant of malaria susceptibility and severity. consistent with the negative correlation observed between FH All children in malaria-endemic countries are potentially and the sequestration index (PfHRP2:parasitemia). susceptible to P. falciparum infection, but not all of these infec- Although total body parasite load is a strong predictor of tions will result in symptomatic malaria. The likelihood of an severity, it is not the only determinant [20]. e Th rapidity of par - infection causing symptomatic disease is dependent on para- asite growth in the circulation may also contribute to severity if site density [19]. As we only included children seeking hospital Table 2. Linear Regression Model to Predict Blood Lactate Concentration Model Variable No. Coefficient SE P AIC -11 Age Age 138 –0.11 0.015 3.02 × 10 263 -9 Log PfHRP2 Log PfHRP2 100 0.23 0.036 6.7 × 10 166 FH FH 138 0.002 0.00077 .0094 301 -8 Age + log PfHRP2 + FH Age 100 –0.073 0.012 7.4 × 10 131 -8 Log PfHRP2 0.18 0.030 3.5 × 10 FH 0.0021 0.00063 .0011 Age, PfHRP2, and FH were assessed individually or combined in a multivariate model to predict ln (lactate). Abbreviations: AIC, Akaike Information Criterion; FH, Factor H. 4 • OFID • van Beek et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy166/5056423 by guest on 16 October 2019 3. Parente R, Clark SJ, Inforzato A, et al. Complement Factor H in host defense and the consequent pro-inflammatory responses occur faster than immune evasion. Cell Mol Life Sci 2017; 74:1605–24. regulatory and protective responses, which are necessary to 4. Haralambous E, Dolly SO, Hibberd ML, et  al. Factor H, a regulator of comple- ment activity, is a major determinant of meningococcal disease susceptibility in limit immunopathology. Our study design precluded analysis UK Caucasian patients. Scand J Infect Dis 2006; 38:764–71. of nonsurvivors, so we cannot exclude the possibility that high 5. Józsi M. Factor H family proteins in complement evasion of microorganisms. FH levels modulated the risk of death. Front Immunol 2017; 8:1–8. 6. Simon N, Lasonder E, Scheuermayer M, et  al. Malaria parasites co-opt human e m Th ain limitation of our study is that we used convales- Factor H to prevent complement-mediated lysis in the mosquito midgut. Cell cent plasma samples, which we believe are representative of the Host Microbe 2013; 13:29–41. 7. Kennedy AT, Schmidt CQ, Thompson JK, et  al. Recruitment of Factor H as a pre-infection status of our subjects. We cannot exclude that dif- novel complement evasion strategy for blood-stage Plasmodium falciparum infec- ferences in FH levels could represent a residual effect of infec- tion. J Immunol 2015; 1501581. 8. Rosa TF, Flammersfeld A, Ngwa CJ, et  al. The Plasmodium falciparum blood tion, although we think this is unlikely because of the lack of stages acquire Factor H family proteins to evade destruction by human comple- association with PfHRP2 concentrations. A  large prospective ment. Cell Microbiol 2016; 18:573–90. 9. Simon N, Friedrich O, Kappes B. Quantification of human complement Factor study would be required to confirm that natural variation in H binding to asexual malaria blood stages by an enzyme-linked immunosorbent FH levels definitely does predict susceptibility, severity, and assay. Vaccine 2018; 1–3. mortality from malaria. Further studies will be needed to clar- 10. Roestenberg M, McCall M, Mollnes TE, et  al. Complement activation in experimental human malaria infection. Trans R Soc Trop Med Hyg 2007; ify the relative roles of complement activation and regulation 101:643–9. in malaria, but interference with parasite FH binding might be 11. Ceesay SJ, Casals-Pascual C, Erskine J, et  al. Changes in malaria indices between 1999 and 2007 in the Gambia: a retrospective analysis. Lancet 2008; considered as an adjunctive approach to limit parasite growth, 372:1545–54. enhance parasite clearance, and tackle drug resistance. 12. Walther M, Jeffries D, Finney OC, et al. Distinct roles for FOXP3 and FOXP3 CD4 T cells in regulating cellular immunity to uncomplicated and severe Plasmodium Acknowledgments falciparum malaria. PLoS Pathog 2009; 5:e1000364. 13. Walther M, de Caul A, Aka P, et al. HMOX1 gene promoter alleles and high HO-1 e a Th uthors would like to thank Ilse Jongerius (Sanquin Research) for levels are associated with severe malaria in Gambian children. PLoS Pathog 2012; critically reading the manuscript. 8:e1002579. Financial support. This work was supported by the European Union’s 14. Cunnington AJ, Bretscher MT, Nogaro SI, et al. Comparison of parasite seques- seventh Framework Program (EC-GA No. 279185; EUCLIDS, www. tration in uncomplicated and severe childhood Plasmodium falciparum malaria. J euclids-project.eu); the UK Medical Research Council (MRC) and the UK Infect 2013; 67:220–30. Department for International Development (DFID) under the MRC/DFID 15. Pouw RB, Brouwer MC, Geissler J, et al. Complement Factor H-related protein 3 Concordat agreement, which is also part of the EDCTP2 programme sup- serum levels are low compared to Factor H and mainly determined by gene copy ported by the European Union (MR/L006529/1 to A.J.C.); and core fund- number variation in CFHR3. PLoS One 2016; 11:e0152164. ing from the UK MRC to the MRC Unit The Gambia at London School of 16. de Paula PF, Barbosa JE, Junior PR, et  al. Ontogeny of complement regulatory proteins—concentrations of Factor H, factor I, c4b-binding protein, proper- Hygiene and Tropical Medicine. din and vitronectin in healthy children of different ages and in adults. Scand J Potential conifl cts of interest. All authors: no reported conflicts of Immunol 2003; 58:572–7. interest. All authors have submitted the ICMJE Form for Disclosure of 17. Hendriksen ICE, Mwanga-Amumpaire J, von Seidlein L, et al. Diagnosing severe Potential Conflicts of Interest. Conflicts that the editors consider relevant to falciparum malaria in parasitaemic African children: a prospective evaluation of the content of the manuscript have been disclosed. plasma PfHRP2 measurement. PLoS Med 2012; 9:e1001297. 18. Mangano VD, Modiano D. Host genetics and parasitic infections. Clin Microbiol References Infect 2014; 20:1265–75. 1. Marsh K, Forster D, Waruiru C, et  al. Indicators of life-threatening malaria in 19. Mwangi TW, Ross A, Snow RW, et al. Case definitions of clinical malaria under African children. N Engl J Med 1995; 332:1399–404. different transmission conditions in Kilifi District, Kenya. J Infect Dis 2005; 2. Silver KL, Higgins SJ, McDonald CR, Kain KC. Complement driven innate 191:1932–9. immune response to malaria: fuelling severe malarial diseases. Cell Microbiol 20. Cunnington AJ, Walther M, Riley EM. Piecing together the puzzle of severe 2010; 12:1036–45. malaria. Sci Transl Med 2013; 5:211ps18. Factor H Levels in P. falciparum Malaria • OFID • 5

Journal

Open Forum Infectious Diseases – Oxford University Press

Published: Jul 1, 2018

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