Immunogenetic Control of Antibody Responsiveness in a Malaria Endemic Area Danielle Carpenter, Hind Abushama, Sándor Bereczky, Anna Färnert, Ingegerd Rooth, Marita Troye-Blomberg, Rupert J. Quinnell, and Marie-Anne Shaw ABSTRACT: This study builds upon the established genetic control of antimalarial immune responses and prior association studies by using a family-based approach, transmission disequilibrium testing, to identify immune response genes that influence antibody responses to Plasmodium falciparum infection in an endemic Tanzanian population. Candidate polymorphisms are within the interleukin-1 (IL-1) gene cluster, the IL-10 promoter, Major histocompatibility complex class II and III, the 5q31-q33 region, and the T-Cell Receptor beta variable ABBREVIATIONS HLA Human leukocyte antigen IL Interleukin MHC Major histocompatibility complex
INTRODUCTION Malaria is a significant cause of worldwide mortality and morbidity. Individuals living in endemic areas experience persistent subclinical malaria infection, but only a minority develops severe disease. These variations in disease pattern are attributable to a number of different factors, which include the genetic background of both host and pathogen. There is now a significant body of evidence to indicate that the genes affecting the immune response can influence the outcome of malaria infection, and the capacity to mount a humoral response [1].
From the Institute of Integrative and Comparative Biology, University of Leeds, Leeds, England (D.C., H.A., R.J.Q., M.-A.S.); Infectious Diseases Unit, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden (S.B., A.F.); Nyamisati Malaria Research, Rufiji, National Institute for Medical Research, Dar-es-Salaam, Tanzania (I.R); and Department of Immunology, Stockholm University, Stockholm, Sweden (M.T.-B.). Address reprint requests to: Dr. Danielle Carpenter, Academic Unit of Anaesthesia, 8.11, Clinical Sciences Building, St. James University Hospital, Leeds, York LS9 7TF, UK; Tel: ⫹44 113 206 5274; Fax: ⫹44 113 206 4140; E-mail:
[email protected]. Received September 8, 2006; revised November 28, 2006; accepted December 1, 2006. Human Immunology 68, 165–169 (2007) © American Society for Histocompatibility and Immunogenetics, 2007 Published by Elsevier Inc.
region. There was a significant association between the IL1RN alleles and total IgE. Weak evidence for association was present between polymorphisms in the IL10 promoter region and both anti-P falciparum IgE and IgG4 antibodies. Human Immunology 68, 165–169 (2007). © American Society for Histocompatibility and Immunogenetics, 2007. Published by Elsevier Inc. KEYWORDS: P falciparum; IL1; IL10; IgE; IgG; TDT; association
SNP TCRBV VNTR
Single nucleotide polymorphism T-Cell Receptor beta variable Variable number of tandem repeats
In this paper, we investigate the genetic control of antimalaria antibody responsiveness to subclinical infection within a malaria-endemic area. The importance of antibody responses during malaria infection has long since been observed, with the production of specific IgG antibodies required for the acquisition of functional immunity to malaria; however, protection is subclass dependent. Malaria infection in humans has also been associated with an increase in total and malaria-specific IgE production [2]. Malaria-specific IgE has previously been associated with severity of malaria disease [2] and with parasitemia [3], although recent results from the current population demonstrate that high malaria-specific IgE levels in asymptomatic individuals are associated with reduced risk for subsequent clinical episodes [4]. The significance of the elevated total IgE in severe malaria infection is unknown [5]. There have been few candidate gene studies of immune responses in malaria. Control of parasitemia, and total IgE in nonparasitic populations, have previously been linked to chromosome 5q31-q33, which contains gene coding for the cytokines IL-4 and IL-13 [6, 7]. 0198-8859/07/$–see front matter doi:10.1016/j.humimm.2006.12.002
166
Polymorphisms in the IL-1 gene cluster have been implicated in the control of both malaria and IgE levels [8 –10], and the down regulatory cytokine IL-10 is also involved in the differential control of IgE and IgG4 levels [11]. Human leukocyte antigen (HLA) class II restriction of the cellular response to specific erythrocytic stage antigens have been demonstrated to exist and reveal associations with a number of different HLA class II alleles [12]. Although the allied T-cell receptor has yet to be closely investigated with respect to malaria, it is an attractive candidate due to its variant nature and essential role in the immune response to malaria. In the current study, we have investigated the role of polymorphisms in a number of candidate genes for control of antibody responses to malaria in a well-studied Tanzanian population by using family-based association methods. Candidate genes (IL1, IL10, MHC class II and III, IL4, IL13, and TCRBV) were chosen for their potential role in the control of both antibody responses and malaria infection. MATERIALS AND METHODS Patients The fishing village of Nyamisati, 150 km south of Dar-es-Salaam, Tanzania is a holoendemic area for malaria, with transmission increasing during the two rainy seasons (April to June and November to December), the predominant malaria species being Plasmodium falciparum. There is no cerebral malaria observed in the village. The study population is of Bantu origin and has previously been described [4]. To enable family-based analysis, parental information was collected and 167 extended pedigrees compiled, composed of two or more generations and containing 303 nuclear families and 1469 individuals. Venous blood was collected in 1999 —1050 samples, of which 860 samples were from related individuals and 190 from unrelated individuals—with informed consent and approval of the local ethics committees. Human genomic DNA was extracted from the frozen venous blood by using standard procedures of salting out, followed by phenol-chloroform extraction. Genotyping SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) by using previously published methods on the following polymorphisms. The observed heterozygosity is also presented for each locus as a measure of information provided by the polymorphism: IL1B⫺511 (heterozygosity: 0.417); IL10⫺592 (heterozygosity: 0.49) and IL10⫺1082(heterozygosity: 0.495); TNF⫺308 (heterozygosity: 0.152) and LTA⫺368 (heterozygosity:
D. Carpenter et al.
0.499); IL13 (heterozygosity: 0.375); and BV8S3 (heterozygosity; 0.22), BV24S1 (heterozygosity: 0.478), BV2S1 (heterozygosity: 0.22), BV15S1 (heterozygosity: 0.167), and BV3S1 (heterozygosity: 0.376). VNTRs were typed by polymerase chain reaction (PCR) and agarose gel electrophoresis: IL1RNVNTR (heterozygosity: 0.238) and IL4VNTR (heterozygosity: 0.43). HLA class II loci were typed using sequence-specific oligonucleotide probes to detect polymorphisms in HLA-DQB1 (heterozygosity: 0.839), HLA-DQA1 (heterozygosity: 0.838), and HLA-DRB1 (heterozygosity: 0.87). Genotype frequencies at all SNP and VNTR loci did not differ significantly from those expected under Hardy-Weinberg equilibrium, with the exception of BV2S1. The genotyping at this locus was repeated and genotypes confirmed. Known haplotypes were determined from family data for IL10 polymorphisms. Serology The levels of total IgE antibodies and of antimalarial IgE and IgG antibodies were determined by ELISA as described previously [4]. P falciparum specific assays used lysates of infected erythrocytes (P falciparum laboratory line F32) [2, 4]. Antimalarial IgG4 antibodies were determined using lysates of infected erythrocytes (P falciparum laboratory line NF54 3D7). Thirty-three individuals manifested a clinical episode at the time of sampling; antibody levels did not differ significantly between individuals with and without clinical disease. Frequency distributions of antibody levels were positively skewed, so data were log transformed before analysis. Antibody levels were adjusted for age and sex effects by multiple regression. Heritability of the adjusted values was estimated using SOLAR [13]. Statistical Analysis Associations between antibody levels and alleles at candidate loci, together with IL-10 haplotypes, were tested by a quantitative transmission disequilibrium test [14] with the program QTDT version 2.1.3 [14]. To control for any population stratification, we tested the withinfamily association in a model including environmental, polygenic heritability, and additive major locus variance components. A multiallelic test was performed for multiallelic loci, thereby reducing multiple testing and generating a single p value per locus. To control for any nonnormality, empirical p values were calculated by permutation. Uncorrected p values are presented. However, since eight independent loci were tested (i.e., not in linkage disequilibrium with another typed locus), only results with p ⬍ 0.00625 (equivalent to 0.05 divided by 8) were considered significant. Linkage disequilibrium between adjacent loci was calculated on
Immunogenetic Control of Antibody Responsiveness in a Malaria Endemic Area
unrelated individuals genotyped for all loci in the haplotype, with the program EH [15]. RESULTS Relatedness of Phenotypes Studied There were significant positive correlations between all measured antibodies, with the exception being between specific IgE and specific IgG4, where the lowest levels were observed (Table 1). The heritability of each trait (⫾SE) was 47% ⫾ 8% for total IgE, 25% ⫾ 6% for anti-P falciparum IgE, 25% ⫾ 7% for anti-P falciparum IgG, and 44% ⫾ 8% for anti-P falciparum IgG4 (all p ⬍ 0.001). Table 2 presents a summary of the quantitative transmission disequilibrium testing. The most significant finding was an association between IL1RNVNTR and total IgE levels, with an uncorrected multiallelic p value for this association of p ⫽ 0.0006. IL1RNVNTR*1 was significantly associated negatively with total IgE levels (p ⫽ 0.0002), whereas IL1RNVNTR*2 and *3 were positively associated (p ⫽ 0.043 and p ⫽ 0.009, respectively). Thus, our data suggests that there is significant association between IL1RNVNTR*1 and total IgE levels. There was no association with IL1B, and no significant linkage disequilibrium between IL1B⫺511 and IL1RNVNTR (␦ ⫽ 0.049, p ⬎ 0.05). There was weak evidence for a negative association between IL10⫺592*A and total IgE (p ⫽ 0.041), and a negative association between IL10⫺1082*A and anti-P falciparum IgG4 antibodies (p ⫽ 0.042). Analysis of two-locus IL10 haplotypes demonstrated association with both P falciparum IgE (p ⫽ 0.05) and IgG4 (p ⫽ 0.039), with a negative association between the haplotype IL10⫺1082*A-IL10⫺592*A and both anti-P falciparum IgE and IgG4 (p ⫽ 0.048 and p ⫽ 0.007, respectively). There was significant linkage disequilibrium between IL10⫺1082*A-IL10⫺592*A (␦ ⫽ 0.652, p ⬍ 0.0001). Some nominally significant associations were observed between MHC class II alleles and IgE responses (HLADQA1*0301 for total IgE, p ⫽ 0.072, and HLADQA1*0101 positively and HLA-DQA1*0401 negatively for specific IgE, p ⫽ 0.002 and p ⫽ 0.051, respectively), and between TNF and specific IgG, with a TABLE 1 Correlation of antibody levels IgE IgE specific IgG IgG4 a
1 0.25a 0.206a 0.116a IgE
Correlation is significant to p ⬍ 0.01.
1 0.124a 0.042 IgE specific
1 0.206a IgG
167
TABLE 2 Quantitative transmission disequilibrium testinga
Locus
Total IgE
P falciparum specific IgE
IL1B⫺511 IL1RNVNTR IL10⫺1082 IL10⫺592 DQB1 DQA1 DRB1 TNF⫺308 LTA⫺368 IL13arg130gln IL4VNTR BV8S3 BV24S1 BV2S1 BV15S1 BV3S1
NS 0.0006 NS 0.041 NS 0.047 NS NS NS NS NS NS NS NS NS NS
NS NS NS NS NS 0.018 NS NS NS NS NS NS 0.035 NS NS NS
Anti-P falciparum IgG
Anti-P falciparum IgG4
NS NS NS NS NS NS NS 0.011 NS NS NS NS NS NS 0.042 NS
NS NS 0.042 NS NS NS NS NS NS NS NS NS NS NS NS NS
Abbreviations: P falciparum ⫽ Plasmodium falciparum; NS ⫽ not significant (p ⬎ 0.05). a The values presented here are the single locus global scores calculated using quantitative transmission disequilibrium testing.
negative association between TNF⫺308*A and anti-P falciparum IgG levels. These associations were weak and were not significant after adjustment for multiple testing. There was no evidence for a role of polymorphisms in IL4 and IL13. There was association between the TCRBV region and specific anti-P falciparum antibodies with TCRBV24S1*G, demonstrating an association with specific IgE and TCRBV15S1*G with specific IgG. DISCUSSION The data provide evidence for a significant association between IL1RNVNTR*1 and total IgE levels. The best method of adjusting p values is unclear in a study such as this with multiple linked markers and correlated phenotypes. However, this association remains significant after adjustment for the number of uncorrelated markers (n ⫽ 8, p ⫽ 0.0048) and after adjustment for all markers and phenotypes (n ⫽ 64, p ⫽ 0.038). The nature of the involvement of the IL1 gene cluster with levels of antibody responses remains to be established. Since IL1 is a potent proinflammatory cytokine and is elevated in severe malaria, it is possible that a locus in this region directly controls antibody responses in malaria, in particular IgE. Polymorphisms within IL1RN have recently been associated with asthma [10] and with IgE levels [10]. It is also possible that a locus in the IL1 gene cluster is involved in control of parasite
168
levels, reflected in alterations of both specific and total antibodies. Polymorphisms in the IL1 gene cluster have been investigated for associations to both severe and mild malaria [8, 9], with a polymorphism in IL1B (IL1B⫹3953*T) being associated with both parasitemia [8] and severe malaria [9], although no previous associations have been observed with IL1RN [8, 9]. There was some evidence for an association between IL10 SNPs and haplotypes and Th2 antibodies. The haplotype IL10⫺1082*A-IL10⫺592*A was negatively associated with specific IgE and IgG4, and IL10⫺592*A was negatively associated with total IgE. Associations between IL10 production and IL10 SNPs and haplotypes have previously been demonstrated, with the major haplotype containing IL10⫺592*A (IL10⫺1082*AIL10⫺819*T-IL10⫺592*A) associated with low IL-10 production [16]. Our observations thus suggest that low production of IL-10 associated with a downregulatory haplotype results in reduced production of Th2-related antibodies in malaria infection. IL-10 has been reported to demonstrate both potentiating and inhibitory effects on IgE production and to increase IgG4 production in vitro [11]. The functional importance of IL-10 mediated upregulation of IgG4 in malaria infection is unclear. IL-10 is a major antiinflammatory cytokine, and a low IL-10/TNF-␣ ratio has been associated with malarial anemia [17, 18], whereas high levels of IL-10 in cases of severe malaria are associated with poor parasite clearance [19]. IL10 haplotypic associations have been investigated in the context of severe malaria, with strong association being observed by case-control analysis but not supported by transmission disequilibrium analysis [20]. We found only weak evidence for a role of polymorphisms within the MHC class II and class III regions in controlling antibody responses in malaria. Class II alleles may be more important in the control of responses to defined antigens, including vaccine candidates [12]. The weak evidence from QTDT for an association between TNF⫺308*A and high anti-P falciparum IgG antibodies, hence a protective phenotype, conflicts with previous studies on severe malaria where the TNF⫺308*A allele is a susceptibility allele [1]. However, there is a little evidence that TNF⫺308*A haplotypes may be associated with protection to mild malaria [1]. There was weak evidence for some associations between alleles within the TCRBV region and specific IgG and IgE antibodies. Although the interpretation of this data is complicated by the presence of strong linkage disequilibrium across the TCRBV region in this population and elsewhere, the associations would not survive correction for multiple testing [21]. However, our results do suggest that there may be some role for TCRBV in subclinical malaria infection and suggest that further
D. Carpenter et al.
studies of the TCRBV region would be relevant and informative. In summary, we have investigated a number of candidate genes for control of antibody responsiveness to malaria and present good evidence for an association between polymorphisms in the IL1 gene cluster and levels of total IgE. Whether this association reflects a functional role for the polymorphisms in the IL-1 gene cluster controlling malaria infection requires further investigation. We have also identified weak associations between the single loci within the IL10 promoter region and also with the haplotype IL10⫺1082*A-IL10⫺592*A, with both anti-P falciparum IgE and IgG4 antibodies, supporting the role of IL-10 as a differential regulator of IgG4 and IgE production. The data also provide limited evidence of roles for the MHC and TCRBV in subclinical malaria infection but not for the 5q31-q33 region. Observations further our understanding of genes controlling an individual’s immune response to malaria infection, relevant for malaria vaccine design.
REFERENCES 1. Kwiatkowski DP: How malaria has affected the human genome and what human genetics can teach us about malaria. Am J Hum Genet 77:171, 2005. 2. Perlmann H, Helmby H, Hagstedt M, Carlson J, Larsson PH, Troye-Blomberg M, Perlmann P: IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria. Clin Exp Immunol 97:284, 1994. 3. Perlmann P, Perlmann H, Looareesuwan S, Krudsood S, Kanop S, Matsumoto Y, Brittenham G, Trpye-Blomberg M, Aikawa M: Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria. Am J Trop Med Hyg 62: 373, 2000. 4. Bereczky S, Montgomery SM, Troye-Blomberg M, Rooth I, Shaw M-A, Farnert A: Elevated anti-malarial IgE in asymptomatic individuals is associated with reduced risk for subsequent clinical malaria. Int J Parasitol 34:935, 2004. 5. Nyakeriga MA, Troye-Blomberg M, Bereczky S, Perlmann H, Perlmann P, ElGhazali G: Immunoglobulin E (IgE) containing complexes induce IL-4 production in human basophils: effect of Th1-Th2 balance in malaria. Acta Trop 86:55, 2003. 6. Rihet P, Traore Y, Abel L, Aucan C, Troare-Leroux T, Fumoux F: Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31q33. Am J Hum Genet 63:498, 1998. 7. Ober C, Hoffjan S: Asthma genetics 2006: the long and winding road to gene discovery. Genes Immun 7:95, 2006.
Immunogenetic Control of Antibody Responsiveness in a Malaria Endemic Area
8. Gyan B, Goka B, Cvetkovic JT, Perlmann H, Lefvert AK, Akanmori B, Troye-Blomberg M: Polymorphisms in interleukin-1 and interleukin-1 receptor antagonist genes and malaria in Ghanaian children. Scand J Immunol 56: 619, 2002. 9. Walley AJ, Aucan C, Kwiatkowski D, Hill AVS: Interleukin-1 gene cluster polymorphisms and susceptibility to clinical malaria in a Gambian case-control study. Eur J Hum Genet 12:132, 2004. 10. Pattaro C, Heinrich J, Werner M, de Marco R, Wjst M: Association between interleukin-1 receptor antagonist gene and asthma-related traits in a German adult population. Allergy 61:239, 2006. 11. Jeannin P, Lecoanet S, Delneste Y, Gauchat JF, Bonnefoy JY: IgE versus IgG4 production can be differentially regulated by IL-10. J Immunol 160:3555, 1998. 12. May J, Meyer CG, Kun JFJ, Lell B, Luckner D, Dippmann AK, Bienzle U, Kremsner PG: HLA class II factors associated with Plasmodium falciparum merozoite surface antigen allele families. J Infect Dis 179:1942, 1999. 13. www.sfbr.org/solar/index/html. 14. Abecasis GR, Cardon LR, Cookson WOC: A general test of association for quantitative traits in nuclear families. Am J Hum Genet 66:279, 2000. 15. linkage.rockefeller.edu/software/ch. 16. Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP: Cytokine (TNF alpha, LT alpha and IL-10) polymor-
17.
18.
19.
20.
21.
169
phisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes Immun 1:185, 2000. Kurtzhals JAL, Adabayeri V, Goka BQ, Akanmori BD, Oliver-Commey JO, Nkrumah FK, Behr C, Hviid L: Low plasma concentrations of interleukin-10 in severe malarial anaemia compared with cerebral and uncomplicated malaria. Lancet 351:1768, 1998. Othoro C, Lal AA, Nahlen B, Koech D, Orago AS, Udhayakumar V: A low interleukin-10 tumour necrosis factor-alpha ratio is associated with malaria anaemia in children residing in a holoendemic malaria region in Western Kenya. J Infect Dis 179:279, 1999. Hugosson E, Montgomery SM, Premji Z, TroyeBlomberg M, Björkman A: Higher IL-10 levels are associated with less effective clearance of Plasmodium falciparum parasites. Parasite Immunol 26:111, 2004. Wilson JN, Rockett K, Jallow M, Pinder M, Sisay-Joof F, Newport M, Newton J, Kwiatkowski D: Analysis of IL10 haplotypic associations with severe malaria. Genes Immun 6:462, 2005. Donaldson IJ, Shefta J, Lawson CA, Bushnell JR, Morgan AW, Isaacs JD, Carpenter D, Shaw MA, Rooth I, Quinnell RJ, Zumla AM, Ollier WER, Chintu CZ, Muyinda GP, Hill AVS, Boylston AW: Unique TCR -subunit variable gene haplotypes in Africans. Immunogenetics 53:884, 2002.
