Abstract
The aim of this study was to investigate the possible influence of the tumor necrosis factor alpha (TNFA) gene promoter polymorphisms and HLA class II genes on the susceptibility to or development of human brucellosis. TNFA genotypes (at positions −308 and −238) were determined in 59 patients with brucellosis and 160 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. There were no significant differences between the patients and the controls for the TNFA-238 genotypes. However, when the overall TNFA-308 genotype distribution of the brucella patients was compared with that of the control subjects, a significant skewing was observed (P = 0·02). The TNFA-308.1/2 genotype was present at significantly higher frequency in the total patient as a whole compared with control subjects (30% versus 15%; P = 0·01, odds ratio (OR) 2·49, 95% confidence interval (CI) 1·16–5·33). No statistically significant differences in the distribution of HLA-DRB1 or DQB1 alleles were observed between brucella patients and control subjects. Stratification to correct for interdependence of TNFA-308.2 and HLA-DR3 alleles confirmed that, in spite of their strong linkage disequilibrium, the association of TNFA-308.2 with brucellosis was independent of HLA-DR3.
Keywords: HLA class II, human brucellosis, polymorphism, TNF
Introduction
Brucellosis is a zoonosis transmittable to humans, caused by Brucella spp. infection. The disease exists worldwide, especially in the Mediterranean basin, the Middle East, India and Central and South America. In the USA, where the disease is far less frequent, changes in the predominant species have been observed throughout the past decades, the most frequently isolated species being B. melitensis [1]. Besides environmental factors and pathogen strain differences, it is generally accepted that host genetic factors are major determinants of susceptibility to or outcome of infectious diseases in humans. Candidate gene studies have implicated several immunogenetic polymorphisms in human infectious diseases, HLA and cytokine genes being the more relevant ones [2].
Macrophages play a fundamental role in the control of Brucella spp. infection, mainly through the secretion of cytokines such as interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) [3,4]. TNF-α production seems to be necessary for full expression for macrophage anti-brucella activity [5]. In addition to being important in resistance to brucella, TNF-α might be associated with immunopathology, depending on the timing, levels and persistence of its production by the host in response to Brucella infection.
Variation in the TNFA promoter region has been associated with severe forms of malaria, leishmaniasis, meningococcal infection and leprosy [6–9], diseases with high serum TNF-α levels. As a result, it has been proposed that these associations reflect genetic variability in TNF-α production which influences the clinical outcome of infectious diseases. We therefore decided to investigate the contribution of the TNFA gene promoter region polymorphisms and HLA class II genes to susceptibility to or development of clinical forms of B. melitensis infection, in a population from the South of Spain, where the disease is endemic.
Materials and methods
The present study included 59 patients with brucellosis from the Infectious Diseases Unit at the hospital Carlos Haya in Málaga, Spain. Their mean age was 45·5 years (range 17–74 years), 31 (52%) were women and 28 (40%) men. A control group was composed of 160 healthy volunteers matched for age and sex, living in the same area as the patients. The diagnosis of brucellosis was established by isolation of B. melitensisin blood culture (70%), and in the other cases (30%) diagnosis was based on a compatible clinical picture together with the presence of high titres of specific antibodies or a four-fold increase or greater of the initial titres in two paired serum samples drawn 2–4 weeks apart. High titres were considered to be 1/160 for Wright's seroagglutination test or 1/320 for Coomb's anti-brucella test. Those patients with focal forms of the disease comprised a subgroup of 23 (39%) patients, the most common complication being osteoarticular (67%), followed by genitourinary (23%), and to a lesser extent hepatobiliary (7%) and pulmonary (3%).
DNA was isolated from anti-coagulated peripheral blood mononuclear cells (PBMC) using standard methods. TNFA −308 and −238 genotyping was carried out by double polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with amplification-created restriction sites, as described [10]. HLA-DRB1 and DQB1 alleles were typed using a commercially available kit (Inno-Lipa HLA-DRB and DQB; Innogenetics, Zwijndrecht, Belgium) based on reverse hybridization with sequence-specific oligonucleotides following PCR amplification (reverse PCR-SSO). Data were analysed by χ2 test with Yates' correction or Fisher's exact test using the Stalcalc program (Epi Info, version 6.0; USD, Stone Mountain, GA). P < 0·05 was considered significant. The magnitude of associations was estimated as odds ratios with 95% confidence intervals (OR, 95% CI).
Results
Comparison of TNFA genotype frequencies between brucella patients and controls
The distribution of the TNFA promoter genotypes in the patient groups and in the controls is shown in Table 1. In the control population, the TNFA allele and genotype frequencies were consistent with the Hardy–Weinberg equilibrium. No differences in the distribution of the TNFA −238 genotypes were detected between the groups under study. However, when the overall TNFA −308 genotype distribution of the brucella patients was compared with that of the control subjects, a significant skewing was observed (P = 0·02, by χ2 test from 3 × 2 contingency table) (Table 1). The TNFA-308.1/2 genotype was present at significantly higher frequency in the total patient as a whole compared with control subjects (30% versus 15%; P = 0·01, OR 2·49, 95% CI 1·16–5·33). No differences in TNFA −308 genotype distribution was observed between patients with or without focal forms of the disease.
Table 1.
Distribution of the TNFA −308 and −238 genotypes in control subjects, brucella patients overall and brucella patients stratified by the presence of focal forms of the disease
| Controls | Brucella patients | Non-focal forms | Focal forms | ||||||
|---|---|---|---|---|---|---|---|---|---|
| n = 160 | (%) | n = 59 | (%) | n = 36 | (%) | n = 23 | (%) | ||
| TNFA −308 | 1/1 | 134 | (84) | 41 | (70) | 26 | (72) | 15 | (65) |
| 1/2 | 24 | (15) | 18 | (30) | 10 | (28) | 8 | (35) | |
| 2/2 | 2 | (1) | – | – | – | – | – | – | |
| TNFA −238 | G/G | 132 | (82.5) | 47 | (79) | 28 | (78) | 19 | (82) |
| G/A | 28 | (17.5) | 12 | (21) | 8 | (22) | 4 | (18) | |
| A/A | – | – | – | – | – | – | – | – | |
Comparison of HLA frequencies between brucella patients and controls
Table 2 shows phenotype frequencies for DRB1 and DQB1 in brucella patients and controls subjects. No statistically significant differences in the distribution of HLA-DRB1 or DQB1 alleles were observed between brucella patients overall and control subjects, or between patients with or without focal forms of the disease (data not shown).
Table 2.
HLA-DRB1 and DQB1 phenotype frequencies in brucella patients and controls
| Controls | Brucella patients | |||
|---|---|---|---|---|
| n = 160 | (%) | n = 59 | (%) | |
| HLA-DRB1 | ||||
| 01 | 37 | (23) | 14 | (13) |
| 15 | 35 | (16) | 14 | (20) |
| 16 | 8 | (5) | 4 | (7) |
| 03 | 32 | (20) | 12 | (20) |
| 04 | 42 | (26) | 10 | (17) |
| 11 | 38 | (24) | 8 | (13) |
| 12 | 2 | (1) | 1 | (2) |
| 13 | 35 | (22) | 18 | (30) |
| 14 | 6 | (4) | 1 | (2) |
| 07 | 47 | (29) | 19 | (32) |
| 08 | 6 | (4) | 7 | (12) |
| 09 | 4 | (2) | 4 | (7) |
| 10 | 11 | (7) | 2 | (3) |
| HLA-DQB1 | ||||
| 02 | 75 | (47) | 26 | (44) |
| 0301 | 52 | (32) | 15 | (25) |
| 0302 | 33 | (21) | 6 | (10) |
| 0303 | 7 | (4) | 6 | (10) |
| 0304 | 1 | (1) | 1 | (2) |
| 0401 | – | – | 1 | (2) |
| 0402 | 7 | (4) | 7 | (12) |
| 0501 | 43 | (27) | 16 | (27) |
| 0502 | 7 | (4) | 5 | (8) |
| 0503 | 7 | (4) | 1 | (2) |
| 0601 | 7 | (4) | 2 | (3) |
| 0602 | 29 | (18) | 11 | (19) |
| 0603 | 20 | (12) | 11 | (19) |
| 0604 | 5 | (3) | 4 | (7) |
Interaction between TNFA and HLA alleles
The existence of linkage disequilibrium between alleles at TNF and HLA loci is well known [11], therefore we examined the possibility that the TNF–brucellosis observed associations were primary, or secondary to HLA class II antigen associations. TNFA-308.2 allele was in linkage disequilibrium with HLA-DRB1*03 (OR = 7·03; P = 0·0007) and, because of this association, stratification was necessary to rule out an eventual confounder effect of HLA-DR3 to the susceptibility to brucella infection. This method allows the division of the study population into HLA-DR3-negative and -positive individuals. Then, to estimate the total contribution of a risk factor in a stratified analysis, the Mantel–Haenszel procedure was used as described [12]. It resulted in a weighted Mantel–Haenszel odds ratio for TNFA-308.2 of 2·54 (95% CI 1·13–5·60; P = 0·02)(Table 3). Likewise, the study population was divided into TNFA-308.2-negative and -positive individuals. This stratification indicated that HLA-DR3 was not individually associated with brucellosis (combined OR = 0·80; 95% CI 0·33–1·90) and that the observed disease associations with TNFA −308 genotypes appear to be independent of HLA class II antigens (Table 3).
Table 3.
Odds ratios (OR) of the TNFA −308 and HLA-DR3 genotypes for susceptibility to human brucellosis
| OR between genotypes | Subgroup | OR | 95% CI |
|---|---|---|---|
| TNFA-308.1/2 and 2/2 versus -308.1/1 | HLA-DR3-negative | 1·96 | 0·71–5·34 |
| HLA-DR3-positive | 4·20 | 0·85–23·1 | |
| Combined (Mantel–Haenszel) | 2·49 | 1·09–5·60 | |
| HLA-DR3/x versus -DRx/x | TNFA-308.2-negative | 0·60 | 0·16–1·97 |
| TNFA-308.2-positive | 1·20 | 0·30–4·89 | |
| Combined (Mantel–Haenszel) | 0·80 | 0·33–1·90 |
The Mantel–Haenszel odds ratio is the weighted mean of the OR of the risk factor in two or more strata. It is used to calculate the Mantel–Haenszel OR if no statistical interaction between the risk factors is present. The weighted Mantel–Haenszel OR for TNF-308.2 was of 2·49 (95% CI 1·09–5·60; P = 0·02) and for HLA-DR3 was 0·80 (0·33–1·90; P = not significant).
Discussion
We have found that TNFA −308 genotypes influence the susceptibility to brucellosis. The fact that after correction for the presence of HLA-DR3 the OR of the TNFA-308.2 allele was 2·54 (P = 0·01) indicates that the TNFA-308.2 allele contributes independently to the susceptibility to brucellosis (Table 3). This study adds to a growing body of evidence showing that this polymorphism, directly or by linkage with other polymorphisms, is important in infectious diseases. Previous studies have linked TNFA-308.2 allele with cerebral malaria, mucocutaneous leishmaniasis, fatal meningococcal disease and lepromatous leprosy [6–9]. The fact that all these conditions are associated with high serum TNF-α levels, along with the evidence from transfection studies indicating that the TNFA-308.2 allele could act to increase constitutive and inducible levels of TNF-α [13], prompted the idea that this variation predisposes to overproduce TNF-α which, in turn, would favour the development of the above-mentioned conditions. However, other reporter gene studies have not been able to reproduce the effect of this polymorphism on up-regulation of TNF-α production [14,15]. In addition, Conway et al. [16] found that scarring trachoma was also associated both with TNFA-308.2 polymorphism and with elevated TNF-α levels in tear fluid; however, detection of TNF-α in tear fluid samples was not associated with particular TNFA promoter genotypes. Furthermore, it has been reported that the levels of TNF-α induced through stimulation of PBMC with Mycobacterium leprae were higher in patients with the paucibacillary tuberculoid form of the disease than in those patients with the multibacillary lepromatous form [17], in spite of the latter condition being associated with high circulating levels of TNF-α. In the same way, a lower capacity to secrete TNF-α upon ex vivo stimulation of whole blood with meningococcal endotoxin has been demonstrated among relatives of non-survivors from severe meningococcal disease than in relatives of survivors [18].
In spite of the known immunopathological aspects of TNF-α, high levels of this cytokine may reflect an advanced stage of disease, and the data suggest that other mechanisms in relation with TNF-α physiology may be acting on the pathogenesis of at least some complicated forms of infectious disease. In a murine model of Brucella infection it has been demonstrated that TNF-α plays an important role in enhancing IL-12 production in vitro and in vivo, which is the key cytokine controlling the induction of acquired cellular immunity against Brucella and other intracellular pathogens [4]. Interestingly, direct evidence has been provided that a secreted Brucella factor specifically inhibits TNF-α expression at the mRNA level on human, but not murine, macrophage-like cells [19]. Therefore, it is possible that individuals bearing TNFA-308.2 allele are more sensitive to down-regulation by such a factor, making them more susceptible to develop disease upon Brucella infection. Thus, it is tempting to speculate that the pathogenic mechanism underlying the development of complicated forms of infectious diseases may be more related to an inability to secrete suitable levels of TNF-α at some crucial points in the natural history of the infective process, allowing intracellular survival, multiplication and spreading of pathogens, than to an increased capacity to secrete high levels of TNF-α.
Individuals with different types of HLA might differ in their susceptibility or resistance to infectious pathogens [20]. Very little information is available on the importance of the HLA genes in brucellosis. Although increased frequencies of the HLA-DR4 allele have been reported in patients with brucellosis [21], we found no differences between patients and controls for this allele. This discordance may be due to the different typing techniques employed, since we used DNA typing in contrast to serological typing. Studies have been carried out to try to associate HLA antigens with focal forms of the disease, however, and in agreement with the results of the present study, no clear associations have been found [22,23].
In conclusion, our study suggests that the TNFA −308 genotypes may be involved in the susceptibility to brucellosis, although we realise that larger studies in different populations will be needed to confirm these observations.
Acknowledgments
We thank Yasmina Beraún and Lourdes Velázquez for excellent technical assistance. This work was supported in part with funds from P.A.I. (CTS-104 and CTS-180), Junta de Andalucía, Spain.
References
- 1.Chomel BB, DeBess EE, Mangiamele DM, et al. Changing trends in the epidemiology of human brucellosis in California from to 1973 to 1992: a shift toward foodborne transmission. J Infect Dis. 1994;170:1216–33. doi: 10.1093/infdis/170.5.1216. [DOI] [PubMed] [Google Scholar]
- 2.Hill AVS. The immunogenetics of human infectious diseases. Ann Rev Immunol. 1998;16:593–617. doi: 10.1146/annurev.immunol.16.1.593. [DOI] [PubMed] [Google Scholar]
- 3.Zhan Y, Liu Z, Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms. Infect Immun. 1996;64:2782–6. doi: 10.1128/iai.64.7.2782-2786.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Zhan Y, Cheers C. Control of IL-12 and IFN-γ production in response to live or dead bacteria by TNF and other factors. J Immunol. 1998;161:1447–53. [PubMed] [Google Scholar]
- 5.Jiang X, Leonard B, Benson R, Baldwin CL. Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide. Cell Immunol. 1993;151:309–19. doi: 10.1006/cimm.1993.1241. 10.1006/cimm.1993.1241. [DOI] [PubMed] [Google Scholar]
- 6.McGuire W, Hill AVS, Allsopp CEM, Greenwood BM, Kwiatkowski D. Variation in the TNF-a promoter region associated with susceptibility to cerebral malaria. Nature. 1994;371:508–11. doi: 10.1038/371508a0. [DOI] [PubMed] [Google Scholar]
- 7.Cabrera M, Shaw M-A, Sharples C, et al. Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. J Exp Med. 1995;182:1259–64. doi: 10.1084/jem.182.5.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Nadel S, Newport MJ, Booy R, Levin M. Variation in the tumor necrosis factor-alpha gene promoter region may be associated with death from meningococcal disease. J Infect Dis. 1996;174:878–80. doi: 10.1093/infdis/174.4.878. [DOI] [PubMed] [Google Scholar]
- 9.Roy S, McGuire W, Mascie-Taylor CGN, et al. Tumor necrosis factor promoter polymorphism and susceptibility to lepromatous leprosy. J Infect Dis. 1997;176:530–2. doi: 10.1086/517282. [DOI] [PubMed] [Google Scholar]
- 10.Vinasco J, Beraún Y, Nieto A, et al. Polymorphism at the TNF loci in rheumatoid arthritis. Tissue Antigens. 1997;49:74–78. doi: 10.1111/j.1399-0039.1997.tb02715.x. [DOI] [PubMed] [Google Scholar]
- 11.Wilson AG, de Vries N, Pociot F, di Giovine FS, van der Putte LBA, Duff GW. An allelic polymorphism within the human tumor necrosis factor α promoter region is strongly associated with HLA-A1, B8 and DR3 alleles. J Exp Med. 1993;177:557–60. doi: 10.1084/jem.177.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Rood MJ, van Krugten MV, Zanelli E, et al. TNF-308A and HLA-DR3 alleles contribute independently to susceptibility to systemic lupus erythematosus. Arthritis Rheum. 2000;43:129–34. doi: 10.1002/1529-0131(200001)43:1<129::AID-ANR16>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
- 13.Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor α promoter on transcriptional activation. Proc Natl Acad Sci USA. 1997;94:3195–9. doi: 10.1073/pnas.94.7.3195. 10.1073/pnas.94.7.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Brinkman BM, Zuijdeest D, Kaijzel EL, Breedveld FC, Verweij CL. Relevance of the tumor necrosis factor alpha (TNF alpha) -308 promoter polymorphism in TNF alpha gene regulation. J Inflamm. 1995;46:32–41. [PubMed] [Google Scholar]
- 15.Stuber F, Udalova IA, Book M, et al. -308 tumor necrosis factor (TNF) polymorphism is not associated with survival in severe sepsis and is unrelated to lipopolysaccharide inducibility of human TNF promoter. J Inflamm. 1995;46:42–50. [PubMed] [Google Scholar]
- 16.Conway DJ, Holland MJ, Bailey RL, et al. Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF alpha) gene promoter and with elevated TNF alpha levels in tear fluid. Infect Immun. 1997;65:1003–6. doi: 10.1128/iai.65.3.1003-1006.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Barnes PF, Chatterjee D, Brennan PJ, Rea TH, Modlin RL. Tumor necrosis factor production in patients with leprosy. Infect Immun. 1992;60:1441–6. doi: 10.1128/iai.60.4.1441-1446.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Westendorp RGJ, Langermans JAM, Huizinga TWJ, et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet. 1997;349:170–3. doi: 10.1016/s0140-6736(96)06413-6. 10.1016/s0140-6736(96)06413-6. [DOI] [PubMed] [Google Scholar]
- 19.Caron E, Gross A, Liautard JP, Dornand J. Brucella species release a specific, protease-sensitive, inhibitor of TNF-α expression, active on human macrophage-like cells. J Immunol. 1996;156:2885–93. [PubMed] [Google Scholar]
- 20.Hill A, Allsopp C, Kwiatkowski D, et al. Common West African HLA antigens are associated with protection from severe malaria. Nature. 1991;352:595–600. doi: 10.1038/352595a0. [DOI] [PubMed] [Google Scholar]
- 21.Pareja E, Bonal FJ, Paule P, Salvatierra D, Garrido F. HLA antigens in brucellosis. Exp Clin Immunogenet. 1985;2:1–3. [PubMed] [Google Scholar]
- 22.Alarcón G, Bocanegra T, Gotuzzo E, Hinostrosa H, Carrillo C, Espinoza L. Reactive arthritis associated with brucellosis. HLA studies. J Rheum. 1981;8:621–5. [PubMed] [Google Scholar]
- 23.Alarcón G, Gotuzzo E, Hinostrosa H, Carrillo C, Bocanegra T, Espinoza L. HLA studies in brucellar spondilytis. Clin Rheum. 1985;4:312–4. doi: 10.1007/BF02031614. [DOI] [PubMed] [Google Scholar]