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Departments of Epidemiology, Medicine
Pediatrics, University of Alabama at Birmingham, Birmingham
Department of Scientific Affairs, Berlex Laboratories, Seattle, Washington
Antigen presentation and immune activation are essential to the effective control of infectious diseases. In 485 North American adolescents at high risk for genital Chlamydia trachomatis infection, we found 2 human leukocyte antigen variants (DRB1*03-DQB1*04 and DQB1*06) to be associated with recurrent Chlamydia infection (adjusted relative odds [RO], >2.0; P < .01, for both variants). A G-C-C haplotype corresponding to variants at IL10 (encoding interleukin-10 [IL-10]) promoter positions -1082, -819, and -592 was underrepresented in individuals with recurrent infection (RO, 0.59; P = .046). These genetic associations were independent of nongenetic factors, including number of sex partners, race, sex, duration of follow-up, and human immunodeficiency virus type 1 seropositivity. Consistent with the observed IL10 association, cervical secretions in female adolescents without the IL10 G-C-C haplotype had elevated IL-10 concentrations after Chlamydia infection, which may reflect involvement of a Chlamydia-specific mechanism for genetically mediated, differential IL-10 expression in the genital tract.
Sexually transmitted Chlamydia trachomatis infection is diagnosed in 3 million people each year in the United States alone [1, 2]. Those with the highest prevalence of Chlamydia infection are 1524-year-old adolescents and youth, who represent 25% of the sexually experienced population [2]. Spontaneous clearance of C. trachomatis from the lower genital tract in nearly 20% of Chlamydia infections clearly suggests that the host immune response often controls this infection before the onset of clinical consequences such as pelvic inflammatory disease (PID) [3].
Specific immune responses to infection almost invariably begin with the process of antigen presentation by highly polymorphic and redundant major histocompatibility complex (MHC) molecules, which are also known as HLAs. Subsequent immune activation, regulation, and production of effectors (both cellular and humoral) are orchestrated by cooperating or competing cytokines and chemokines that are known to be expressed at varying levels as the infection progresses [4, 5]. These sequential events can be highly individualized, and coinfection with multiple pathogens can further complicate the effectiveness of immune reactions.
From a population perspective, frequent and diverse associations of HLA and cytokine gene variants with myriad inflammatory and infectious diseases reflect differential immune responses to specific pathogens [69]. Chlamydia infection appears to be no exception: both murine-model studies and epidemiological studies have suggested that multiple MHC class II antigen-presentation molecules influence the development of protective immunity [1013]. Class I variants in HLA genes have also been associated with incident Chlamydia infection [14], chlamydial PID [15], and trachomatous scarring [16]. Recognition of the effects of human genetic variation may provide useful guidance for more targeted experimental studies and ultimately may have therapeutic and prognostic implications. To define host genetic influences on recurrent Chlamydia infection in adolescents and to glean insights into the functional relevance of observed immunogenetic relationships, we examined the individual and joint effects of HLA and cytokine gene variants.
SUBJECTS AND METHODS
Study population.
The enrollment of adolescents in 15 US cities and the methods for quarterly follow-up, sample collection, and measurements of genital-tract pH and serum estradiol level (in female subjects) in the Reaching for Excellence in Adolescent Care and Health (REACH) study have been described in detail elsewhere [14, 1719]. Overall, 548 adolescents (1219 years old), mostly female (76%) and African American (71%), were recruited between 1996 and 2000; data and specimens from 485 adolescents (1318 years old) were available for the present study. This study conforms to the procedures for informed consent approved by institutional review boards at all sponsoring organizations and to human-experimentation guidelines set forth by the United States Department of Health and Human Services.
Testing for Chlamydia infection.
At enrollment and every 6 months thereafter, specimens of urine and cervical and anorectal swabs were screened for incident Chlamydia infection, by use of ligase chain reaction (LCR) (LCx STD system; Abbott Laboratories) [20]. Test results generated from a central laboratory were returned to clinicians at variable time points (up to a 6-month interval). Treatment could be initiated even before test results became available, because local testing was also performed for all sexually active participants.
DNA samples and genotyping of HLAs.
DNA samples were extracted from 25 million peripheral blood mononuclear cells (PBMCs) for polymerase chain reaction (PCR)based HLA genotyping, which resolved HLA-A, -B, and -C variants to 2-digit (group) specificity and resolved DRB1 and DQB1 to 4-digit (allele) specificity [14, 18]. HLA-BHLA-C and DRB1-DQB1 haplotypes were assigned manually, on the basis of well-documented patterns of linkage disequilibrium (LD) seen in representative, general North American (US) populations [2124].
Classification of T-helper type 1 (TH1) and TH2 cytokine gene polymorphisms.
A panel of 16 common single-nucleotide polymorphisms (SNPs) (table 1) from 7 genes (interleukin 2 [IL2], IL4, IL4R, IL6, IL10, IL12B, and tumor necrosis factor ) representing both TH1 and TH2 pathways in the cytokine network were typed by PCR with sequence-specific primers [19]. Validation of techniques and individual genotyping followed procedures recommended by the manufacturer (Department of Transplantation Immunology, University of Heidelberg; Pel-Freez Clinical Systems) and the 13th (2002) International Histocompatibility Workshop and Congress [19, 25, 26]. When multiple SNPs within a single gene were tested, their cis and trans relationships were directly established by PCR for all except 2 at the IL6 locus. The insertion/deletion variants in the IL12B promoter sequence corresponded to 2 alleles (longer [L] and shorter [S]) defined by fragment sizes that differed by 4 bp after PCR amplification and automated denaturing gel electrophoresis on the ALFexpress system (Amersham Pharmacia Biotech) [26, 27].
Quantification of interleukin-10 (IL-10) by ELISA.
As described elsewhere [20], the IL-10 concentration in cervical secretions was quantified by high-sensitivity ELISA (Biosource International). A value of 0 was assigned to any sample (in triplicate) with all readings below the lower detection limit (0.2 pg/mL). Occasionally, cervical specimens had to be discarded when blood contamination was suspected. IL-10 concentrations in peripheral blood were not measured in the present study, since earlier work has documented a lack of correlation between cervical and plasma IL-10 concentrations [28].
Statistical analyses.
Associations of nongenetic and genetic factors with recurrent Chlamydia infection (i.e., positive test results obtained more than once by LCR, with intervening negative tests, during the study period) were performed using Statistical Analysis Software (version 9.0; SAS Institute). Univariate association analyses focused on the relationship of major HLA and cytokine gene variants (alleles and haplotypes with frequencies of 5% in the study population) to recurrence of Chlamydia infection. Independent associations of nongenetic and genetic markers with recurrent Chlamydia infection were tested in multivariable logistic-regression models. Genetic variants showing tight LD were assessed in stratified analyses, to separate the primary from the secondary contributors. A stepwise selection method was used to develop the most parsimonious model for further, more generalizable logistic-regression analyses. Only factors with adjusted (multivariable) P values .05 were accepted as putative contributors, or "markers." Because reported P values were not corrected for multiple comparisons performed, the associations of low-to-medium magnitude (relative odds [RO], 0.30.8 or 1.23.0; P > .001) reported in the present study require cautious interpretation and assessment of internal and external consistency. In this regard, we used alternative multivariable analytic approaches to define the robustness of the genetic associations and performed nonparametric testing (i.e., Mann-Whitney U test) of IL-10 in cervical secretions, to provide confirmatory results. Further analyses targeted a small group of persistently infected subjects with positive chlamydial LCR test results at 2 consecutive visits and/or PID.
RESULTS
Overall characteristics of the study population.
As shown in earlier analyses [14], the 485 adolescents from the REACH study (mean age at enrollment, 17 years) were mostly female (74%), African American (70%), HIV-1 seropositive (68%), and representative of the entire REACH cohort (n = 548), except for a slightly narrower range of age (1318 years). These participants were considered to be at high risk for Chlamydia infection, HIV-1 infection, and several other infections, through sexual activity and drug use during the study period between 1996 and 2000. The median follow-up was 35 months (range, 656 months), with a mean ± SD of 6 ± 2 chlamydial LCR tests/subject.
At baseline, 133 adolescents (27%) had a record of Chlamydia infection before study entry. A new Chlamydia infection was diagnosed only once in 82 (17%) of the subjects and was diagnosed more than once in another 117 subjects (24%). All Chlamydia-positive subjects received a prescription from their physicians for appropriate antichlamydial therapy, but adherence to therapy could not be reliably assessed. Among the multiply infected subjects, persistent infection (at 2 consecutive visits; n = 27) and recurrent infection (2 Chlamydia infections separated by a Chlamydia-negative visit; n = 90) could not be analyzed separately, because virtually all persistently infected subjects also had recurrent infection, when Chlamydia infection before enrollment was counted as an additional infection. Subsequent comparative analyses began with the 90 subjects with recurrent infection. Analyses of disease complications (e.g., PID) were not considered to be informative, since only 12 (8%) of 156 female subjects with at least 1 infection had PID.
Baseline, nongenetic predictors of recurrent Chlamydia infectionunivariate analyses.
Compared with participants who had no incident Chlamydia infection during the study period (n = 286), higher proportions of the 90 who had recurrent infection were African American (RO, 1.70; P = .062) and female (RO, 1.72; P = .077) and had 2 sex partners during the 3 months preceding first detection of Chlamydia infection (RO, 2.08; P = .005) (table 2). Subjects with recurrent infection also were more likely to have a longer duration of follow-up (RO, 1.41/year; P = .003). Social and clinical characteristics not associated with recurrent Chlamydia infection (P .10 in all tests) included (1) age at enrollment, (2) age at first sexual activity, (3) HIV-1 seropositivity, (4) exposure to highly active antiretroviral therapy for HIV-1 infection, (5) CD4+ T cell count at baseline, and (6) consumption of alcohol, recreational drugs, or birth control pills. Age and HIV-1 seropositivity were nevertheless examined further in multivariable analyses, because of the dual implications of these factors for sexual behavior and immunological status.
HLA associations with recurrent Chlamydia infectionunivariate analyses.
Comparisons of individuals with recurrent infection and those with no incident Chlamydia infection were performed for 12 HLA-A, 13 HLA-B, 8 HLA-C, 10 DRB1, and 5 DQB1 allele groups present in >25 subjects (5%). Similar analyses were applied to 12 HLA-BHLA-C haplotypes and 12 DRB1-DQB1 haplotypes. Two of these (HLA-A*36 and DQB1*06) showed positive associations with recurrent Chlamydia infection (univariate RO, 2.56 [P = .040] and 1.80 [P = .018], respectively) (table 2), and Cw*16 showed a negative association (RO, 0.42; P = .031). As is often the case, the associations with the DQB1*06 and DRB1*15-DQB1*06 haplotypes coincide, because of tight LD. Two other HLA variants (B*58-Cw*06 and DRB1*03-DQB1*04) also showed borderline associations (P < .10), and they were not excluded at this stage.
Association of cytokine gene variants with recurrent Chlamydia infectionunivariate analyses.
Among 16 common SNPs plus 1 insertion/deletion variation at IL2, IL4, IL4R, IL6, IL10, IL12B, and TNF loci, the ancestral [19] T-G haplotypedefining variants at IL2 positions -330 and +166 showed a weak, positive association with recurrent infection (RO, 2.37; P = .037); however, this association diminished (RO, 1.34; P > .15) upon adjustment for race and sex. The IL10 G-C-C haplotype at promoter positions -1082, -819, and -592 in the proximal promoter region was negatively associated with recurrent Chlamydia infection (RO, 0.61; P = .044) (table 2). Genotyping of 2 upstream IL10 promoter SNPs (-3575A/T and -2763A/C) in a subset (n = 377; 78%) of subjects ruled out any likely contribution by the distal promoter region to the effect of the G-C-C haplotype: frequencies of all G-C-C haplotypes were similarly elevated in the recurrently infected subjects and in others. The absence of association of recurrent Chlamydia infection with 2 other IL10 haplotypes (A-C-C and A-T-A) implied that -1082G homozygosity and heterozygosity could completely capture the effect of the IL10 G-C-C haplotype, with -1082A/A homozygosity predictably showing the reciprocal relationship (RO, 1.64; P = 0.044). No other cytokine gene alleles or haplotypes were associated with recurrent Chlamydia infection (tabulations are available from J.T.).
Homozygosity and heterozygosity in relation to Chlamydia infection.
Homozygous and heterozygous genotypes of common genetic markers were also compared (data not shown). Homozygosity and heterozygosity for DQB1*06 and the IL10 G-C-C haplotype showed similar relationships to recurrent Chlamydia infection. Further analyses of overall heterozygosity or homozygosity at each locus (including HLA) did not suggest any additional effects.
Individual associations defined by multivariable logistic-regression analyses.
Few genetic and nongenetic factors remained predictive of recurrent Chlamydia infection in multivariable analyses (table 2). Specifically, age, race, sex, and HIV-1 seropositivity were dismissed as nongenetic predictors (adjusted P > .20), but number of sex partners (adjusted RO, 2.36; P = .002) and duration of follow-up (RO, 1.59/year; P < .001) remained as independent predictors in the reduced (most parsimonious) model (table 2). Of the genetic markers suggested in univariate analyses (A*36, Cw*16, B*58-Cw*06, DRB1*03-DQB1*02, DQB1*06, and IL10 G-C-C), all showed only modest changes in RO, and adjusted P values remained .05 in multivariable analyses.
Alternative univariate and multivariable analyses.
Subjects who experienced just a single Chlamydia infection (n = 109) formed a third, intermediate group between those with recurrent infection and those who were infection-free at any time during follow-up visits. In an alternative analysis of these 3 subject categories (table 3), race, sex, age, number of sex partners, and duration of follow-up all had statistically significant (P .050) proportional odds (PO), ranging from 1.41 to 2.44. Three of the 6 previously highlighted genetic markers (DRB1*03-DQB1*04, DQB1*06, and IL10 G-C-C) also remained significantly associated in univariate analyses (PO, 1.73, 1.76, and 0.71, respectively; P .05 for all).
In further multivariable analyses (table 3), race, sex, number of sex partners, duration of follow-up, the DRB1*03-DQB1*04 haplotype, and DQB1*06 alone were again independently associated with an increased likelihood of recurrent Chlamydia infection (P = .002.023), whereas the IL10 G-C-C haplotype was independently associated with a decreased likelihood (P = .019). Overall, >70% of all subjects had 1 of these 3 genetic markers; the individual marker frequencies ranged from 13% to 54%.
Additional analyses of female subjects with persistent Chlamydia infection.
A group of 36 female subjects were defined as persistently infected, on the basis of either testing positive for infection at 2 consecutive visits or having clinical evidence of PID. Compared with 143 female adolescents who had no incident or prior infection (table 4), the persistently infected female subjects were characterized by a longer mean follow-up, increased presence of unfavorable HLA markers (i.e., DRB1*03-DQB1*04 and DQB1*06), and reduced frequency of the IL10 G-C-C haplotype (adjusted P = .007.082).
Genital-tract IL-10 concentrations in relation to Chlamydia infection and IL10 promoter genotypes.
IL-10 ELISA was performed at least twice for 296 female subjects. In the comparison of earliest available ELISA results adjusted for sample volume [20], the median IL-10 concentration measured during 394 Chlamydia-negative visits (45 pg/mL) was lower than the median IL-10 concentration measured during 134 Chlamydia-positive visits (69 pg/mL) (P = .005). For a subset of 96 adolescents with a Chlamydia-negative visit followed by a Chlamydia-positive visit, the median IL-10 concentration in cervical secretions was higher after infection (61 pg/mL) than at the initial Chlamydia-negative visit (34 pg/mL) (P = .074). An increase in IL-10 concentration was also seen at a second Chlamydia-positive visit (61 pg/mL) (figure 1), whereas subjects who cleared their infection had a median IL-10 concentration (37 pg/mL) similar to that measured at the earlier Chlamydia-negative visit (data not shown).
In the 96 adolescents who acquired Chlamydia infection between 2 visits, IL-10 concentrations in cervical secretions could be further stratified by IL10 promoter genotype. In this analysis, an increase in IL-10 concentration in Chlamydia-infected subjects was most often detected in those without the G-C-C haplotype (figure 1). More specifically, median IL-10 concentration (69 pg/mL) in the 51 individuals without the G-C-C haplotype was somewhat higher than that (45 pg/mL) in the 45 G-C-C-carriers (P = .056). The same trend persisted at the subsequent visit among 31 subjects in whom Chlamydia infection was detected again. In contrast, at 2 randomly spaced Chlamydia-negative visits among 360 subjects, IL-10 concentrations did not differ by the presence or absence of the G-C-C haplotype (P = .34.73) (data not shown). Further adjustment for vaginal pH, serum estradiol levels, use of oral contraceptives, and HIV-1 infection or other infrequent infections (e.g., bacterial vaginosis) did not reveal additional correlates of IL-10 concentrations. For example, the median (5.0) and interquartile range (IQR) (5.06.0) of vaginal pH were identical between Chlamydia-negative and Chlamydia-positive visits in all subjects; the medians (29 vs. 37 pg/mL) and IQRs (1592 vs. 1677 pg/mL) of serum estradiol levels were quite similar (P > .70, based on data available for 33%52% of visits).
DISCUSSION
Genital Chlamydia infection occurs at substantial prevalence in adolescents and young adults [2932]. Despite the availability of both behavioral interventions [33] and effective antimicrobial regimens, recurrent Chlamydia infection can be seen in 5%38% of patients each year after treatment [31, 3436]. Such recurrent or persistent infection can lead to reproductive sequelae [37], along with increased risk for spreading infection to others. In the population in the present study, the rate of recurrent or persistent Chlamydia infection fell within the reported range, and multiple host factors appeared to influence recurrent infection in adolescents.
After controlling for the potential effects of age, sex, race, number of sex partners, and coinfection with other pathogens (e.g., HIV-1), we identified 2 HLA variants and a single IL10 haplotype as putative, independent markers of recurrent Chlamydia infection. Collectively, these variants are common, appearing in approximately three-quarters of the study population (table 4). More importantly, these findings are largely consistent with recognized roles of HLA class II antigens (including DRB1 and DQB1) [3841] and IL-10 (see below) in Chlamydia infection. Minor associations of other HLA variants with recurrent Chlamydia infection were also expected: several HLA class I variants had occasionally been implicated in earlier analyses of PID [15] and trachomatous scarring [16] after Chlamydia infection. Thus, the antigen-presenting function of HLA molecules may play a central role in the effective control of Chlamydia infection.
In the absence of multifactorial influences on Chlamydia infection and related outcomes, genetic associations reported here and elsewhere [3841] are relatively modest (RO, 0.30.8 or 1.23.0) and require verification of one type or another. Nonetheless, the repeated association of DQB1*06 and IL10 genotype with both recurrent Chlamydia infection (in adolescents) and later sequelae (in adults) [3841] points to the possibility that late-stage disease manifestations (e.g., tubal factor infertility ) result from recurrent Chlamydia infection rather than from a single episode of aberrant infection. Increased presence of IL-10, an anti-inflammatory cytokine, was also persistently observed in the genital tract after Chlamydia infection, suggesting that hyperimmune activation could be further ruled out as the cause of Chlamydia-related, late-stage disease.
Unlike HLA variants that encode amino acid substitutions in antigen-presenting molecules, cytokine gene variations occur more often in promoter and other regulatory (e.g., 3 untranslated region and enhancer/silencer) sequences. The SNPs and insertion/deletion variants studied here have been previously associated with various diseases and patterns of differential cytokine expression, either in vivo or in vitro after stimulation (reviewed in [8] and [9]). For example, IL6 -174G is associated with high IL-6 production [42], and IL4 -590C is associated with reduced mRNA transcription [43]. Other cytokine gene variants, including IL2 -330G/T, TNF -308G/A, and the insertion/deletion variation in the IL12B promoter, also have been found to differ in their functional properties and influences on disease outcomes in experimental and/or epidemiological studies [42, 44, 45]. Despite previous observations of functional relationships with these cytokine gene alleles and haplotypes, their association with recurrent Chlamydia infection was rather limited in this adolescent cohort.
The association of the IL10 polymorphism with Chlamydia infection may be related to HLA function, as well. Chlamydia infection has been specifically shown to reduce HLA class I expression through the induction of IL-10 [46]. This may, in part, account for the malfunction of CD8+ T cells that is accompanied by compromised cell-mediated immune response to C. trachomatis [4749]. HLA class II expression is also affected by IL-10 [50], mostly through inhibition of interferon (IFN) function [50, 51] and partially through decreased expression of IFN- itself after Chlamydia infection [52]. The inhibitory effect of IL-10 is more prominent on HLA-DR, as is evidenced by the inverse correlation between IL-10 concentration and HLA-DR expression [53, 54], by the posttranslational endocytosis of HLA-DR induced by IL-10 [55], and by IFN-mediated augmentation of HLA-DR and not HLA-DQ expression [56, 57]. Thus, the joint yet independent associations of IL10 and HLA polymorphisms with recurrent Chlamydia infection are consistent with known immunological pathways [1013], as well as with earlier epidemiological findings [11, 12, 3840].
At the molecular level, a 60-kDa heat shock protein (CHSP60) from C. trachomatis has been shown to induce prominent IL-10 secretion not only by PBMCs from subjects with C. trachomatisassociated TFI [58] and PID [52] but also in tissues from subjects with newly diagnosed Chlamydia-associated arthritis [59]. IL10 knockout animals show enhanced TH1 type immune responses and less severe outcomes after Chlamydia infection [60], whereas elevated IL-10 concentration is considered to be a potential risk factor for persistence of or complications from Chlamydia infection [39, 47, 51, 61]. Our identification of the IL10 G-C-C haplotype as a favorable factor (tables 2 and 3) associated with lower genital-tract IL-10 concentration after Chlamydia infection (figure 1) is further consistent with findings from a more recent study, in which IL10 -1082G, -819C, and -592C on an extended haplotype correlated with low IL-10 production by PBMCs stimulated with bacterial lipopolysaccharides [62]. However, in several other studies, the G-C-C haplotype has appeared to represent a high producer genotype [42, 63], suggesting that relationships between IL10 genotypes and IL-10 production (universally measured in vitro with various stimuli) can be complicated. Distinction between local (e.g., in genital tract) and systemic (e.g., in PBMCs) IL-10 production [28], along with the use of different stimuli, may account for some of the reported discrepancies. More importantly, our work indicates that immunological outcomes, including IL-10 concentrations, may well vary according to the combination of host genotype and the specific pathogen involved. Further experimental testing is necessary to determine whether genetic polymorphisms influence susceptibility to infection by altering critical interactive pathways between antigen presentation and subsequent immune regulation.
Acknowledgments
We thank investigators and staff (listed in [64]) of the Adolescent Medicine HIV/AIDS Research Network (19942001) and the youth who participated in the Reaching for Excellence in Adolescent Care and Health (REACH) project, for their valuable contributions to various aspects of this work. We are further indebted to E. Lobashevsky, W. Shao, A. D. Myracle, and C. A. Rivers, for excellent technical assistance.
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