Free Access Interleukin 18 and human immunodeficiency virus type I infection in adolescents and adults W. Song, Departments of Epidemiology,Search for more papers by this authorC. M. Wilson, Pediatrics and Medicine, University of Alabama at Birmingham, Birmingham, AL, USA, andSearch for more papers by this authorS. Allen, Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, GA, USASearch for more papers by this authorC. Wang, Departments of Epidemiology,Search for more papers by this authorY. Li, Medicine, University of Alabama at Birmingham, Birmingham, AL, USA, andSearch for more papers by this authorR. A. Kaslow, Departments of Epidemiology, Medicine, University of Alabama at Birmingham, Birmingham, AL, USA, andSearch for more papers by this authorJ. Tang, Corresponding Author Medicine, University of Alabama at Birmingham, Birmingham, AL, USA, andDr Jianming ‘James’ Tang, Department of Medicine, University of Alabama at Birmingham, 1665 University Boulevard, RPHB 624 A, Birmingham, AL 35294–0022, USA.E-mail: jtang@uab.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Interleukin (IL)-18, a proinflammatory cytokine, has been recognized recently as an important factor in both treated and untreated patients with human immunodeficiency virus type 1 (HIV-1) infection. Consistent with all earlier reports, our quantification of serum IL-18 concentrations in 88 HIV-1 seropositive, North American adolescents (14–18 years old) revealed a positive correlation with cell-free HIV-1 viral load at two separate visits (Spearman\'s r = 0·31 and 0·50, respectively, P   0·01 for both), along with a negative correlation with CD4+ T cell counts (r = –0·31 and −0·35, P   0·01 for both). In additional analyses of 66 adults (21–58 years old) from Zambia, HIV-1 seroconversion was associated uniformly with elevated IL-18 production (P   0·0001). These epidemiological relationships were independent of other population-related characteristics, including age, gender and ethnicity. In neither study population could serum IL-18 concentrations be associated with the IL-18 gene (IL18) promoter genotypes defined by five major single nucleotide polymorphisms. Collectively, these findings suggest that circulating IL-18 rather than the IL18 genotype may provide a useful biomarker for HIV-1-related events or outcomes. Introduction As a close relative of interleukin (IL)-1β, IL-18 [formerly known as interferon (IFN)-γ-inducing factor] is a proinflammatory cytokine produced primarily by activated macrophages, dendritic cells and Kupffer cells [1-3]. Bioactive IL-18 is derived from a precursor protein, which is cleaved by the IL-1β-converting enzyme (ICE or caspase 1) [4]. A well-documented function of IL-18 is its ability to augment type 1 T-helper (TH1) responses, often in concert with IL-12 and through the production of IFN-γ[2, 5, 6]. In studies of human immunodeficiency virus type 1 (HIV-1) infection, the presumed anti-viral effects of IL-18 have been seen with bulk peripheral blood mononuclear cells (PBMCs) [7] but not with certain monocytic cell or T cell lines [8-10]. Serum IL-18 concentrations are elevated in patients with advanced diseases (AIDS) or with symptomatic HIV-1 infection [11, 12]. Serum IL-18 concentrations decrease in patients after highly active anti-retroviral therapy (HAART) [13, 14], while high IL-18 concentrations predispose HIV-1 seropositive patients to therapeutic failure or complications such as lipodystrophy [14, 15]. The IL-18 gene (IL18) encoding human IL-18 is mapped to chromosome 11q22.2–22·3 [16]. Genetic variations (mainly single nucleotide polymorphisms) within the human IL18 promoter and the non-coding exon 1 sequences also seem to account for differential IL-18 expression [17] and susceptibility to various infectious diseases [18]. In the context of HIV-1 infection, our work here aimed to define virological, immunological and genetic correlates of serum IL-18 concentrations in adolescent and adult populations. The 154 subjects studied here came from two parent projects. First, 88 HIV-1 seropositive, North American adolescents were selected randomly from the Reaching for Excellence in Adolescent Care and Health (REACH) study supported by the National Institute of Child Health and Development (NICHD) [19, 20]. Their ages ranged from 14 to 18 years at enrolment. HIV-1 viral load (RNA copies/ml) and CD4+ T cell counts (cells/µl) were quantified prospectively (every 3 months) in certified laboratories between 1996 and 2000 [21] to guide and/or monitor patient care, especially in relation to effective treatment, including HAART. T cell activation, as measured by CD8+ CD38+ T cells, was found to be a reliable predictor of CD4+ T cell loss in HIV-1 seropositive patients from this cohort [22]. Secondly, HIV-1 seropositive and seronegative adults came from the Zambia–Emory HIV-1 Research Project (ZEHRP) supported by National Institute of Allergy and Infectious Diseases (NIAID) [23, 24]. They were enrolled initially as part of a prospective programme for voluntary counselling and testing on a quarterly basis between 1996 and 2003. In this work, analyses began with 22 seroconverters, who were seronegative at enrolment and became seropositive at a subsequent follow-up visit. This selection was based solely on availability of serum specimens from both pre- and post-infection visit intervals. Cross-sectional analyses included 22 adult Zambians with prevalent HIV-1 infection (seropositive at enrolment) and another 22 subjects who were persistently seronegative despite the high risk of heterosexual HIV-1 transmission, as judged by self-reported sex behaviour and relatively high viral load (  10 000 HIV-1 RNA copies/ml of plasma) in their cohabiting partners already infected with HIV-1. In the absence of anti-retroviral therapy, HIV-1 viral load served as a key outcome measure in Zambians, while HIV-1 genotyping confirmed the predominance of clade C HIV-1 infection in this cohort [25]. Both parent studies (REACH and ZEHRP) and this substudy conformed to the procedures for informed consent approved by institutional review boards at all sponsoring organizations and to human experimentation guidelines set forth by the US Department of Health and Human Services. Serum samples frozen at −80°C were retrieved from central repositories for quantification of IL-18 concentration by sandwich ELISA using a commercial kit (MBL Ltd, Nagoya, Japan; marketed by R&D Systems, Minneapolis, MN, USA). These assays followed strictly the procedures recommended by the manufacturer, with all samples being tested in duplicate after a 1 : 10 dilution. The optical density at 450 nm (OD450) was measured using Synergy HT Autoreader (Bio-Tek Instruments, Inc., Winoosk, VT, USA). Conversion of OD450 to IL-18 concentration (in pg/ml) was based on a standard curve. The intra- and interassay coefficients of variation for IL-18 ELISA were 5·0–10·8% and 5·2–10·1%, respectively, which were consistent with results obtained from our testing samples. Additional ELISA assays (R&D Systems) also measured other cytokines, chemokines and related products [e.g. monocyte chemoattractant protein (MCP-1) (CCL2), regulated upon activation normal T cell expressed and secreted (RANTES) (CCL5), angiogenin and epidermal growth factor] that are often present at high concentrations in peripheral blood, but in this study none of these could account for our primary findings. Genomic DNA was extracted from PBMCs or whole blood using the QiaAmp blood kit (Qiagen Inc., Chatsworth, CA, USA) and stored in Tris-EDTA (TE) buffer [10 mM Tris-HCl, pH 8·0, 2 mM ethylenediamine tetraacetic acid (EDTA)] at a concentration of 100 ng/µl. An 820-base pairs (bp) fragment (nucleotides 15215–16034 in GenBank sequence AP002884) was amplified by polymerase chain reaction (PCR) and sequenced bi-directionally using the BigDye chemistry and automated capillary electrophoresis (Applied Biosystems, Foster City, CA, USA) in order to detect single nucleotide polymorphisms (SNPs). Linkage disequilibria (LD) between individual SNPs were determined and visualized using the haploview version 3·2. Alleles showing strong LD (D′   0·90) were analysed together as haplotypes for their respective associations with serum IL-18 concentrations. Sequencing was also performed for the IL18 gene in three chimpanzees (Pan troglodytes) (courtesy of Dr P. N. Fultz, Department of Microbiology, University of Alabama at Birmingham, AL, USA) in order to define the ancestral state of common SNPs in human populations. Routine procedures in the statistical analysis software (SAS) package, version 9·1 (SAS Institute, Cary, NC, USA) were used for descriptive and comparative analyses. For each cohort, serum IL-18 concentrations (pg/ml) were tested for relationships to categorical variables including gender, ethnicity, age group, status of HIV-1 infection, levels of HIV-1 viraemia, response to anti-retroviral therapy and IL18 genotypes (alleles and haplotypes). The Wilcoxon rank test and the Mann–Whitney U-test were applied to comparisons of paired and unpaired IL-18 measures, respectively. All categorical factors associated with IL-18 concentrations (univariate P ≤ 0·05) were retained as covariates in Spearman\'s rank correlation tests, in which IL-18 concentrations served as the independent variable, with HIV-1 viral load and CD4+ T cell counts being considered as dependent variables. Overall, the 88 adolescents (56 females and 32 males) with seroprevalent HIV-1 infection had a mean age of 17·6 years [standard deviation (s.d.) = 1·3 years]. Serum IL-18 concentrations in these adolescents, as quantified by ELISA, ranged from a median of 427 pg/ml [interquartile range (IQR) = 273–526 pg/ml] at visit 1 to a median of 366 pg/ml (IQR = 230–479 pg/ml) at visit 2 (Table 1). The mean time intervals between the two visits were 8·2 months for the 42 untreated patients and 9·7 months for the 46 treated patients. During these selected visit intervals, the clinically prescribed, anti-retroviral therapy regimens were effective for the 46 treated patients, as HIV-1 viral load (mean ± s.d.) declined from 4·20 ± 0·56 log10 before treatment to 1·89 ± 1·32 log10 after treatment (P   0·0001 by Wilcoxon\'s signed-rank test). Reduction in viral load was also accompanied by an increase in CD4+ T cell counts from 447 ± 174 cells/µl (before treatment) to 657 ± 251 cells/µl (after treatment) (P   0·0001). Table 1. Serum interleukin (IL)-18 concentrations in North American adolescents infected with human immunodeficiency virus type 1 (HIV-1). IL-18 concentration is expressed as pg/ml and P-values as shown are based on Mann–Whitney U-tests. IQR, interquartile range (25th−75th percentile points); n.a., not applicable. Restricted to the 42 patients not receiving treatment. Viral load is expressed as HIV-1 RNA copies per ml of plasma. Restricted to the 46 treated patients. P   0·0001 by Wilcoxon\'s signed-rank tests of paired IL-18 measurements obtained before and after therapy. The HIV-1 seropositive adolescents were stratified sequentially by ethnicity, gender, age group and HIV-1-related outcome measures (Table 1). At the two separate visits, IL-18 concentrations varied clearly by HIV-1 viral load (P = 0·01–0·02 by the Mann–Whitney U-test), CD4+ T cell counts (P = 0·01–0·02) and anti-viral treatment (P   0·0001 by Wilcoxon\'s signed-rank test) (Table 1) but not by ethnicity, gender, age or CD8+ CD38+ T cell counts. For example, in the 46 adolescent patients with a narrow pre- to post-treatment interval (mean = 9·7 months), median IL-18 concentrations decreased from 405 pg/ml (IQR = 198–494 pg/ml) at the pretreatment visit to 299 pg/ml (IQR = 204–399 pg/ml) at the post-treatment visit (P   0·001). In a linear model, IL-18 correlated positively with HIV-1 viral load [Spearman\'s correlation coefficient (r) = 0·31, P = 0·004] (Fig. 1a) and negatively with CD4+ T cell counts (Spearman\'s r = −0·31, P = 0·004) (Fig. 1b) at the pretreatment visit. At the next visit, when 46 patients had received and responded to anti-retroviral therapy, the correlation between IL-18 and viral load became stronger (Spearman\'s r = 0·50, P   0·0001), while correlation between IL-18 and CD4+ T cell counts remained stable (Spearman\'s r = −0·35, P   0·001). Meanwhile, IL-18 showed no clear correlation with CD8+ CD38+ T cell percentages (P ≥ 0·06 in all tests). Correlation between human immunodeficiency virus type 1 (HIV-1)-related outcome measures with serum interleukin (IL)-18 concentrations (pg/ml) in 88 HIV-1 seropositive adolescents (Reaching for Excellence in Adolescent Care and Health project). (a)Log10 HIV-1 RNA concentration (viral load) has a positive correlation with IL-18 concentrations [Spearman\'s correlation coefficient (r) = 0·31, P   0·01]. (b) CD4+ T cell counts has a negative correlation with IL-18 concentrations (Spearman\'s r = −0·31, P   0·01). These analyses were based on the earliest visit without anti-retroviral therapy. The dotted line on each axis indicates the mean value. Further analyses of data from a subsequent visit, when 46 (52%) of the patients received anti-retroviral therapy, led to similar findings (see text). The IL-18 concentrations in the 66 Zambians (31 females and 35 males; mean age = 31·5 years and s.d. = 9·1 years) were quite different to those seen in adolescents. In particular, HIV-1 seropositive Zambians had much higher median IL-18 concentrations (813 pg/ml) (IQR = 579–1365 pg/ml) when compared with untreated, HIV-1 seropositive adolescents (median = 427 pg/ml at visit 1) (P   0·001 by the Mann–Whitney U-test). Within the adult Zambian population, however, serum IL-18 concentration did not differ by gender or age group (≥ 40 versus   40 year). For the 22 seroconverters, the mean time interval between the two studied visits was 9·5 months. IL-18 concentrations increased by about twofold (Fig. 2a), from a median of 375 pg/ml (IQR = 252–479 pg/ml) before HIV-1 infection to a median of 777 pg/ml (IQR = 544–1137 pg/ml) after HIV-1 infection. The increase in IL-18 concentrations following HIV-1 infection was almost universal, being seen in all but one (4·5%) seroconverters. The linear correlation between IL-18 concentration and HIV-1 viral load at the post-seroconversion visit (Spearman\'s r = 0·40, P = 0·06 due to the small sample size) in Zambians was similar in magnitude to that observed in HIV-1-infected adolescents (Fig. 1). Serum interleukin (IL)-18 concentrations (pg/ml) in adult Zambians stratified by human immunodeficiency virus type 1 (HIV-1) infection. The interquartile ranges (boxes), medians (bars within boxes) and entire ranges (closed vertical bars) are shown for several patient groups. In (a), results were obtained from 22 adults before and after HIV-1 infection. In (b), results were obtained from 22 high-risk individuals without HIV-1 infection (persistently seronegative) and another 22 subjects with prevalent HIV-1 infection (seropositive at first visit). The P-values as shown are based on Wilcoxon\'s rank test of paired IL-18 measures (a) and the Mann–Whitney U-test of unpaired IL-18 results (b). For secondary, cross-sectional analyses, the 22 high-risk seronegative Zambians were compared with 22 seroprevalent individuals at a single visit (Fig. 2b). Median IL-18 concentrations in the subjects with prevalent HIV-1 infection were nearly 70% higher than those in the seronegatives (833 versus 498 pg/ml), with IQRs of 605–1708 and 336–629 pg/ml, respectively (P = 0·002). When the 22 persistently seronegatives were compared further with the 22 seroconverters at the preinfection visit, modest differences (33%, P = 0·09) between their respective median IL-18 concentrations exceeded that of interassay variability (ranging from 8% to 13%). Duration of HIV-1 infection did not seem to bring additional changes in IL-18 concentrations, as seroprevalent Zambians with much longer duration of HIV-1 infection did not differ from the newly infected seroconverters in their respective IL-18 concentrations (P = 0·31). Within individuals, IL-18 concentrations were highly predictable between visits, as reflected by Spearman\'s rank correlation coefficients in both study populations. For example, after statistical adjustment for age, gender and time interval between visits, IL-18 concentrations before and after HIV-1 infection had a strong correlation between visits in the 22 Zambian seroconverters (adjusted r = 0·70, P   0·001). In the 42 HIV-1 seropositive adolescents not receiving anti-retroviral therapy, IL-18 concentrations at the first and second visits also showed a clear correlation (r = 0·78, P   0·0001). Similarly, correlation was seen with the two separate measures of IL-18 concentrations at the pre- and post-treatment visits (r = 0·67, P   0·0001) for the 46 treated adolescents. The strengths of these intervisit correlations were similar to those of HIV-1 viral load or CD4+ T cell counts in the various subgroups of patients (adjusted r = 0·51–0·89, P   0·001 for all analyses). IL18 promoter genotypes and their relationships to circulating IL-18 concentrations and HIV-1 infection DNA sequencing was successful for 63 Zambians (adults), 58 African Americans (adolescents) and 24 others (adolescent European Americans and Hispanic Americans); all five common SNPs were detected in the 820-bp region (Fig. 3). Their positions, relative to the major transcription start site, corresponded to nucleotides −656, −607, −137, +113 and +127, respectively [17]. The first two, with reference sequence (rs) numbers rs1946519 and rs1946518 in the dbSNP database, showed very tight linkage disequilibrium (D′ = 0·99) and met the most stringent definition of a haplotype block (haploblock) [26]. Similarly, the three downstream SNPs, designated as rs187238, rs360718 and rs360717, formed another haploblock as a result of tight linkage disequilibrium in pairwise tests (D′   0·95). Interleukin 18 gene (IL18) polymorphisms. DNA sequencing has revealed five major single nucleotide polymorphisms (SNPs) within an 820 base pairs (bp) region (nucleotides 15215–16034 in GenBank sequence AP002884). These SNPs form two haplotype blocks (haploblocks) based on the strengths of linkage disequilibrium (a). For each SNP site, the sequence found exclusively in three chimpanzees is shown in bold. Relative nucleotide positions (pos.) refer to distances from the transcription start site (+1). Frequencies of three major haplotypes in the first block vary greatly (P   0·001) between North American (African American and other) and Zambian populations (b), while haplotypes in the second block are distributed more equally. Haplotypes formed between the first two SNPs (block 1) showed population-specific differences in their frequencies (P = 0·001), while others formed among the other three SNPs (block 2) were distributed more equally among the three ethnic groups (native African, African American and other) (Fig. 3). None of the SNP alleles and their intrablock haplotypes could be associated with serum IL-18 concentrations in any aggregate or stratified analyses, but the extended and second most common haplotype T-A-C-G-T (in the order of 5′−3′) was associated with slightly lower IL-18 concentrations in the 42 adolescent (REACH) patients not receiving therapy for any of the two visits (P = 0·03 and 0·07 at visits 1 and 2, respectively). This association was not confirmed in analysis of the treated adolescents (P = 0·85 at the pretreatment visit 1) or adults (Zambians) (P   0·50), while no relationships were found between IL18 variants and HIV-1-related outcomes (seroconversion, viral load and CD4+ T cell counts) (data not shown). Three additional SNPs (rs5744225, rs5744226 and rs5744227) recorded in various public databases (e.g. National Center for Biotechnology Information and HapMap) were also confirmed in our study populations, with minor allele frequencies ranging from 0·007 to 0·009. Three novel SNPs were also seen in multiple individuals (n = 4–7). However, these SNPs were too rare to withstand any informative association analyses. When compared with IL18 sequences from three chimpanzees, all but one SNP (rs1946519) found in humans had the major allele sequences that most probably represented the ancestral states, i.e. identical between chimpanzees and humans. The minor allele (T) of rs1946519 matched all chimpanzee sequences, suggesting a preferential expansion of the alternate allele (G) in humans, as represented by the study populations. Our studies of two populations that differed starkly in age (adolescent versus adult), geography (United States versus Zambia), HIV-1 subtypes (clade B versus clade C) and access to anti-retroviral therapy produced consistent evidence that serum IL-18 concentrations correlate reciprocally with the two most common measures of clinical importance (viral load and CD4+ T cell count). Overall, positive correlation between serum IL-18 and HIV-1 viral was accompanied by negative correlation between serum IL-18 and CD4+ T cell count, which was corroborated further by increased serum IL-18 concentrations associated with HIV-1 seroconversion, along with decreased serum IL-18 concentrations following effective anti-retroviral therapy. These findings are highly consistent with those from five earlier studies of treated and untreated patient populations in Canada and Europe (Denmark, Italy, Norway and Poland) [11-15]. Mechanisms underlying these epidemiological relationships between serum IL-18 and HIV-1 infection are not well understood. On one hand, IL-18-mediated augmentation of IFN-γ production can help control HIV-1 infection, but this function can be abolished by two high-affinity isoforms of IL-18-binding protein (IL−18-BP) [27]. On the other hand, IL-18 is also known to induce the production of TH2 cytokines (IL-4 and IL-13) by certain cells [28, 29]. The dual role of IL-18 in TH1 and TH2 Immune responses can be complicated or compromised by HIV-1 infection when HIV-specific CD4+ and CD8+ T cells are often defective in co-producing IL-2 (a T cell growth factor) and IFN-γ[30, 31]. Presumably, elevated IL-18 concentrations following HIV-1 infection can result from the well-documented phenomenon of hyper-immune activation [22, 32] and progressive T cell malfunction [33-35]. Additional longitudinal data from carefully selected patient groups may help elucidate the exact role of IL-18 and timing of its response to HIV-1 infection. The relationships between IL-18 and other, more classic cytokines [e.g. IFN-γ, IL-2, IL-4, IL-5 and tumour necrosis factor (TNF)-α] are of broad interest. In studies of cardiovascular diseases, IL-18 is already associated with other proinflammatory cytokines, including IL-1β and TNF-α[36, 37]. More recent work suggests that IL-18 triggers the production of intercellular adhesion molecule 1 (ICAM-1), followed by release of soluble ICAM-1 into the bloodstream [38]. In other studies of HIV-1 infection, it is evident that acute infection leads to elevated gene expression of IL-1β, IL-6, IL-8, IL-10 and TNF-α[39, 40]. The persistent correlation of IL-18 with HIV-1-related outcomes may also imply the involvement of other cytokines and related products. However, unlike most of the classic cytokines that circulate (often transiently) at low concentrations (typically 100 pg/ml) in the peripheral blood [41], IL-18 is always present at a much higher concentration in serum from HIV-infected patients. It is also clear that within individuals, serum IL-18 concentration at one visit can be highly predictable of the concentrations at the next visit, regardless of substantial changes associated with HIV-1 seroconversion or effective anti-retroviral therapy. As high IL-18 concentrations are further predicative of therapeutic failure or other complications in HIV-1 patients [13, 15, 42], it may be useful to consider serum IL-18 as another informative biomarker for HIV-related outcomes. The organization and sequence variation in the IL18 gene that encodes human IL-18 has been well characterized [17, 43, 44]. Our analyses here suggest that the five common SNPs, located in the 5′ untranscribed region and in the transcribed but non-coding exon 1, belong to two haploblocks. The −137, +113, and +127 SNPs (rs187238, rs360718 and rs360717, respectively) within the second block have been associated with autoimmune, inflammatory and infectious diseases in Caucasians, Japanese and Chinese [17, 18, 45, 46]. However, our work did not reveal any clear association between IL18 genotypes and serum IL-18 concentrations or HIV-1-related outcomes. The limitation of statistical power can be a possible explanation, as our sample size was small or modest for each cohort. Alternatively, genetic determinants of IL-18 expression may lie in other regions of the IL18 sequences not being tagged by the few SNPs defined in our study populations. 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Association of interleukin-18 gene single-nucleotide polymorphisms with susceptibility to inflammatory bowel disease. Tissue Antigens 2005; 65: 88– 92. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Please check your email for instructions on resetting your password. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. Can\'t sign in? Forgot your username? Enter your email address below and we will send you your username If the address matches an existing account you will receive an email with instructions to retrieve your username