Genes and Immunity (2009) 10, 487–494 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09 $32.00 www.nature.com/gene ORIGINAL ARTICLE Age- and gender-specific modulation of serum osteopontin and interferon-a by osteopontin genotype in systemic lupus erythematosus SN Kariuki1, JG Moore1, KA Kirou2,MKCrow2, TO Utset1 and TB Niewold1 1Section of Rheumatology, University of Chicago, Chicago, IL, USA and 2Mary Kirkland Center for Lupus Research, Hospital for Special Surgery, New York, NY, USA Osteopontin (OPN) is a multifunctional cytokine involved in long bone remodeling and immune system signaling. Additionally, OPN is critical for interferon-a (IFN-a) production in murine plasmacytoid dendritic cells. We have previously shown that IFN-a is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variants of OPN have been associated with SLE susceptibility, and one study suggests that this association is particular to men. In this study, the 3 0 UTR SLE-risk variant of OPN (rs9138C) was associated with higher serum OPN and IFN-a in men (P ¼ 0.0062 and P ¼ 0.0087, respectively). In women, the association between rs9138 C and higher serum OPN and IFN-a was restricted to younger subjects, and risk allele carriers showed a strong age-related genetic effect of rs9138 genotype on both serum OPN and IFN-a (Po0.0001). In African- American subjects, the 5 0 region single nucleotide polymorphisms, rs11730582 and rs28357094, were associated with anti- RNP antibodies (odds ratio (OR) ¼ 2.9, P ¼ 0.0038 and OR ¼ 3.9, P ¼ 0.021, respectively). Thus, we demonstrate two distinct genetic influences of OPN on serum protein traits in SLE patients, which correspond to previously reported SLE-risk variants. This study provides a biologic relevance for OPN variants at the protein level, and suggests an influence of this gene on the IFN-a pathway in SLE. Genes and Immunity (2009) 10, 487–494; doi:10.1038/gene.2009.15; published online 2 April 2009 Keywords: systemic lupus erythematosus; interferon-a; osteopontin; age; gender; autoantibodies Introduction IFN-a is clustered in SLE families in both healthy and SLE-affected members, suggesting that high serum IFN-a Systemic lupus erythematosus (SLE) is a prototype is a heritable risk factor for SLE.7 In follow-up studies, systemic autoimmune disorder, typically involving the we have demonstrated two SLE-associated genetic skin, musculoskeletal, renal and hematologic organ polymorphisms, IRF5 and PTPN22, predispose to high systems. Susceptibility to SLE likely arises from a serum IFN-a in SLE patients,8,9 however, much of the complex network of gene–gene and gene–environment genetic variation resulting in heritability of serum IFN-a interactions.1 Although many genetic loci have been remains to be discovered. linked to disease susceptibility, much work remains to Osteopontin/secreted phosphoprotein 1 (OPN) is a integrate these loci into the pathophysiology of the phosphorylated extracellular matrix protein with disease. Recent studies suggest a role for interferon-a a variety of functions, including wound healing, bone (IFN-a) in SLE pathogenesis. IFN-a is a pleiotropic type I formation and remodeling,10 as well as immunological IFN with the potential to break self tolerance by functions such as T-cell activation, Th1 differentiation, activating antigen-presenting cells after uptake of self B-cell activation,11 and macrophage activation and material.2 Serum IFN-a levels are elevated in many SLE chemotaxis.12 Studies have demonstrated high levels of patients, and elevations may correlate with disease OPN in biopsies of inflamed tissues in SLE and other activity.3,4 In addition, a number of patients treated with autoimmune diseases,13,14 and variants of the OPN gene recombinant human IFN-a for malignancy and chronic have been associated with SLE susceptibility.15–17 In a viral hepatitis have developed de novo SLE, which study of 394 Italian SLE patients, two single nucleotide typically resolves after the IFN-a is discontinued.5,6 We polymorphisms (SNPs) in OPN were associated with have previously shown that abnormally high serum SLE, including rs11439060 in the 50 flanking region (odds ratio (OR) ¼ 2.35, P ¼ 0.006) and rs9138 in the 30 UTR (OR ¼ 1.57, P ¼ 0.00094).16 Linkage disequilibrium Correspondence: Dr TB Niewold, Section of Rheumatology, between these two SNPs was low (r2 ¼ 0.23), and logistic University of Chicago, 5841 S Maryland Ave., MC 0930, Chicago, regression models supported an independent genetic IL 60637, USA. E-mail: [email protected] association between each of these SNPs and SLE. Received 8 December 2008; revised and accepted 03 March 2009; A follow-up study including 707 European-American published online 2 April 2009 and 434 African-American SLE patients did not find Osteopontin genotype modulates serum cytokines SN Kariuki et al 488 significant associations between 50 SNPs and SLE, older SLE patients.19 Given the role of OPN in long bone although the rs11439060 variant was not genotyped remodeling and the potential for age-related influences directly.17 This study replicated the association between on OPN expression, we also looked for age-related rs9138 and SLE, although surprisingly the association trends in serum IFN-a and serum OPN levels related to was particular to the male patients in their cohort (13% of OPN genotype. Cytokines were studied as quantitative the SLE patients studied). The previous Italian study did traits in analyses stratified by OPN SNPs, gender and not separate patients by gender for analysis, however, the previously described high IFN-a age groups.19 SLE- male/female ratio in that study was similar (14% male). associated autoantibodies were studied in the patients as The potential influence of OPN genetic variants on categorical traits, analyzed as outcome variables in serum OPN levels is not well described to date. One logistic regression models using OPN SNPs as predictor previous study found that the SLE-associated rs9138 C variables. allele in the 30 UTR was associated with increased serum OPN levels in healthy controls, however, the same association was not seen in the SLE patients.16 In murine models, OPN is essential for IFN-a production down- Results stream of the endosomal Toll-like receptor 9 in plasma- rs9138 C allele was associated with increased IFN-a in male cytoid dendritic cells, likely via interaction with the SLE patients MyD88 adaptor protein.18 Plasmacytoid dendritic cells We analyzed serum IFN-a data in the 40 male SLE are thought to be the major IFN-a producing cells in the patients stratified by OPN genotype at each of the 9 body, and this striking impact of OPN on the murine genotyped SNPs. Diagrams showing the haplotype Toll-like receptor/IFN-a system suggests a potential structure between the nine SNPs in each ancestral relevance for OPN in IFN-a production in human background are shown in Figure 1, and allele frequencies disease. are shown in Supplementary Table 1. The rs9138 C allele In this study, we investigate the association of nine was associated with increased serum IFN-a activity in OPN SNPs with serum IFN-a activity and serum OPN male patients in a dose–response fashion (P ¼ 0.0087; levels in SLE patients, to determine whether genetic Figure 2a). rs9138 is the most consistently replicated SLE- variation at the OPN locus is associated with altered risk SNP in previous case–control genetic studies,15–17 serum levels of IFN-a, OPN or autoantibodies in SLE and this is the SNP which demonstrated a preferential patients in vivo. SNPs previously associated with SLE15–17 association with SLE in men in one previous study.17 and tag SNPs collectively representing 495% of the Other OPN SNPs did not show significant associations known variation in the gene region were included in the with serum IFN-a activity in male patients (P40.4 for study. other SNPs, data not shown). Male patients of all Given that a gender-specific genetic association ancestral backgrounds were analyzed in aggregate, as between the rs9138 SNP in OPN and SLE was detected the number of male subjects was too small to support in one study,17 we separated male and female patients for separate analyses in each ancestral background. Pub- these analyses. Additionally, we have previously shown lished work supports the idea that rs9138 C is a risk that younger SLE patients have higher serum IFN-a than factor for SLE in male subjects of both European and African-American European-American 123456789 123456789 511016 0423 0 48 34 60 58 16 16 75 7 0 11 2 20 21 14 6 47 51 43 10 25 20 20 27 44 6 4 40 24 77 75 10 12 21 78 9 0 0 9 1 53 21 11 10 19 7 31 1 7 9 27 13 11 2 19 9 9 8 13 2 0 5 11 5 9 Hispanic-American 1. rs10516800 2. rs11730582 1234567 89 3. rs28357094 16 9 46 13 3 6 50 0 4. rs2853749 13 19 12 2 22 12 9 5. rs11728697 23 76 0 3 37 51 7 4 3 3 11 6. rs6532040 0 23 7 0 7. rs1126772 2 13 9 8. rs9138 1 4 0 9. rs7655182 Figure 1 Haplotype structures observed in osteopontin (OPN) gene in each ancestral background studied. Darker gray shading in the box plot indicates higher r2 values, and a gene diagram showing the location of each studied single nucleotide polymorphism (SNP) is shown above the box plot. Pairwise r2 values in each square are shown as r2 Â 100, calculated using Haploview 4.0 software. Genes and Immunity Osteopontin genotype modulates serum cytokines SN Kariuki et al 489 Figure 3 Serum interferon-a (IFN-a) activity in female patients stratified by age and osteopontin (OPN) rs9138 genotype.