Age- and Gender-Specific Modulation of Serum Osteopontin and Interferon-Α by Osteopontin Genotype in Systemic Lupus Er

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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

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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. P value for a difference in serum IFN-a by OPN genotype in the p23-year- old group calculated as the probability that a horizontal line was a better fit of the data than a semi-log line with a non-zero slope between genotype categories using the extra sum-of-squares F test. P value for a difference in serum IFN-a in AC genotype p23 years old vs AC genotype in 423 years old calculated by Mann–Whitney U-test. Each dot represents a unique patient; lines and error bars show the median and interquartile range.

significant relationships were seen between any of the OPN SNPs and serum IFN-a (P40.15 for all SNPs, data not shown). The rs9138 C allele has been associated with SLE in cohorts composed of 86% female subjects previously,16 however, there was no significant relationship between serum IFN-a and rs9138 in the female patients, in contrast to the above result in the male patients (Figure 2b). We undertook additional analysis to explore the possibility of an age-related genetic effect in female patients at rs9138. Given the role of OPN in bone remodeling and long bone growth,10 we hypothesized Figure 2 Serum interferon-a (IFN-a) in male and female systemic that the OPN gene may exhibit age-specific regulation, lupus erythematosus (SLE) patients stratified by osteopontin (OPN) and that OPN may be differentially expressed in younger rs9138 genotype. (a) Shows male SLE patients stratified by OPN patients. We stratified the female patients by age, using genotype, P value calculated as the probability that a horizontal line cutoffs established in our previous work defining the was a better fit of the data than a semi-log line with a non-zero slope 19 between genotype categories using the extra sum-of-squares F test. highest IFN-a age group (age range 12–23). In this Each dot represents a unique patient; lines and error bars show the analysis, we saw that the rs9138 C allele was associated median and interquartile range. (b) Shows all female SLE patients with increased serum IFN-a in a dose–response fashion stratified by OPN genotype, P value by Mann–Whitney U-test. in the younger patients (Figure 3; P ¼ 0.018). There was a strikingly strong age-related difference in serum IFN-a comparing younger vs older patients with the AC African-American ancestry, with a similar OR in each genotype at rs9138, which was not present in subjects ancestral background.17 with the AA genotype (Figure 3; P ¼ 0.0001 for higher serum IFN-a in subjects aged p23 years with AC Age-related effect of rs9138 C allele on serum IFN-a in female genotype vs subjects aged 423 years with AC genotype, SLE patients P ¼ 0.87 for an age-related difference in serum IFN-a in We next examined serum IFN-a activity in the 283 female subjects with AA genotype). When patients are analyzed SLE patients in relation to each of the 9 OPN SNPs. No separately by ancestry, similar age-related trends in

Genes and Immunity Osteopontin genotype modulates serum cytokines SN Kariuki et al 490 serum IFN-a are seen in each ancestral background in patients with rs9138 AC and AA genotypes (Supplementary Figure 1).

Serum OPN and IFN-a were correlated, and serum OPN levels show a similar relationship with OPN genotype We next measured serum OPN levels in the same sample in which IFN-a was measured for 180 SLE patients of representative age, sex and OPN genotype. There were no differences in OPN levels between male and female patients, although both male and female patients had higher serum OPN levels than the healthy unrelated controls (Figure 4a). Comparing OPN levels to IFN-a levels in the same sample, the two cytokines were weakly correlated in the overall cohort (Spearman’s rho ¼ 0.18, P ¼ 0.016). When patients were separated by age and gender, there was an OPN/IFN-a correlation in 53 women aged p23 years with OPN data (Spearman’s r ¼ 0.30, P ¼ 0.032) and there was a nonsignificant similar tendency in the 29 male patients with OPN data (Spearman’s rho ¼ 0.28, P ¼ 0.15; combining female patients aged p23 years and male patients, Spearman’s rho ¼ 0.33, P ¼ 0.0037). This relationship was not present in the 98 female patients aged 423 years (Spearman’s rho ¼ 0.09, P ¼ 0.37). We then examined serum OPN levels in patients stratified by gender, age in women and genotype at the rs9138 SNP to determine if serum OPN demonstrated similar age- and gender- related influences in relation to this SNP. Male SLE patients showed a dose–response increase in serum OPN in relation to the rs9138 C allele, similar to the pattern seen with IFN-a in male patients (P ¼ 0.0062; Figure 4b). Female patients did not show any overall difference in serum OPN when stratified by rs9138 genotype (Figure 5a). However, after stratification of the cohort by age, the subjects p23 years old showed a dose–response relationship between rs9138 C and higher serum OPN (P ¼ 0.021; Figure 5b). Older patients showed no statistical difference in serum OPN levels related to rs9138 genotype. Similar to the IFN-a analyses, younger patients with the AC genotype at rs9138 showed a large difference in serum OPN when compared to older patients with the AC rs9138 genotype (Po0.0001), and Figure 4 Serum osteopontin (OPN) in the overall systemic lupus erythematosus (SLE) cohort and in male SLE patients stratified by patients with the AA genotype did not show any age- OPN rs9138 genotype. (a) Shows serum OPN in men, women and related difference in serum OPN levels (Figure 5b). healthy unrelated controls, with P values by Mann–Whitney U-test. Similar age-related patterns of serum OPN are seen in (b) Shows serum OPN in male SLE patients stratified by OPN relation to the AC and AA genotypes of rs9138 in each genotype, P value calculated as the probability that a horizontal line ancestral background (Supplementary Figure 2). was a better fit of the data than a semi-log line with a non-zero slope between genotype categories using the extra sum-of-squares F test. Each dot represents a unique patient; lines and error bars show the rs9138 C allele is enriched in younger female SLE patients median and interquartile range. Comparing allele frequencies at rs9138 in female SLE patients, there was a nonsignificant tendency toward increased frequency of the C allele in those p23 years old at the time of sample vs those 423 years old at the this finding requires replication in larger independent time of sample (European-American OR ¼ 1.48, P ¼ 0.25; cohorts. African-American OR ¼ 1.35, P ¼ 0.53; Hispanic-Ameri- can OR ¼ 1.50, P ¼ 0.41). The modified Breslow–Day test 50 region flanking OPN was associated with anti-RNP for OR homogeneity20 across ancestral backgrounds was antibodies in African-American patients not significant (w2 ¼ 1.99 with 2df, P ¼ 0.37), indicating no We have previously shown associations between SLE- significant heterogeneity of genetic effect in this sub- specific autoantibodies and serum IFN-a activity,7,8 and analysis. Thus, we also performed a combined analysis our group and others have demonstrated associations across all ancestries, which demonstrated a marginally between SLE-specific autoantibodies and SLE risk significant enrichment of the rs9138 C allele in the alleles.21,22 We tested for autoantibody-OPN genotype younger subjects (OR ¼ 1.49, w2 ¼ 4.22 with 1df, relationships in our cohort using logistic regression P ¼ 0.040). Power is limited with our sample size, and modeling, and we found two SNPs in the 50 region

Genes and Immunity Osteopontin genotype modulates serum cytokines SN Kariuki et al 491 with risk of SLE, and the association of the À156 indel was independent of the rs9138 C allele in this previous study.16 We saw similar nonsignificant trends toward association of these two 50 SNPs with anti-RNP antibodies in the other ancestral backgrounds. Anti-RNP antibodies were found at lower frequency in other ancestral backgrounds (62% in African-American vs 18% in European-American and 41% in Hispanic-Amer- ican), and it is possible that we were underpowered to detect this association in other ancestral backgrounds. The other SNPs, including rs9138, did not show any significant relationships with autoantibody traits in our SLE cohort. There was no significant linkage disequilibrium between the 50 flanking SNPs, rs11730582 and rs28357094, and the 30 UTR SNP, rs9138 (r2o0.2 for all pairwise comparisons in all ancestral backgrounds; Figure 1). The prevalence of anti-RNP antibodies did not differ by age, and there was no evidence for an age- or sex-related effect at these two 50 flanking region SNPs (data not shown). Genotype at the two 50 SNPs was not associated with statistical differences in serum IFN-a or serum OPN in the overall cohort, in African-American patients, or in patients stratified by anti-RNP antibodies (P40.25 for each SNP in each of the above analyses). Although anti-RNP antibodies have been associated with higher IFN-a in SLE patients,7 we were likely underpowered to detect an overall difference in serum IFN-a in our cohort due to these alleles. There were no significant differences in serum OPN comparing subjects with anti-RNP antibodies to those lacking anti-RNP antibodies (P ¼ 0.90 in overall cohort, P ¼ 0.73 in African- American subjects).

Discussion In this study, we demonstrate an association of the OPN rs9138 variant with two cytokine traits, serum IFN-a activity and serum OPN. The SLE-risk rs9138 C allele is associated with higher levels of both cytokines, suggest- Figure 5 Serum osteopontin (OPN) in female systemic lupus ing that these cytokines may mediate SLE susceptibility erythematosus (SLE) patients. (a) Shows female SLE patients related to this locus. Strikingly, this effect at rs9138 was stratified by OPN rs9138 genotype, all P values 40.05. (b) Shows observed in a small cohort of male SLE patients with an female SLE patients stratified by age and OPN rs9138 genotype. age range of 8–68 (mean age ¼ 29, s.d. ¼ 18), whereas the P value for a difference in OPN levels by OPN genotype in the p23- same effect appears to be restricted to female SLE year-old group calculated as the probability that a horizontal line patients p23 years old. The male SLE cohort is not large was a better fit of the data than a semi-log line with a non-zero slope between genotype categories using the extra sum-of-squares F test. enough to perform age stratification, and it is possible P value for a difference in OPN levels in AC genotype p23 years that a similar age-related effect is driving the relationship old vs AC genotype in 423 years old calculated by Mann–Whitney we demonstrate in male patients. However, a difference U-test. between men and women in OPN regulation is also possible, and interestingly a recent large-scale genetic association study found that risk of SLE due to OPN genotype at rs9138 was specific to male patients.17 We flanking OPN, rs11730582T and rs28357094 G, were would hypothesize that reanalysis of the case–control associated with anti-ribonucleoprotein (anti-RNP) genetic data in female patients by age may demonstrate autoantibodies in African-American ancestry patients an association between the rs9138 C allele and SLE (P ¼ 0.0038, OR ¼ 2.9, 95% confidence interval (CI) ¼ susceptibility in women aged p23 years. Our current 1.4–6.1 for rs11730582 and P ¼ 0.021, OR ¼ 3.9, 95% data suggest that the previously reported SLE-risk allele CI ¼ 1.1–11.8 for rs28357094). The rs11730582T allele rs9138 C is enriched in younger female SLE patients, in was common in African-American subjects, and thus whom the genetic effect on cytokine profiles is observed. had a lower P value despite also having a lower OR than This preliminary observation requires validation in the rare rs28357094 G allele. These two SNPs are in LD larger case–control datasets, and if confirmed would with an insertion/deletion (À156 À/G, rs11439060) in support the idea that OPN variants predispose to SLE via European ancestry which has previously been associated their impact on cytokine traits.

Genes and Immunity Osteopontin genotype modulates serum cytokines SN Kariuki et al 492 We have previously demonstrated age-related trends Medicine (TRIDOM) at the University of Chicago and the in serum IFN-a activity in SLE patients and their first Hospital for Special Surgery (HSS) Lupus Registry. A degree relatives,19 and this study would suggest that total of 323 SLE patients (283 female, 40 male) were OPN genotype contributes to this phenomenon at least in studied. Patients in the study were of African-American women. Physiologic reasons for age-specific expression (n ¼ 146), European-American (n ¼ 108) and Hispanic of OPN are unclear, however, OPN plays a role in long ancestries (n ¼ 69). All patients met four or more of the bone remodeling,10 and the period of long bone devel- American College of Rheumatology criteria for the opment during adolescence coincides with the age range diagnosis of SLE.26 To standardize the IFN-a assay, 141 in which we see differences in serum OPN due to OPN healthy donor serum samples were used, as previously genotype. Additionally, work in both murine and human described.7 The study was approved by the institutional in vitro systems suggests that estrogen can stimulate review boards at all institutions, and informed consent OPN expression via the nonclassical estrogen receptor- was obtained from all subjects in the study. related receptor-a (ERR-a) pathway.23,24 It is possible that age-related differences in estrogen signaling in the Reporter cell assay for IFN-a female SLE patients could underlie some of the age- The reporter cell assay for IFN-a has been described in related differences in OPN we have observed. detail elsewhere.7,27 Reporter cells were used to measure The mechanism by which OPN genotype modulates the ability of patient sera to cause IFN-induced serum IFN-a is also unclear, although murine data gene expression. The reporter cells (Wistar Institute, suggest an essential role for OPN in IFN-a production Susan Hayflick (WISH) cells, ATCC no. CCL-25 by plasmacytoid dendritic cells.18 The rs9138 C SLE-risk (American Type Culture Collection, Manassas, VA, USA variant in the 30 UTR is associated with increased serum http://www.atcc.org) were cultured with 50% patient OPN levels in our study. We favor the hypothesis that sera for 6 h, and then lysed. mRNA was purified from both intracellular and extracellular OPN levels are cell lysates, and cDNA was made from total cellular regulated by the 30 UTR region, possibly by variation in mRNA. cDNA was then quantified using real-time PCR mRNA polyadenylation or stability. Other influences, using an Applied Biosystems 7900HT PCR machine with such as promotion of gene expression due to increased the SYBR Green fluorophore system. Forward and estrogen/ERR-a signaling, could cooperate with in- reverse primers for the genes MX1, PKR and IFIT1, creased mRNA stability to result in higher protein levels. which are known to be highly and specifically induced Increased OPN levels in the setting of the risk variant by IFN-a, were used in the reaction.7 GAPDH was could subsequently augment downstream signaling from amplified in the same samples to control for background endosomal Toll-like receptors, resulting in greater IFN-a gene expression. production, and the correlation between serum IFN-a Real-time PCR data analysis and serum OPN, which we observed in certain patient The amount of PCR product of the IFN-a-induced gene subgroups. was normalized to the amount of product for the The association between 50 flanking SNPs and anti- housekeeping gene GAPDH in the same sample. The RNP antibodies in the African-American subjects is relative expression of each of the three tested IFN- interesting, and this will be important to replicate and induced genes was calculated as a fold increase extend to other ancestral backgrounds. Anti-RNP anti- compared to its expression in WISH cells cultured with bodies are strongly associated with higher serum IFN-a media alone. Healthy unrelated donor sera (n ¼ 141) in SLE patients,7 and the nucleic acid component of anti- were tested in the WISH assay to establish a normal RNP antibody immune complexes is thought to activate value for IFN-a activity, and the mean and standard the endosomal Toll-like receptor pathway of IFN-a deviation (s.d.) of IFN-a-induced gene relative expres- production.25 Our data suggest two distinct influences sion induced by healthy donor sera were calculated. The of OPN genotype on the IFN-a system in SLE, one ability of patient serum samples to cause IFN-induced variant in the 30 UTR which likely influences OPN gene expression in the reporter cells was then compared production and subsequently IFN-a production in to the mean and s.d. induced by healthy unrelated donor plasmacytoid dendritic cells, and a second variant which serum. The number of s.d. above healthy donors for each is associated with anti-RNP antibodies capable of gene was calculated to generate a serum IFN-a activity activating the endosomal Toll-like receptor pathway. score as described in reference.7 Both of these variants have been previously associated with SLE susceptibility, suggesting that the protein Osteopontin ELISA phenotypes and patterns of association we present relate OPN was measured in serum samples of 180 of the SLE to underlying disease pathogenesis and susceptibility. patients of representative age, sex and genotype within The complex patterns of association we demonstrate may the cohort using the Assay Designs, Ann Arbor, MI, USA be helpful in reconciling previous case–control genetic human OPN enzyme-linked immunosorbent assay studies, as differences in age, gender or autoantibody (ELISA), per manufacturer instructions. This ELISA kit composition of the cases could have influenced the has been shown to have good performance character- outcome of previous studies. istics when used to measure OPN in human serum.28 Healthy unrelated donor samples were tested (n ¼ 15) and performed as expected (mean ¼ 1.71 ng mlÀ1, Materials and Methods s.d. ¼ 1.02 ng mlÀ1). Patients and samples Measurement of autoantibodies Serum and genomic DNA samples were obtained from Antibodies to anti-Ro, anti-La, anti-Sm and anti-RNP the Translational Research Initiative in the Department of were measured by ELISA methods in the University of

Genes and Immunity Osteopontin genotype modulates serum cytokines SN Kariuki et al 493 Chicago and the Hospital for Special Surgery samples in Doctoral Fellowship Award, Arthritis National Research the respective clinical laboratories. Standard clinical lab Foundation Scholar Award. cutoff points were used to categorize samples as positive or negative. Anti-dsDNA antibodies were measured using Crithidia lucidae immunofluorescence at both sites, and detectable fluorescence was considered positive. References

Genotyping 1 Harley JB, Kelly JA, Kaufman KM. Unraveling the genetics of Individuals in the HSS registries were genotyped at the systemic lupus erythematosus. Springer Semin Immunopathol 2006; 28: 119–130. previous SLE-associated SNPs rs9138, rs11730582 and 2 Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. 15–17 rs28357094, as well as the tag SNPs rs10516800, Induction of dendritic cell differentiation by IFN-alpha in rs2853749, rs11728697, rs6532040, rs1126772 and systemic lupus erythematosus. Science 2001; 294: 1540–1543. rs7655182 to cover 495% of the variation in the gene 3 Hooks JJ, Moutsopoulos HM, Geis SA, Stahl NI, Decker JL, region. Genotyping was performed using ABI Notkins AL. Immune interferon in the circulation of patients Taqman (Applied Biosystems, Inc., Foster City, CA, with autoimmune disease. N Engl J Med 1979; 301: 5–8. USA) Assays-by-Design primers and probes on an ABI 4 Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK. 7900HT PCR machine. Diagrams showing the haplotype Activation of the interferon-alpha pathway identifies a structure between the nine SNPs in each ancestral subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum background are shown in Figure 1, generated using 2005; 52: 1491–1503. Haploview 4.0 software. None of the observed genotype 5 Ronnblom LE, Alm GV, Oberg KE. Possible induction of frequencies at the studied SNPs deviated significantly systemic lupus erythematosus by interferon-alpha treatment from the expected Hardy–Weinberg proportions (P40.01 in a patient with a malignant carcinoid tumour. J Intern Med for all SNPs in each ancestral background; Supplemen- 1990; 227: 207–210. tary Table 1). 6 Niewold TB, Swedler WI. Systemic lupus erythematosus arising during interferon-alpha therapy for cryoglobulinemic vasculitis associated with hepatitis C. Clin Rheumatol 2005; 24: Statistical analysis 178–181. To analyze categorical data, w2 distribution was used, and 7 Niewold TB, Hua J, Lehman TJ, Harley JB, Crow MK. High nonparametric Mann–Whitney U was used to compare serum IFN-alpha activity is a heritable risk factor for systemic quantitative IFN-a and serum OPN ELISA data between lupus erythematosus. Genes Immun 2007; 8: 492–502. two genotype subgroups. Analysis of quantitative data 8 Niewold TB, Kelly JA, Flesch MH, Espinoza LR, Harley JB, across multiple genotype subgroups, which demon- Crow MK. Association of the IRF5 risk haplotype with high strated a consistent dose–response trend, was done using serum interferon-alpha activity in systemic lupus erythema- a semi-log linear model, with equal weighting of tosus patients. Arthritis Rheum 2008; 58: 2481–2487. 9 Kariuki SN, Crow MK, Niewold TB. The PTPN22 C1858T genotype categories on the x axis and log-distributed polymorphism is associated with skewing of cytokine profiles PCR data on the y axis. Genotype categories were toward high interferon-alpha activity and low tumor necrosis arrayed on the x axis by increasing SLE-risk designation, factor alpha levels in patients with lupus. Arthritis Rheum and P values were calculated as the probability that a 2008; 58: 2818–2823. horizontal line (null hypothesis) was a better fit of the 10 Denhardt DT, Noda M. Osteopontin expression and function: data than a semi-log line with a non-zero slope between role in bone remodeling. J Cell Biochem Suppl 1998; 30–31: genotype categories using the extra sum-of-squares 92–102. F test. The modified Breslow–Day test of OR homo- 11 Lampe MA, Patarca R, Iregui MV, Cantor H. Polyclonal geneity as corrected by Tarone is performed as described B cell activation by the Eta-1 cytokine and the development of systemic autoimmune disease. J Immunol 1991; 147: in.20 Data for the cross-ancestral analysis of rs9138 allele 2902–2906. frequencies in younger vs older female patients are 12 Scatena M, Liaw L, Giachelli CM. Osteopontin: a multifunc- analyzed using an allelic test with a fixed-effects model. tional molecule regulating chronic inflammation and vascular Logistic regression models were tested using the alleles disease. Arterioscler Thromb Vasc Biol 2007; 27: 2302–2309. of OPN SNPs as predictor variables in an additive model 13 Masutani K, Akahoshi M, Tsuruya K, Tokumoto M, Ninomiya with the presence or absence of each SLE-associated T, Kohsaka T et al. Predominance of Th1 immune response in autoantibody as the outcome variable. All P values diffuse proliferative lupus nephritis. Arthritis Rheum 2001; 44: shown in the paper are uncorrected for multiple 2097–2106. comparisons. 14 Chabas D, Baranzini SE, Mitchell D, Bernard CC, Rittling SR, Denhardt DT et al. The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease. Science 2001; 294: 1731–1735. Acknowledgements 15 Forton AC, Petri MA, Goldman D, Sullivan KE. An osteopontin (SPP1) polymorphism is associated with systemic Grant support and disclosures: SN Kariuki – none; JG lupus erythematosus. Hum Mutat 2002; 19:459. Moore – none; KA Kirou – patent pending for interferon 16 D’Alfonso S, Barizzone N, Giordano M, Chiocchetti A, Magnani C, Castelli L et al. Two single-nucleotide polymorph- assay; MK Crow – NIH AI059893, Alliance for Lupus 0 0 Research, Mary Kirkland Center for Lupus Research, isms in the 5 and 3 ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus. Arthritis Lupus Research Institute, and patent pending for Rheum 2005; 52: 539–547. interferon assay; TO Utset – Lupus Clinical Trials 17 Han S, Guthridge JM, Harley IT, Sestak AL, Kim-Howard X, Consortium, Genentech; TB Niewold – NIH CTSA K12 Kaufman KM et al. Osteopontin and systemic lupus erythe- Scholar Award RR025000-02, NIAID Clinical Research matosus association: a probable gene-gender interaction. PLoS Loan Repayment AI071651, Arthritis Foundation Post- ONE 2008; 3: e0001757.

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