Drs. Feng and Wu contributed equally to this work.
Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus
Version of Record online: 31 AUG 2006
Copyright © 2006 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 54, Issue 9, pages 2951–2962, September 2006
How to Cite
Feng, X., Wu, H., Grossman, J. M., Hanvivadhanakul, P., FitzGerald, J. D., Park, G. S., Dong, X., Chen, W., Kim, M. H., Weng, H. H., Furst, D. E., Gorn, A., McMahon, M., Taylor, M., Brahn, E., Hahn, B. H. and Tsao, B. P. (2006), Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus. Arthritis & Rheumatism, 54: 2951–2962. doi: 10.1002/art.22044
- Issue online: 31 AUG 2006
- Version of Record online: 31 AUG 2006
- Manuscript Accepted: 16 MAY 2006
- Manuscript Received: 27 JAN 2006
- NIH. Grant Number: R01-AR-43814
- Southern California Chapter of the Arthritis Foundation
- Skirball Foundation
- Meyer Young Investigator award
To study 5 type I interferon (IFN)–inducible genes (LY6E, OAS1, OASL, MX1, and ISG15) in patients with systemic lupus erythematosus (SLE) and to correlate expression levels with disease activity and/or clinical manifestations.
Peripheral blood cells were obtained from 48 SLE patients, 48 normal controls, and 22 rheumatic disease controls, and total RNA was extracted and reverse transcribed into complementary DNA. Gene expression levels were measured by real-time polymerase chain reaction, standardized to a housekeeping gene, and summed to an IFN score. Disease activity was determined by the Safety of Estrogens in Lupus Erythematosus: National Assessment–Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) composite.
Each gene was highly expressed in SLE patients compared with normal controls (P ≤ 0.0003) or disease controls (P ≤ 0.0008 except for MX1). IFN scores were positively associated with the SELENA-SLEDAI instrument score (P = 0.001), the SELENA-SLEDAI flare score (P = 0.03), and the physician's global assessment score (P = 0.005). Compared with patients without nephritis, lupus nephritis patients had higher IFN scores (overall P < 0.0001), especially during active renal disease. IFN scores were weakly associated with neurologic manifestations. Elevated IFN scores were positively associated with the current presence of anti–double-stranded DNA (anti-dsDNA) antibodies (P = 0.007) or hypocomplementemia (P = 0.007). LY6E expression levels distinguished active from inactive lupus nephritis (P = 0.02) and were positively associated with proteinuria (P = 0.009).
The 5 IFN-inducible genes were highly expressed in SLE patients, and increased levels were correlated with disease activity defined by several methods. IFN scores, or LY6E levels, were elevated in lupus nephritis patients, especially during active renal disease, and in patients with anti-dsDNA antibody positivity and hypocomplementemia. IFN scores, or LY6E levels, may be useful as a biomarker for lupus nephritis therapy.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune dysregulation resulting in the production of antinuclear and other autoantibodies, generation of circulating immune complexes, and activation of the complement system. The disease course of SLE is heterogeneous, affecting different individuals with a wide range of manifestations. Unpredictable flares and improvements may be observed. There is no specific single diagnostic test for SLE, and therapy is typically initiated after signs of organ damage appear. To monitor disease activity and allow earlier and more appropriate treatment, there is increasing interest in the identification of biomarkers for SLE (1, 2).
A link between type I interferon (IFN) and SLE in humans and mice has been established by a series of studies (3–7). Global profiling of gene expression in peripheral blood mononuclear cells (PBMCs) has consistently shown up-regulation of IFN-inducible genes (referred to as an IFN expression signature) in SLE patients compared with normal controls (8–11). The IFN expression signature has been correlated with disease activity in one study of pediatric lupus patients (9) and associated with more severe clinical manifestations in another study (8). Preliminary reports of recent microarray analyses using peripheral blood cells also suggested that an IFN signature was associated with current Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (12) scores (13) and might predict elevated SLEDAI scores at the next clinic visit (14). However, microarray studies are semiquantitative and are not easily compared if performed on different platforms, and different groups have reported up-regulation of distinct and different sets of IFN-inducible genes. In addition, most of the microarray studies have not been confirmed by quantitative real-time polymerase chain reaction (PCR) (10).
Type I IFNs (but not type II IFN) have been shown to be the predominant stimulus for the IFN expression signature in SLE patients (15, 16). Quantitative real-time PCR measurements of expression levels of 3 type I IFN–inducible genes (PRKR, IFI44, and IFIT1) in PBMCs from SLE patients have been used to derive IFN scores, which have recently been associated with increased disease activity measured by the SLEDAI 2000 update (SLEDAI-2K), higher titers of anti–double-stranded DNA (anti-dsDNA), low complement levels, and more renal involvement (15). Among these 3 genes, PRKR and IFI44 expression levels were elevated significantly in lupus patients compared with healthy donors or rheumatoid arthritis (RA) patients.
Differential expression of ∼20 type I IFN–inducible genes in peripheral blood cells from SLE patients and healthy controls has been reported (for review, see ref.10). The expression of 14 type I IFN–inducible genes, including LY6E, OAS1, OAS2, OASL, IFIT1, IFIT4, IFI44, STAT1, ISG15, MX1, MX2, PLSCR1, XIAPaf1, and IRF7, reported by at least 2 independent microarray studies of IFN expression signatures (8–11), was measured in our pilot study of 25 SLE patients and 25 normal controls. Using factor analysis (17), we found that 5 genes explained 98% of the total variation for these 14 genes. These 5 genes (LY6E, OAS1, OASL, MX1, and ISG15) were subsequently studied for differential expression between SLE patients and controls, and studied further to correlate their expression levels with disease activity and clinical features.
PATIENTS AND METHODS
Patients and controls.
This study was approved by the Institutional Review Board (protocol no. 04-08-039). Forty-eight SLE patients, 48 normal controls, and 22 disease controls (14 patients with RA and 8 patients with Wegener's granulomatosis [WG]) were enrolled (Table 1). SLE patients fulfilled the 1997 updated revised criteria of the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) (18, 19), RA patients fulfilled the 1987 revised criteria of the ACR (20), and WG patients fulfilled the 1990 ACR criteria (21). All patients were followed up at the University of California, Los Angeles (UCLA) Medical Center. Normal controls were selected from a pool of healthy volunteers at the UCLA Medical Center with an effort to match ethnicity, age, and sex of the lupus patients (Table 1). Subjects with a current or recent infection were excluded.
|SLE patients (n = 48)||Normal controls (n = 48)||Disease controls (n = 22)†|
|Age, years||41 ± 2 (20–76)||39 ± 2 (20–65)||44 ± 3 (20–69)|
|Disease duration, years||10.1 ± 1.3 (0.1–37)||–||–|
|PGA score (possible scores 0–3)||1.1 ± 0.1 (0–3)||–||–|
|SELENA-SLEDAI score (possible scores 0–105)||5.7 ± 0.7 (0–20)||–||–|
Among our lupus patients at the time of study, only 1 was receiving pulse steroid therapy, 21 were receiving antimalarials (400 mg/day for 20 patients and 300 mg/day for 1 patient), and 28 were receiving oral prednisone (1–10 mg/day for 18 patients and 11–40 mg/day for 10 patients). For each SLE patient, disease activity at the time of blood donation was assessed by their rheumatologists with the use of the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA)–SLEDAI composite, including the SELENA-SLEDAI instrument, the physician's global assessment, and the SELENA-SLEDAI flare score (22, 23). These clinical data were verified by one of the authors (JMG) by reviewing the medical records and including the laboratory test results in the final SELENA-SLEDAI composite scores. Historic lupus-specific clinical manifestations and laboratory results for the presence of autoantibodies were obtained by chart review.
Sample collection and RNA processing.
A 10–15-ml sample of blood was collected in BD Vacutainer tubes containing ACD Solution A (BD Biosciences, Manchester, UK) or in PAXgene tubes (Qiagen, Valencia, CA). When using BD Vacutainer tubes, total RNA was extracted immediately using TRIzol (Invitrogen, Carlsbad, CA). When the blood was drawn into PAXgene tubes, total RNA was extracted using the PAXgene 96 Blood RNA kit according to the manufacturer's protocol. A 1–2-μg aliquot of total RNA was reverse transcribed into complementary DNA (cDNA) using Omniscript reverse transcriptase (Qiagen). All RNA and cDNA samples were stored at −70°C before use.
Measuring IFN-inducible gene expression.
We assessed expression of 14 type I IFN–inducible genes that were identified in our pilot study of 25 SLE patients and 25 normal controls as being significantly increased in lupus patients compared with controls. The 14 genes were lymphocyte antigen 6 complex, locus E (LY6E), 2′,5′-oligoadenylate synthetase 1, 40/46 kd (OAS1), 2′,5′-oligoadenylate synthetase 2, 69/71 kd (OAS2), 2′,5′-oligoadenylate synthetase–like (OASL), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), IFIT4, interferon-induced protein 44 (IFI44), signal transducer and activator of transcription 1, 91 kd (STAT1), interferon-α–inducible protein (clone IFI-15K) (ISG15), myxovirus resistance 1 (MX1), MX2, phospholipid scramblase 1 (PLSCR1), XIAP-associated factor 1 (XIAPaf1), and interferon regulatory factor 7 (IRF7). These gene products are involved in regulation of lymphocyte activation, cell death, signal transduction, antiviral effects, or immunomodulation of type I IFN effects (24–31).
TaqMan gene expression assays (Applied Biosystems, Foster City, CA) were used to measure gene expression. Assay identification numbers for LY6E, OAS1, OAS2, OASL, IFIT1, IFIT4, IFI44, STAT1, ISG15, MX1, MX2, PLSCR1, XIAPaf1, and IRF7 were Hs00158942_m1, Hs00242943_m1, Hs00159719_m1, Hs00388714_m1, Hs00356631_g1, Hs00155468_m1, Hs00197427_m1, Hs00234829_m1, Hs00192713_m1, Hs00182073_m1, Hs00159418_m1, Hs00275514_m1, Hs00213882_m1, and Hs00185375_m1, respectively. Five of these 14 type I IFN–inducible genes were subsequently studied for differential expression between SLE patients and controls in an enlarged sample, and studied further to correlate their expression levels with disease activity and clinical features.
Experiments were performed in duplicate for each sample in 96-well plates using the Applied Biosystems 7700 real-time PCR system. Fluorogenic probes were labeled at the 5′ end with FAM and at the 3′ end with MGB nonfluorescent quencher. Reactions were performed in a 25-μl reaction volume in TaqMan Universal PCR Master Mix (Applied Biosystems). Fluorescent signal detection used ROX as the internal passive reference dye. Cycling times and temperatures were as follows: initial denaturation was carried out for 10 minutes at 95°C, followed by 40 cycles of denaturation at 95°C for 15 seconds and combined primer annealing/extension at 60°C for 1 minute. Data were displayed using SDS software, version 1.9 (Applied Biosystems). Human ribosomal protein, large, P0 was used as the housekeeping gene to normalize cellular RNA amounts (assay identification no. Hs99999902_m1; Applied Biosystems). Relative IFN-inducible gene expression values for each sample were normalized to expression of human ribosomal protein, large, P0.
Expression levels of 14 type I IFN–inducible genes (LY6E, OAS1, OAS2, OASL, IFIT1, IFIT4, IFI44, STAT1, ISG15, MX1, MX2, PLSCR1, XIAPaf1, and IRF7) in peripheral blood samples from 25 SLE patients were used to conduct principal component analysis to identify correlated groups of genes (collectively referred to as factors) for the purpose of data reduction (17). Weighted sums of squared correlations of these 14 genes were used to derive eigenvalues to assess the amount of variance explained by each component factor. The first component explains the greatest amount of variance, and each subsequent factor explains decreasing proportions of variance and is independent of the other factors. These eigenvalues were plotted against factor numbers, yielding the scree plot (32). A single IFN-inducible gene within each factor was selected to explain the greatest amount of variance while having the least amount of correlation with other factors.
Calculation of IFN scores.
IFN scores were calculated as described in previous studies (15, 16). The mean and SD level of each IFN-inducible gene in the normal control (ctr) group (meanctr and SDctr) were used to standardize expression levels of each gene for each study subject. The standardized expression levels were subsequently summed for each patient to provide an IFN expression score:
where i = each of the 5 IFN-inducible genes, Gene iSLE = the gene expression level in each SLE patient, and Gene ictr = the gene expression in controls. The mean IFN score in SLE patients was 54.0, with a range of −3.2 to 401.6, and in normal controls, the mean IFN score was 0, with a range of −3.1 to 12.7.
Data were analyzed with GraphPad Prism, version 3.0 software (GraphPad Software, San Diego, CA). Because of extremely elevated IFN-inducible gene expression levels in certain patients, the continuous variable data were not normally distributed. All values were therefore expressed as medians with 25th and 75th percentiles and interquartile range (IQRs), and comparisons were made using the nonparametric Mann-Whitney test. Comparisons among 3 groups were conducted using the nonparametric Kruskal-Wallis test. Correlation between groups was evaluated using the Spearman test. P values less than 0.05 were considered significant.
Identification of 5 representative IFN-inducible genes.
Fourteen type I IFN–inducible genes (LY6E, OAS1, OAS2, OASL, IFIT1, IFIT4, IFI44, STAT1, ISG15, MX1, MX2, PLSCR1, XIAPaf1, and IRF7), reported by at least 2 independent microarray studies of IFN expression signatures in SLE (8–11), were selected for real-time PCR measurements of expression levels in peripheral blood samples from 25 SLE patients and 25 matched normal controls. Compared with the normal controls, the SLE patients had higher expression of these 14 genes that showed a wide range of pair-wise correlations (Spearman's r = 0.37–0.91; data not shown). The expression data from these 25 SLE patients were evaluated by principal components analysis to identify correlative groups of IFN-inducible genes (also known as factors) to reduce the complexity of the data (17). The scree plot of the eigenvalues against the factor numbers (32) showed that the first 5 factors could account for 72%, 14%, 7%, 3%, and 2%, respectively, of the variance observed in the expression data for the 14 genes. These 5 factors explained a cumulative 98% of the variance. We selected 1 IFN-inducible gene from each of the 5 factors as being representative IFN-inducible genes for the enlarged study described below.
Increased IFN-inducible gene expression in SLE patients.
Type I IFN–inducible gene expression in peripheral blood cells from 48 SLE patients, 48 normal controls, and 22 rheumatic disease controls (14 RA patients plus 8 WG patients) was determined by quantitative real-time PCR. Compared with the normal controls, the SLE patients were not significantly different in terms of age, sex, or ethnic distributions (Table 1). As shown in Figure 1, all 5 genes (LY6E, OAS1, OASL, MX1, and ISG15) were more highly expressed in peripheral blood samples from SLE patients compared with those from normal controls (P ≤ 0.0003 for all comparisons) and compared with those from rheumatic disease controls (14 RA patients plus 8 WG patients) (P ≤ 0.0008 for all comparisons except for MX1, which was not significant). One RA patient expressing the highest level of each of these 5 genes had moderate disease activity (Disease Activity Score in 28 swollen and 28 tender joints [DAS28] = 4.2) (33), while the other 13 patients (4 with high activity, 5 with moderate activity, 1 with mild activity, and 3 with disease in remission) had expression levels similar to those of normal controls at the time of blood drawing. All 8 WG patients had inactive disease at the time of blood drawing.
Association of IFN scores with disease activity as assessed by SELENA-SLEDAI and physician's global assessment in SLE patients.
To assess whether expression levels of type I IFN–inducible genes are related to disease activity, we compared IFN scores in SLE patients with varying levels of disease activity, as assessed by the SELENA-SLEDAI composite (22, 23), including the SELENA-SLEDAI instrument, the SELENA-SLEDAI flare system, and the physician's global assessment at the time blood was obtained. When patients were divided into 3 groups according to their SELENA-SLEDAI instrument scores (0–4, 5–12, and >12) (22), there was a significant difference between the groups (P = 0.001 by Kruskal-Wallis test); patients with active disease (SLEDAI scores of 5–12) or with severe disease activity (SLEDAI scores >12) had higher IFN scores compared with patients with inactive or mild disease activity (SLEDAI scores of 0–4) (P = 0.009 and P = 0.002, respectively) (Figure 2A). Similar results were obtained using physician's global assessment scores as a measurement of disease activity (overall P = 0.005) (Figure 2B).
When disease activity was defined as stable, mild/moderate flare, or severe flare according to the SELENA-SLEDAI flare system (34), we found that patients with severe flares had higher IFN scores than did patients with mild/moderate flares (P = 0.02) or those without flare (P = 0.02) (Figure 2C). LY6E expression, the highest among the 5 tested genes correlating with IFN scores (r = 0.91), also showed an association with SLEDAI scores (P = 0.001) and physician's global assessment scores (P = 0.005) (Figures 2D and E).
Association of IFN scores with anti-dsDNA antibody and hypocomplementemia in SLE patients.
In these SLE patients, hypocomplementemia, anti-dsDNA antibody positivity, ongoing rash, and arthritis as well as renal involvement contributed to SELENA-SLEDAI scores. Hypocomplementemia was defined as serum levels of C3 or C4 below the normal range. C3 and C4 levels were measured in 44 SLE patients and antibodies to DNA were measured in 42 SLE patients at the time of blood drawing. Eleven patients had concurrent anti-dsDNA antibody positivity and hypocomplementemia.
As shown in Figure 3A, the 15 patients with hypocomplementemia had higher IFN scores (median 58.8 [IQR 36.1, 73.2]) than the 25 patients with normal complement levels (median 5.9 [IQR −0.78, 39.9]) (P = 0.007). C3 levels were negatively correlated with IFN scores (r = −0.43, P = 0.006) (Figure 3C). Similarly, C4 levels showed a negative trend of correlation with IFN scores (r = −0.27, P < 0.1). The 23 patients with anti- dsDNA antibody positivity also had higher IFN scores (median 50.1 [IQR 14.0, 77.8]) than the 21 patients without anti-dsDNA antibody positivity (median 3.57 [IQR −0.78, 30.3]) (P = 0.007) (Figure 3B). Circulating levels of IgG anti-dsDNA showed a positive trend toward correlation with IFN scores (r = 0.38, P = 0.07) (Figure 3D). There were no significant differences in IFN scores between patients with and without ongoing rash or between patients with and without arthritis (Table 2).
|No. of patients||Median (IQR)||No. of patients||Median (IQR)|
|Renal||7||134.8 (71.6, 165.3)||41||9.6 (0.5, 50.1)||0.001|
|Neurologic||1||31.3||47||14.8 (0.9, 64.7)||ND|
|Arthritis||17||3.6 (−0.6, 50.1)||31||33.4 (4.4, 75.0)||NS|
|Hematologic||3||43.3 (36.1, 78.7)||45||13.1 (0.5, 63.2)||NS|
|Serositis||2||25.8 (12.5, 39.2)||46||21.9 (1.3, 65.5)||NS|
|Rash†||16||20.0 (1.1, 64.0)||32||21.9 (1.1, 63.4)||NS|
|Mucosal ulcer||4||7.0 (2.3, 24.9)||44||29.6 (1.1, 64.0)||NS|
|Renal||21||63.2 (33.4, 134.8)||27||1.6 (−1.0, 29.6)||<0.0001|
|Neurologic||9||63.2 (31.3, 188.2)||38||11.5 (0.5, 60.4)||0.03|
|Arthritis||44||21.9 (0.5, 64.0)||4||28.2 (11.0, 66.2)||NS|
|Hematologic||22||21.9 (4.3, 69.1)||24||19.9 (0.2, 61.7)||NS|
|Serositis||16||8.1 (1.1, 54.1)||32||29.6 (1.1, 80.0)||NS|
|Rash§||32||29.6 (−0.6, 61.7)||16||9.5 (3.1, 73.9)||NS|
|Mucosal ulcer||24||11.5 (0.2, 64.9)||22||31.8 (3.8, 65.0)||NS|
|Anti-dsDNA||37||39.9 (4.6, 70.0)||10||−0.1 (−2.2, 7.5)||0.005|
|Anti-Sm||10||63.7 (38.2, 81.7)||25||4.6 (−1.1, 39.9)||0.02|
|Anti-Ro/La||12||41.6 (24.4, 73.9)||21||4.6 (−1.8, 30.3)||0.04|
Elevated IFN scores or LY6E expression in patients with lupus nephritis, especially during active renal disease and proteinuria.
Lupus nephritis is one of the most serious manifestations of SLE. In our cohort, nearly 44% of patients had either previous or current lupus nephritis. Subjects were considered to have active renal disease if proteinuria was >0.5 gm/day, hematuria was >5 red blood cells per high-power field (hpf), pyuria was >5 white blood cells/hpf, or cellular casts were present. Infection, kidney stones, or other causes were excluded. These are the same parameters used in the SELENA-SLEDAI.
Patients with nephritis had higher IFN scores (median 63.2 [IQR 33.4, 134.8]) than those without renal manifestations (median 1.6 [IQR −1.0, 29.6]) (P < 0.0001). While IFN scores were significantly elevated in patients with inactive lupus nephritis (P = 0.004), they were even more pronounced in patients with active lupus nephritis (P = 0.0002) (Figure 4A). LY6E expression levels were significantly higher in patients with active renal disease than in those patients with inactive lupus nephritis at the time of blood drawing (P = 0.02) (Figure 4B). In addition, expression levels of LY6E were significantly different in 3 groups of patients stratified by urinary protein level (0 or trace, 1+ or 2+, and 3+ or 4+) (overall P = 0.009) (Figure 4C). Seven patients who had 3+ or 4+ proteinuria had significantly higher levels of LY6E gene expression than patients who had 0 or trace proteinuria in samples obtained at the time of blood drawing (P = 0.004) (Figure 4C). LY6E levels were elevated in patients with anti-dsDNA antibody positivity and hypocomplementemia (data not shown). In summary, these data showed that lupus nephritis patients had elevated IFN scores and/or LY6E expression. These levels were even higher during active renal disease and/or with elevated proteinuria.
IFN scores and other clinical manifestations.
Neurologic disorders comprise other serious complications of SLE. In this cohort, lupus patients with seizures or psychosis were designated as having a neurologic disorder. Nine patients who had ever had neurologic involvement exhibited higher IFN scores (median 63.2 [IQR 31.3, 188.2]) than those without neurologic disorders (median 11.5 [IQR 0.5, 60.4]) (P = 0.03). Other manifestations, such as arthritis, hematologic involvement, serositis, rash, and mucosal ulcers, occurring currently or historically, were not associated with IFN scores (Table 2). In this cohort, IFN scores were elevated in SLE patients with a history of anti-dsDNA antibody positivity (P = 0.005), anti-Sm antibody positivity (P = 0.02), or anti-Ro antibody positivity (P = 0.04) (Table 2).
An IFN signature in SLE has been convincingly established using microarray analyses of blood samples (for review, see ref.10). However, it is not yet entirely clear how up-regulated IFN-inducible gene expression relates to disease activity and/or specific features of lupus. Here we selected a unique set of 5 type I IFN–inducible genes (LY6E, OAS1, OASL, MX1, and ISG15) identified in at least 2 independent microarray studies of IFN signatures, and using real-time PCR, we confirmed elevated expression of each gene in SLE patients compared with normal controls (P ≤ 0.0003) (Figure 1). Although none of these 5 genes were the same as the 3 IFN-inducible genes previously studied by Kirou et al (15), our results replicated their findings that IFN scores (calculated from expression levels of PRKR, IFI44, and IFIT1) were associated positively with disease activity (SLEDAI scores), low complement levels, and renal involvement and were associated negatively with C3 levels.
In our study, IFN scores were also shown to be significantly associated with 2 additional disease activity measures, the SELENA-SLEDAI flare instrument and the physician's global assessment. Elevated IFN scores were associated with lupus nephritis (P < 0.0001) (Figure 4) and neurologic manifestations (P = 0.03) (Table 2), but not with other clinical features (active or inactive), including arthritis, hematologic involvement, serositis, rash, and mucosal ulcers (Table 2). In studies by other investigators, high IFN scores were associated with renal and/or central nervous system or hematologic involvement in SLE (8) or with renal manifestations alone (15). It appears that high IFN scores are consistently associated with renal involvement in SLE. Other IFN score–associated manifestations were variable, which might be explained, at least in part, by clinical heterogeneity in a sample of rather limited size (48–81 SLE patients) (8, 13, 15).
Pulse glucocorticoid therapy in SLE patients has previously been shown to down-regulate IFN-inducible gene expression rapidly and almost completely (9, 15). Among our lupus patients, only 1 received pulse steroid therapy, 21 were receiving antimalarials, and 28 were receiving oral prednisone (1–10 mg/day for 18 patients and 11–40 mg/day for 10 patients). The limited sample size of pulse steroid–treated patients (n = 1) prevents us from commenting on the effect of treatments on expression levels of IFN-inducible genes. Our SLE patients taking either antimalarials or 11–40 mg/day of prednisone at the time of blood drawing had high IFN scores and strong correlations with SLEDAI scores and physician's global assessment scores (r = 0.68–0.71, P < 0.05), while those receiving none of these medications or ≤10 mg/day of prednisone had low IFN scores and no correlations with disease activity. If these medications suppressed expression of the 5 genes, our observations would represent conservative estimates of increased expression of IFN-inducible genes associated with disease activity and lupus nephritis.
SLE is a chronic and potentially fatal autoimmune disease characterized by unpredictable exacerbations and improvements, with variable clinical manifestations. Several biomarkers for SLE activity seem to be valid in some patients, such as anti-dsDNA and complement levels in nephritis. However, no single marker is universally useful. Six overall disease activity measures, the SELENA-SLEDAI, the SLEDAI, the British Isles Lupus Assessment Group (BILAG) (35), the Systemic Lupus Activity Measure Revised (36), the European Consensus Lupus Activity Measure (37), and the Responder Index for Lupus Erythematosus (38), could be used to evaluate clinical conditions as being improved, worsened, or remaining the same (39). In this study, we used the SELENA-SLEDAI composite for measuring overall SLE disease activity, which includes disease activity index scores, physician's global assessment scores, and disease flare scores, with the consideration of treatment changes and fluctuations in clinical features (34). Because the SELENA-SLEDAI tends to focus on some, but not all, clinical features, it may be useful in future studies to evaluate prospectively obtained BILAG data.
The IFN expression signature has been reported in other rheumatic diseases, including dermatomyositis (40) and Sjögren's syndrome (41), suggesting a shared molecular pathway in the pathogenesis of several autoimmune diseases. However, RA (15) and juvenile chronic arthritis (9) do not share this IFN expression signature. Unremarkable expression levels of the 5 IFN-inducible genes in 22 rheumatic disease controls also support the likelihood that the IFN signature is not a global feature of inflammation and systemic autoimmunity (Figure 1). IFN scores for the 4 RA patients who had active disease (DAS28 >5.1) at the time of blood drawing were similar to those of normal controls (33) (data not shown). None of the 8 WG patients had active disease, although a highly elevated MX1 expression level was observed in 1 asymptomatic WG patient who subsequently developed pulmonary and renal involvement 2 weeks after the blood drawing. A longitudinal followup study will help to address the specificity and predictability of these gene expression levels for disease flares.
The type I IFN pathway may have a direct role in the pathogenesis of lupus. In animal models, in vivo delivery of murine IFNα results in a rapid and severe disease with characteristics of lupus in (NZB × NZW)F1 mice (6), and knockout of IFN type I receptors protects from disease (42). When patients with viral hepatitis or tumors were treated with IFNα, nearly one-fourth of them developed antinuclear antibody positivity (5), and a few patients developed SLE (7, 43). Viral infections or other events leading to IFNα induction could be activators of SLE flares (44).
IFNα is mainly produced by plasmacytoid dendritic cells (PDCs), the major IFNα-producing cell type in blood (45). PDC numbers were markedly reduced in the blood of SLE patients, suggesting an accelerated migration of PDCs from blood into tissues (46). Although low in numbers, depletion of PDCs from PBMCs of SLE patients resulted in a >50% reduction of IFNα release upon viral triggering (47). Toll-like receptors (TLRs), especially TLRs 7 and 9, were involved in PDC activation to produce IFNα (48), linking innate immunity to the pathogenesis of SLE, since those receptors bind RNA and DNA that are antigens recognized by autoantibodies characteristic of SLE. Activation of monocytes by IFNα in vivo has been proposed to be a factor in the hyperexpression of IFN signature in some patients (15).
Our data showed that IFN scores were associated positively with SELENA-SLEDAI scores, physician's global assessment scores, and SELENA-SLEDAI flare scores (Figure 2). IFN scores were elevated in patients with renal involvement, especially in those having current renal flares, and in patients who had anti-dsDNA antibody positivity and/or hypocomplementemia at the time of blood drawing. IFN scores correlated negatively with C3 levels (r = −0.43, P = 0.0059; n = 40), but the correlation with C4 levels did not reach statistical significance (r = −0.27, P = 0.09; n = 40). We also observed a trend toward positive correlations between IFN scores and IgG anti-dsDNA levels (r = 0.38, P = 0.07; n = 23) and the erythrocyte sedimentation rate (ESR) (r = 0.39, P = 0.06; n = 24). Of note, the ESR correlated positively with levels of OASL expression (r = 0.50, P = 0.013), but not with expression levels of the other 4 IFN-inducible genes (data not shown). Elevated IFN scores were also associated positively with the ever presence of autoantibodies to dsDNA, Sm, or Ro/La (Table 2). In our sample, the observed association between elevated IFN scores and SELENA-SLEDAI scores was mainly attributable to active renal flares.
Expression levels of these 5 IFN-inducible genes were highly correlated with each other in SLE patients (r = 0.56–0.85, P < 0.0001). However, LY6E exhibited the best correlation with IFN scores (r = 0.91). Of interest, blood levels of LY6E messenger RNA were significantly elevated during active lupus nephritis (Figure 4) compared with those in patients with inactive lupus nephritis (at the time of blood drawing) and compared with those in patients without lupus nephritis. Ly-6E (also known as thymic shared antigen 1 [TSA-1]), a member of the Ly-6 superfamily, is a small glycosyl phosphatidylinositol–linked surface protein expressed on hematopoietic stem cells, lymphocyte subsets (T, B, and natural killer), and nonlymphoid tissues including liver and kidney cells (49). Inbred mouse strains have either Ly-6E.1 or Ly-6A.2 alleles that differ in one amino acid, and in these mice expression levels of Ly-6A/E on lymphocytes appear to be tightly regulated during development and activation of T and B lymphocytes (50, 51).
The function of Ly-6A/E in the immune system is not entirely clear, although it is thought to subserve some functions in cell signaling and/or cell adhesion processes (24, 52). Recently, it was reported that surface expression levels of Ly-6A/E on peripheral lymphocytes (but not on cells of myeloid origin) positively correlate with disease severity in several lupus-prone strains of mice (53). The expression of Ly-6E (TSA-1) in renal proximal tubules is elevated in response to proteinuria, identified by gene expression profiles and confirmed by laser microdissection along with real-time PCR, suggesting a potential role of LY6E in the pathophysiology of renal disease (54). These findings are consistent with our findings of markedly elevated LY6E gene expression in patients with proteinuria compared with other patients (0 or trace proteinuria) (Figure 4C), and they are also consistent with the positive correlation trend between the LY6E expression levels and the estimated Modification of Diet in Renal Disease glomerular filtration rate (r = 0.28, P = 0.07) (data not shown) (55).
In conclusion, IFN-inducible gene expression was elevated in SLE patients in comparison with normal controls. Our data show that the IFN score was associated with 3 instruments measuring disease activity. Expression levels of all 5 genes were highly correlated with the IFN score, with LY6E having the highest correlation. The LY6E expression level (capturing 83% of the IFN score information) exhibited a significant association with the SELENA-SLEDAI score and the physician's global assessment score (Figure 2) and could discriminate active from inactive renal disease (Figure 4). These results suggest that IFN scoring and/or the LY6E expression level might be biomarkers for SLE, especially for renal disease activity. However, in view of the limited sample size of this study, our findings should be considered preliminary. We are extending these studies in an independent longitudinal cohort of SLE patients, as well as testing whether the expression of LY6E may be used as an outcome measure of lupus nephritis therapy.
We thank the patients, healthy volunteers, and rheumatologists at UCLA Medical Center who participated in this study. We thank Marisa Mizus for help in the preparation of the manuscript.
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