Biomarkers in systemic lupus erythematosus: challenges and prospects for the future

Abstract

The search for lupus biomarkers to diagnose, monitor, stratify, and predict individual response to therapy is currently more intense than ever before. This effort is essential for several reasons. First, epidemic overdiagnosis and underdiagnosis of lupus, even by certified rheumatologists, leads to errors in therapy with concomitant side effects which may be more serious than the disease itself. Second, identification of lupus flares remains as much an art as it is a science. Third, the capacity to stratify patients so as to predict those who will develop specific patterns of organ involvement is not currently possible but would potentially lead to preventive therapeutic strategies. Fourth, only one new drug for the treatment of lupus has been approved by the US Food and Drug Administration in over 50 years. A major obstacle in this pipeline is the dearth of biomarkers available to prove a patient has responded to an experimental therapeutic intervention. This review will summarize the challenges faced in the discovery and validation of lupus biomarkers, the most promising lupus biomarkers identified to date, and the promise of future directions.

Keywords

biomarker lupus systemic lupus erythematosus SLE

Introduction

Systemic lupus erythematosus (SLE) is arguably the most serologically and clinically diverse autoimmune disease, with immunopathogenic abnormalities continuously being uncovered and clinical manifestations widely varying in individual patients. Immune dysregulation leads to excess production of autoantibodies and immune complexes, excess complement activation, and insidious tissue inflammation in patients with SLE, which together cause a clinical syndrome with multi-organ involvement and hard-to-predict courses [Crispin et al. 2010b; Rahman and Isenberg, 2008; Tsokos, 2011]. Owing to its complex nature, SLE remains one of the greatest challenges to both investigators and physicians.

Over the past several decades, tremendous enthusiasm and efforts have been devoted to tackling the numerous challenges ranging from understanding the etiopathogenesis of SLE, through the development of diagnostic tests and biomarkers, to improved care for patients with SLE. Although significant progress has been made, there are still many unmet needs in lupus research and patient care. Fundamental to the unmet needs is the lack of reliable lupus biomarkers for diagnosis, monitoring, stratification, and prediction of response to therapy.

Based on scientific and clinical rationale, a biomarker is defined as a measurement, including but not limited to a genetic, biological, biochemical, molecular, or imaging event whose alterations correlate with the pathogenesis and/or manifestations of a disease and can be evaluated qualitatively and/or quantitatively in laboratories [Illei et al. 2004a, 2004b]. Given the rapid advance in the understanding of SLE pathogenesis and development of new technologies, an impressive spectrum of SLE biomarkers has been accrued during the past few years. A comprehensive review of these biomarkers will be beyond the scope of this article; readers can reference several recently published reviews [Ahearn et al. 2012; Herbst et al. 2012]. Instead, this report will focus on select groups of potential biomarkers that have been developed based on or in response to: (1) recent understanding of SLE pathogenesis (e.g. epigenetics, cytokines) or cutting-edge technologies (e.g. proteomics); (2) clinical importance in SLE management (e.g. lupus nephritis); and (3) emerging appreciation of the need of composite panels, rather than individual lupus biomarkers. Finally, we will discuss the current limitations and future directions for discovery and validation of lupus biomarkers.

Biomarkers originated from better understanding of SLE pathogenesis

The pathogenesis of SLE is multifactorial and multistaged, with various genetic, epigenetic, environmental, gender, and immunoregulatory factors contributing to the susceptibility, onset, progress, and prognosis of the clinical disease in a given patient [Crispin et al. 2010b; Manderson et al. 2004; Mok and Lau, 2003; Moser et al. 2009; Sarzi-Puttini et al. 2005]. Genetic factors clearly confer susceptibility of an individual to the development of SLE. In rare cases, the development of SLE is due to the deficiency of a single gene product (e.g. complement C1q) [Pickering et al. 2000]. Much more commonly, variations (single nucleotide polymorphisms, gene copy numbers, etc.) at multiple genetic loci are believed to increase the risk of SLE in a hierarchical interactive manner [Moser et al. 2009; Nath et al. 2004]. Although extensive studies have associated many common genetic variants with SLE [Deng and Tsao, 2010; Flesher et al. 2010], the cumulative effect size of the loci identified so far accounts for only 15–20% of the heritability of SLE [Manolio et al. 2009]. The variations potentially underlying the remaining 75–80% of the heritability appear to be missing.

Epigenetics-related biomarkers

The ‘missing heritability’ has led to a renewed appreciation of epigenetic factors. Because SLE affects predominantly women of child-bearing age, it is widely accepted that female hormones contribute to the development of SLE through mechanisms that are not fully elucidated [Weckerle and Niewold, 2011]. However, epigenetically dysregulated expression of genes located on chromosome X, e.g., the CD40 Ligand (CD40L) gene, may also contribute to the female prevalence of SLE [Lu et al. 2007].

Epigenetics refers to heritable modifications that regulate gene expression without alterations in DNA sequence [Bird, 2007]. Epigenetic effects, which are heritable (but specific to different cells), stable (but reversible), and subject to environmental influences, may account for several perplexing observations such as the incomplete concordance of SLE in monozygotic twins [Hughes and Sawalha, 2011; Javierre et al. 2010; Jeffries and Sawalha, 2011; Shen et al. 2012]. Common epigenetic mechanisms, including DNA methylation, histone modifications, and microRNA-mediated regulation, play important roles in modulating gene expression over the cell cycle, lineage commitment, and cellular function throughout the body [Fraga et al. 2005; Laurent et al. 2010]. The immune system, naturally, is also under tight control at the epigenetic level [Allan et al. 2012; Dai and Ahmed, 2011; Fields et al. 2002; Hughes et al. 2010; Rauch et al. 2009; Renaudineau and Youinou, 2011]. Therefore, aberrant epigenetic regulation may contribute to the complex array of immune abnormalities and influence the disease manifestations in lupus patients (Table 1).

Table 1.

Epigenetic alterations and potential epigenetic biomarkers identified in SLE.

Mechanism	Target	Cell Type	Alteration	Consequence
DNA methylation	ITGAL (CD11a)	CD4 T cells	Hypomethylation	Increased CD11a expression
	CD70 (TNFSF7)	CD4 T cells	Hypomethylation	Increased CD70 expression and B-cell costimulation
	CD154 (CD40L)	CD4 T cells	Hypomethylation	Increased B-cell costimulation
	Perforin	CD4 T cells	Hypomethylation	Increased perforin expression
	KIR family	CD4 T cells	Hypomethylation	Increased KIR expression
	RUNX3	CD4 T cells	Hypermethylation	Dysregulation of ITGAL (CD11a) expression
	MMP9	CD4 T cells	Hypomethylation	Cellular basement membrane breakdown
	CD9	CD4 T cells	Hypomethylation	T-cell activation
Histone modification	Histone H4	Monocytes	Increased acetylation	Increased expression of proinflammatory cytokines
MicroRNA	miR-146a	PBMCs	Underexpression	Type I IFN overproduction
	miR-21	CD4 T cells	Overexpression	Downregulation of DNMT1 (indirect) and thus decreased DNA methylation
	miR-148a	CD4 T cells	Overexpression	Downregulation of DNMT1 (direct) and decreased DNA methylation
	miR-125a	PBMCs	Underexpresssion	Increased KLF expression and thus RANTES overproduction
	miR-126	CD4 T cells	overexpression	Downregulation of DNMT1 and decreased DNA methylation

IFN, interferon; KIR, killer immunoglobulin-like receptor; KLF, Kruppel-like factor; MMP, matrix metalloproteinase PBMC, peripheral blood mononuclear cell; RANTES, regulated on activation normal T cell expression and secreted; RUNX, runt-related transcription factor; SLE, systemic lupus erythematosus

DNA methylation

The expression of a gene is initiated by the access of transcription factors to the specific DNA region. Methylation of the promoter and cytosine-P-guanosine (CpG)-rich regions (CpG islands) of genomic DNA by DNA methyl transferases (DNMTs) prevents the binding of transcription factors and is an important negative regulator of gene expression. Decreased methylation (hypomethylation) of DNA will lead to aberrant gene expression. Global DNA hypomethylation in CD4 T cells has long been observed in SLE, initially in drug-induced SLE and later in idiopathic SLE [Cornacchia et al. 1988; Hughes et al. 2010; Richardson et al. 1990]. It is now known that hydralazine and procainamide inhibit DNA methylation and thus may induce SLE in some individuals [Cornacchia et al. 1988]. Hypomethylation of the regulatory regions of several genes known to be involved in the pathogenesis of SLE, such as CD70 (a costimulatory molecule), CD40L (CD154; also a costimulatory molecule), ITGAL [CD11a; a subunit of lymphocyte function-associated antigen 1 (LFA-1)], perforin (a cytolytic protein), killer immunoglobulin-like receptors (KIRs), interleukin (IL)-10, and IL-13 have been reported [Basu et al. 2009; Kaplan et al. 2004; Liu et al. 2009; Lu et al. 2002, 2005, 2007; Oelke et al. 2004; Zhao et al. 2010a]. Consequently, DNA hypomethylation may cause increased cytokine production and hyperactivity of CD4 T cells and increased immunoglobulin production by B cells. The degree of reduced DNA methylation and overexpression of proteins encoded by the hypomethylated genes in SLE T cells have been reported to correlate with disease activity [Lu et al. 2002].

It has been a puzzle that monozygotic twins are at increased risk to develop SLE but the concordance rate has never reached 100% [Deapen et al. 1992]. A recent genome-wide DNA methylation study showed significant epigenetic variation in leukocytes derived from disease-discordant monozygotic twins; specifically differential methylation of 49 autoimmunity-relevant genes in the white blood cell population between the affected twins and their healthy monozygotic siblings were identified [Javierre et al. 2010]. This study lends support to the possibility that variations in epigenetic modifications may drive the difference in SLE development during the life course of monozygotic twins. Jeffries and colleagues recently conducted a case-control study utilizing high-throughput methylation arrays to scan 27,578 CpG sites in the promoter region of 14,495 genes [Jeffries et al. 2011]. They identified 236 hypomethylated sites (representing 232 genes) and 105 hypermethylated sites (representing 104 genes) in CD4 T cells of SLE patients. A more recent genome-wide study reported that the methylation status of the IL-10 and IL-1R2 genes was significantly reduced in SLE patient samples compared to healthy control samples. Moreover, the SLE patients with hypomethylated IL-10 and IL-1R2 genes appeared to have higher disease activity [Lin et al. 2012]. These studies, taken together, suggest that genome-wide DNA methylation studies may aid in identifying potential biomarkers that may correlate with the pathogenic process and/or disease activity of SLE.

Histone modifications

Histone proteins are the major component of nucleosomes (the basic subunit of chromatin) and help determine which part of the chromatin is accessible for active transcription [Luger et al. 2012; Williamson and Pinto, 2012]. Covalent modification of histone proteins may alter chromatin structure (but not the DNA sequence) and, hence, regulate gene expression at the epigenetic level. For example, acetylation and methylation of a specific lysine residue (lysine 9) on histone 3 (H3K9) have been shown to enhance or repress gene transcription, respectively [Roh et al. 2005; Snowden et al. 2002; Wilson et al. 2009]. Reduced global levels of methylated H3K9 and H3 acetylation have been reported in CD4 T cells of SLE patients [Hu et al. 2008]. In addition, hyperacetylation of histone 4 (H4) and overexpression of several genes have been reported in monocytes of SLE patients [Zhang et al. 2010]. These findings point to widespread variations in histone modifications in immune cells of SLE patients and such changes may serve as potential biomarkers for elucidating the pathogenesis of SLE.

MicroRNAs

MicroRNAs (miRNAs or miR) are recently discovered, short (20–24 base pairs in length), noncoding ribonucleic acids (RNAs) that play important roles in the regulation of gene expression post-transcriptionally [Bartel, 2004; Carthew and Sontheimer, 2009; Fabian et al. 2010]. miRNAs bind to homologous sequences present in messenger RNA (mRNA) transcripts, and regulate gene expression by directly cleaving the target mRNA or effectively blocking the subsequent translation of the target mRNA. The miRNA-mediated regulatory network is extremely complex: a single miRNA may regulate hundreds to over thousands of mRNAs, and a single mRNA may be targeted by multiple miRNA. The production of miRNAs themselves is also under tight genetic as well as epigenetic regulations.

During the last several years, accumulating evidence has indicated that miRNAs are critical not only for the development of the immune system, but also for regulation of adaptive and innate immune responses [Baltimore et al. 2008; Xiao and Rajewsky, 2009]. Therefore, not surprisingly, abnormalities in the expression and functioning of miRNAs have been identified as part of the pathogenesis of autoimmune diseases including SLE [Dai and Ahmed, 2011; Shen et al. 2012] . Dai and colleagues first reported the identification of six miRNAs whose expression was altered in the peripheral blood mononuclear cells (PBMCs) prepared from patients with SLE, but not patients with idiopathic thrombocytopenic purpura [Dai et al. 2007]. Since then, several investigators have conducted and reported studies aimed at detecting and profiling miRNA expression in blood cells (PBMCs, T cells, etc), body fluid (serum, plasma, urine, etc.), and tissues taken from patients with SLE [Dai et al. 2009a; Pan et al. 2010; Tang et al. 2009; Te et al. 2010; Zhao et al. 2010b, 2011].

miR-146a, a miRNA targeting signaling proteins and thus negatively regulating innate immune responses, has been reported to be underexpressed in CD4 T cells of patients with SLE [Tang et al. 2009]. In that study, it was shown that reduced miR-146a expression led to activation of the type 1 interferon (IFN) pathway and that miR-146a levels correlated inversely with SLE disease activity. Another miRNA, miR-125, was also reported to be underexpressed in CD4 T cells of patients with SLE [Zhao et al. 2010b]. Decreased levels of miR-125a appeared to lead to increased production of an inflammatory chemokine RANTES. Pan and colleagues reported that miR-21 and miR-148a were upregulated in CD4 T cells prepared from both patients with SLE and MRL-lpr mice [Pan et al. 2010]. These investigators subsequently showed that miR-148a directly and miR-21 indirectly target DNA methyltransferases 1 (DNMT1), suggesting their role in regulating DNA methylation in SLE CD4 T cells [Pan et al. 2010]. Indeed, overexpression of miR-148a and miR-21 in CD4 T cells resulted in DNA hypomethylation and increased expressed of CD70 and LFA-1. The possibility of intricate cross-regulations of miRNA and DNA methylation has subsequently been confirmed in a study by Zhao and colleagues. They reported that miR-126 was overexpressed in SLE CD4 T cells [Zhao et al. 2011]. It was further shown that miR-126 targets DNMT1 mRNA and reduced DNMT1 protein expression. Similarly, overexpression of miR-126 in CD4 T cells from healthy individuals led to hypomethylation and consequently overexpression of CD11a (a subunit of LFA-1) and CD70 [Zhao et al. 2011].

Recently, systematic, microarray-based studies of miRNA expression have been initiated. Te and colleagues performed a study that investigated the expression profile of miRNA in PBMCs and Epstein–Barr virus-transformed B-cell lines derived from SLE patients with nephritis or without nephritis [Te et al. 2010]. They found that 29 and 50, out of 850 tested, miRNAs were differentially expressed in SLE patients with nephritis of African-American ancestry and of European-American ancestry, respectively. Among these miRNAs, 18 miRNAs were differentially expressed in patients of both racial groups. Another recent study, comparing miRNA expression profiles in PBMCs of SLE patients and healthy controls, showed differential expression of 27 miRNAs out of 365 analyzed [Stagakis et al. 2011]. It was further shown that the levels of miR-21, miR-25, miR-106b, and miR-148b correlated positively with SLE disease activity, whereas the levels of miR-196a and miR-379 negatively correlated with SLE disease activity. This latter finding suggests a potential role for miRNA profiling as disease activity biomarkers for SLE.

In addition to investigation of miRNAs in blood cells, some investigators also attempted to detect cell-free miRNAs in serum and urine samples from patients with SLE [Wang et al. 2011, 2012]. Wang and colleagues reported reduced levels of cell-free miR-146a and miR-155 circulating in the serum of patients with SLE and elevated levels of miR-146a in the urine of patients with SLE, compared to healthy controls. Furthermore, serum miR-146a levels correlated inversely with SLE disease activity and the degree of proteinuria, whereas serum miR-146a and miR-155 levels correlated positively with glomerular filtration rate [Wang et al. 2011]. The same group subsequently conducted a profiling study of circulating miRNAs in patients with SLE, patients with rheumatoid arthritis, and healthy controls. They found that miR-126 was specifically elevated in the blood of patients with SLE, whereas miR-125a-3p, miR-155, and miR-146a were significantly reduced in patients with SLE [Wang et al. 2012]. Several other miRNAs (miR-16, miR-223, miR-451, miR-21) were found to be upregulated in both patients with SLE and patients with rheumatoid arthritis. The bioinformatics exploratory analysis implied that most dysregulated circulating miRNAs may be involved in regulating intracellular signal transduction. Taken together, these results suggest that cell-free miRNAs in body fluids may serve as potential biomarkers for SLE.

Recent studies have identified numerous miRNAs that are dysregulated in human patients with SLE and lupus-prone mice. However, it should be cautioned that the results from different studies are not always consistent or reproducible. These discrepancies may originate from the nature of the studies (cross-sectional, small sample size, etc.) and differences in the patient populations (ethnicity, disease severity, duration, manifestations, cohort size, etc.) and the detection methods used. Nevertheless, miRNAs represent a promising group of novel biomarkers for SLE and warrant further investigation.

Cytokine and chemokine biomarkers

With increasing numbers of cytokines and chemokines identified and understanding of cytokine biology improved, these molecules have emerged as important players in the pathogenesis of SLE or as indirect markers reflecting dysregulated immune responses in SLE [Adhya et al. 2011; Apostolidis et al. 2011; Davis et al. 2011] (Table 2). This emerging role of cytokines and chemokines has in turn sparked growing attention on their potential as biomarkers and therapeutic targets in SLE.

Table 2.

Cytokine and chemokine abnormalities associated with SLE.

Cytokine/Chemokine	Abnormality	Disease Association
IFNα/β	Elevated levels in serum and CSF	CNS and hematological manifestations; fever
IFN signature	Upregulated expression of IFN-regulated genes	CNS (cerebritis), hematologic, and renal manifestations
IFN-inducible chemokines
MCP-1 (CCL2)	Increased levels in serum and/or urine; increased gene expression	Disease activity/flares
RANTES (CCL5)		Lupus nephritis
MIP-3B (CCL19)		NPSLE
IP-10 (CXCL10)		Hypocomplementemia
SIGLEC-1		Autoantibodies (anti-dsDNA, anti-U1-RNP; Ro, Sm)
CXCL1
CXCL16
IFNγ	Elevated serum levels	Disease activity?
IL-17	Elevated serum levels	Disease activity?
IL-6	Elevated levels in serum and urine	Disease activity; anti-dsDNA levels; low serum C3/C4; lupus nephritis
IL-10	Elevated serum levels	Disease activity; anti-dsDNA levels; low serum C3/C4
IL-12	Elevated or decreased levels reported in different studies	Lupus nephritis (elevated levels in serum and urine)
IL-15	Elevated serum levels	Disease pathogenesis?
IL-21	Elevated serum levels	Disease activity; disease pathogenesis?
IL-2	Decreased serum levels	Disease pathogenesis?
IL-1	Elevated serum levels	Disease activity
IL-1 receptor antagonist (IL-1ra)	Decreased serum levels	Lupus nephritis
BAFF (BLys)	Elevated serum levels	Disease activity
TNFα	Elevated levels in serum and kidneys	Disease activity; correlated with serum IFNα levels

BAFF, B-cell activating factor; CNS, central nervous system; CSF, cerebrospinal fluid; IFN, interferon; IL, interleukin; NPSLE, neuropsychiatric systemic lupus erythematosus; SLE, systemic lupus erythematosus; TNF, tumor necrosis factor

Type I IFNs and IFN-inducible genes and chemokines

Numerous studies, both cross-sectional and longitudinal, have identified various cytokines and chemokines that are associated with SLE disease activity and clinical manifestations. Prominent among these candidate biomarkers is the type 1 IFN system (e.g. IFNα and IFNα-inducible genes and proteins) [Crow, 2007; Elkon and Wiedeman, 2012; Obermoser and Pascual, 2010]. As early as in 1970s, IFNα levels were shown to be elevated in the serum of SLE [Hooks et al. 1979; Ytterberg and Schnitzer, 1982]. The role of IFNα, however, was left underappreciated until the recent decade when gene expression profiling techniques and multiplexed serologic tests advanced considerably. Baechler and colleagues pioneered the use of DNA microarray techniques to study gene expression profiles in PBMCs of SLE patients and found a striking pattern of upregulated IFN-inducible genes (termed the ‘IFN signature’) in a subset of SLE patients [Baechler et al. 2003]. They further observed that the IFN signature correlated with more severe disease, such as cerebritis, nephritis, and hematological involvement, in those patients. Other investigators have subsequently reported similar findings, including significant associations of enhanced expression of IFN-inducible genes and/or serum levels of IFN-inducible chemokines with increased disease activity, organ involvement, hypocomplementemia, and the presence of autoantibodies specific for dsDNA and RNA-binding proteins (Ro, U1-RNP, and Sm), in both adult and pediatric SLE patients [Bauer et al. 2006; Bennett et al. 2003; Feng et al. 2006; Kirou et al. 2005; Nikpour et al. 2008; Vila et al. 2007]. Two independent studies utilizing transcriptome analysis and traditional techniques [enzyme-linked immunosorbent assay (ELISA) and flow cytometry] demonstrated that IP-10 and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) were among the most prominent type 1 IFN-regulated genes/proteins [Biesen et al. 2008; Rose et al. 2012]. Increased serum levels of IP-10 and SIGLEC-1 were detected in 50% and 86%, respectively, of patients with active SLE [Rose et al. 2012]. The serum IP-10 and SIGLEC-1 levels correlated positively with disease activity and anti-dsDNA antibody levels, but inversely with serum complement levels [Biesen et al. 2008]. A pathologic role for IFNα in neuropsychiatric SLE (NPSLE) has recently been postulated [Karageorgas et al. 2011]. This postulation is based on an early study in which elevated IFNα levels in the cerebrospinal fluid (CSF) were detected in some patients with NPSLE [Winfield et al. 1983] and a recent study reporting that CSF from patients with NPSLE was capable of inducing higher IFNα production in vitro compared with other disease controls [Santer et al. 2009].

The above-mentioned cross-sectional studies have recently been followed by longitudinal studies. Bauer and colleagues measured IFN-regulated chemokine levels in 267 patients with SLE followed for 1 year (1166 total visits) [Bauer et al. 2009]. Serum levels of CXCL10 (IP-10), CCL2 (MCP-1), and CCL19 (MIP-3B) were found to correlate strongly with lupus activity, rising at flare and decreasing with remission, and to perform better than currently available laboratory tests. To further determine whether changes in the IFN signature expression correlate with clinical outcomes such as disease flares, a Canadian research group investigated the expression levels of selected IFNα-inducible genes, serological variables, and clinical disease activity [as measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)] in 94 SLE patients over a period of 3–12 months [Landolt-Marticorena et al. 2009]. When analyzed cross-sectionally at a single timepoint, expression levels of IFNα-inducible genes were found to be significantly elevated and associated with high SLEDAI scores, active renal disease, decreased C3 levels, and positive anti-dsDNA and anti-RNA-binding protein autoantibodies. However, when followed over time, no significant correlation between changes in IFNα-inducible gene expression and changes in disease activity, C3 levels, or autoantibody levels was observed. Similarly, Petri and colleagues conducted a study combining both cross-sectional and longitudinal analysis of INFα-regulated gene expression in peripheral blood cells of patients with SLE [Petri et al. 2009]. A cross-sectional analysis of 66 patients with SLE showed that higher IFN-response (IFNr) scores (calculated based on the expression of three INFα-regulated genes) were associated with greater disease activity. However, the IFNr scores did not significantly differ in 15 patients who were studied before and during flares. An extended longitudinal follow up of 11 patients also showed little change in IFNr scores over time, even with dynamic disease activity changes. These results demonstrate that although IFNr scores appear to be associated with SLE disease activity overall, the IFNr scores of individual patients do not correlate with changes in disease activity.

Taken together, the candidacy of the IFN gene signature/IFN-inducible proteins as biomarkers for monitoring and/or predicting SLE disease activity needs to be further investigated with large-scale multicenter trials.

Interleukin-17

A growing list of cytokines and soluble forms of cytokine receptors have been identified as potential biomarkers of SLE disease activity in many cross-sectional studies with limited cohort sizes. Among them, cytokines of the IL-17 family have received recent attention [Crispin and Tsokos, 2010a]. The IL-17 family consists of IL-17A, IL-17-B, IL-17C, IL-17D, and IL-17F, with IL-17A being the most prominent and well characterized member of the family [Ouyang et al. 2008]. Elevated serum levels of IL-17A and increased numbers of IL-17-producing cells have been reported in patients with SLE [Crispin et al. 2008; Doreau et al. 2009; Shah et al. 2010; Wong et al. 2000; Yang et al. 2009]. IL-17A is mainly produced by CD4+ T helper 17 (T_H17) cells [Korn et al. 2009], γδ T cells [O’Brien et al. 2009], and CD4-CD8- [double negative (DN)] T cells [Crispin et al. 2008; Crispin and Tsokos, 2010b]. In SLE, T_H17 cells and particularly the DN T cells can infiltrate tissues (e.g. the kidneys) and stimulate stromal cells and other immune cells to produce additional cytokines (e.g. IL-6, granulocyte colony stimulating factor, and granulocyte monocyte colony stimulating factor), thereby promoting tissue inflammation and injury [Crispin et al. 2008; Ouyang et al. 2008; Zhang et al. 2009]. Although elevated serum IL-17 levels have been reported in patients with SLE, the association between elevated IL-17 levels and SLE disease activity is unclear. The potential of IL-17 as an SLE biomarker, therefore, awaits further investigation.

B-cell activating factor

B-cell activating factor [BAFF; also known as B lymphocyte stimulating factor (BLys), TALL-1, or TNFSF13B] is expressed as a transmembrane protein on monocytes, macrophages, and monocyte-derived dendritic cells [Mackay and Schneider, 2009; Moore et al. 1999]. A soluble form of BAFF, cleaved from the cell surface, is biologically active and critical for B-cell growth and survival [Nardelli et al. 2001]. Several cross-sectional studies showed that approximately 30% of SLE patients had significantly elevated circulating levels of BAFF/BLys [Cheema et al. 2001; Zhang et al. 2001]. Elevated BAFF/BLys levels appeared to correlate with increased total IgG and autoantibody (particularly anti-dsDNA) levels [Cheema et al. 2001; Pers et al. 2005], and, in some studies, with increased disease activity (as measured by SLEDAI) [Becker-Merok et al. 2006]. A recent study has shown that excessive productions of IFNγ by activated T cells may be responsible for the induction of BAFF/BLyS production by monocytes and macrophages in lupus patients [Harigai et al. 2008].

To delineate the role of BAFF/BLyS in the long-term immune dysregulation in SLE, Stohl and colleagues conducted a longitudinal observational study in which 68 SLE patients were followed regularly for disease activity (measured using SLEDAI) and serum BAFF/BLyS levels over a period of 147–420 days (median 369 days) [Stohl et al. 2003]. They found that SLE patients exhibited considerable variability in serum BAFF/BLyS levels with 50% of patients having persistently or intermittently elevated serum BAFF/BLyS levels over the follow-up period. However, changes in serum BAFF/BLyS levels did not correlate with changes in disease activity and/or specific organ involvement in individual patients. In another study, Becker-Merok and colleagues studied clinical disease activity, serological variables, and serum BAFF/BLyS levels in 60 patients with RA and 42 patients with SLE, 19 of whom were followed prospectively over a period of approximately 16 months [Becker-Merok et al. 2006]. These investigators found that considerably more SLE patients had significantly higher serum BAFF/BLyS levels than did RA patients. They found that serum BAFF/BLyS levels generally correlated with SLEDAI scores in SLE patients in cross-sectional comparison, but did not correlate with changes in disease activity over time.

Since BAFF/BLyS is not known to have direct or immediate proinflammatory activities, changes in serum BAFF/BLyS levels are unlikely to trigger acute inflammatory reactions and disease manifestations. Therefore, the reported lack of correlations between changes in BAFF/BLyS levels and changes in disease activity in SLE patients might not be surprising. However, it is possible that an increase in disease activity may lag behind increases in circulating BLyS levels due to indirect or ‘delayed’ effects of BAFF/BLyS in the systemic immune-inflammatory reactions of SLE. Petri and other investigators have recently conducted a prospective multicenter study that evaluated 254 SLE patients every 3–6 months over a 2-year period [Petri et al. 2008]. The results showed that plasma BAFF/BLyS levels were associated with anti-dsDNA levels and disease activity [measured using Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA)-SLEDAI]. Interestingly, multivariate analyses showed that a greater increase in the SELENA-SLEDAI score in the current visit was significantly associated with higher BAFF/BLyS levels at the previous visit. Similarly, a greater increase in the BAFF/BLyS level from the previous visit was associated with a greater SELENA-SLEDAI score in the subsequent follow-up visit. These results suggest a ‘delayed’ relationship between circulating BAFF/BLyS levels and SLE disease activity. Most recently, James and colleagues reported that BAFF/BLyS levels were associated with increased lupus disease activity in White but not in African-American patients who had higher BAFF/BLyS levels regardless of disease activity [Ritterhouse et al. 2011].

Other cytokines

Other cytokines, such as IL-6, IL-10, IL-12, IL-15, and IL-21, have also been implicated in the pathogenesis of SLE and proposed as potential biomarkers for SLE disease activity. A study by Chun and colleagues reported that patients with SLE had higher serum levels of IL-1, IL-10, IL-12, and IFNγ, but lower level of IL-2, than healthy controls [Chun et al. 2007]. Moreover, serum IL-6 and IL-10 levels correlated positively with SLEDAI scores and anti-dsDNA titers but negatively with serum C3 and C4 levels. IL-12 is capable of stimulating the differentiation of T cells into T_H1 cells that produce IFNγ. Therefore, IL-12 may be involved in the pathogenesis of SLE indirectly via its effects on cells expressing IL-12 receptors (IL-12R). A recent study reported that patients with SLE have a higher copy number of the IL-12RB gene as compared with healthy controls [Yu et al. 2011]. A higher copy number of the IL-12RB gene may confer increased sensitivity to IL-12 and hence increased IFNγ production by immune cells. IL-27, a new member of the IL-12 cytokine family, can synergize with IL-12 to induce IFNγ production [Awasthi et al. 2007]. It has been reported that serum IL-27 levels were lower in SLE patients than in healthy individuals [Li et al. 2010]. IL-15 and IL-21 are members of the IL-2 cytokine family. IL-15 is involved in the expansion and homeostasis of T cells and also capable of promoting immunoglobulin isotype switching in B cells [Gabay and McInnes, 2009]. Increased serum levels of IL-15 have been reported in SLE patients [Aringer et al. 2001]. Different from IL-15, IL-21 is also involved in B-cell activation and activation-induced death of B cells [Ettinger et al. 2008]. Decreased IL-21R expression on B cells has been associated with high autoantibody production and nephritis in SLE patients [Mitoma et al. 2005].

T-cell-related biomarkers

T cells play crucial effector and regulatory roles in virtually any immune-inflammatory response. T cells of patients with SLE display numerous phenotypic and functional abnormalities, thereby contributing to SLE pathogenesis and serving potentially as a rich source of SLE biomarkers [Hoffman, 2004; Moulton and Tsokos, 2011] (Table 3).

Table 3.

Novel T-cell-related SLE biomarkers.

Type	Observation	Disease association
CD44v3/CD44v6-expressing T cells	Elevated expression by T cells	Disease activity (SLEDAI scores); anti-dsDNA; lupus nephritis
KIR-expressing T cells	Aberrant expression on in T cells	Disease activity (SLEDAI scores)
NKG2D-expression CD4+ T cells	Increased expansion in patients with juvenile-onset SLE	Inverse correlation with disease activity
CD4-CD8- (double negative) T cells	Significant expansion in the peripheral blood and kidneys; capable of producing IL-17	Lupus nephritis
Altered lipid raft-expressing T cells	Increased levels of GM1 and clustered lipid raft in the membrane of SLE T cells	Disease pathogenesis?

IL, interleukin; KIR, killer immunoglobulin-like receptor; SLE, systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index

Unique T-cell subsets

A heightened capacity of T cells to adhere and migrate into inflamed tissue may lead to organ damage in patients with SLE. A recent study has shown that surface expression of CD44 (an adhesion molecule) and its variant isoforms CD44v3 and CD44v6 were elevated on T cells of patients with SLE [Crispin et al. 2010a]. Significantly, T-cell surface levels of CD44v3 and CD44v6 were found to be correlated with the SLEDAI score, positivity of anti-dsDNA, and the presence of lupus nephritis. These results suggest that expression levels of CD44v3 and CD44v6 on T cells may serve as biomarkers for SLE disease activity [Crispin et al. 2010a]. Recently, it has also been reported that killer cell-Ig like receptors (KIR), surface molecules originally identified on natural killer (NK) cells, were aberrantly expressed by T cells of patients with SLE and that the expression of T-cell surface KIR was proportional to the SLEDAI score [Basu et al. 2009]. These studies implicate the potential of frequencies of KIR-expressing T cells and T-cell expression levels as disease activity biomarkers for SLE. Another potential T-cell-relevant disease activity biomarker was suggested by a recent study that reported increased expansion of a subpopulation of CD4+ T cells that express the NKG2D receptor and display suppressive/regulatory activity in patients with juvenile-onset SLE; the frequencies of such ‘suppressive/regulatory’ NKG2D+CD4+ T cells were correlated inversely with disease activity [Dai et al. 2009b].

DN T cells, which lack surface expression of CD4 and CD8, constitute a small population (<5% of T cells) in healthy individuals, but are significantly expanded in patients with SLE [Crispin et al. 2008]. A recent study showed that these cells can secrete cytokines such as IL-17 and IL-1β, induce anti-dsDNA antibody production by autoreactive B cells, and infiltrate the kidneys of patients with lupus nephritis [Crispin et al. 2008]. Thus, the frequency of DN T cells in the peripheral blood might serve as a surrogate biomarker of lupus nephritis.

Other potential T-cell-based biomarkers

Previous studies have demonstrated multiple abnormalities at different levels of the T-cell receptor (TCR) signaling cascade and downstream in T cells derived from patients with SLE [Moulton and Tsokos, 2011]. It has been shown that the fundamental defect may be linked to alterations in the structure and dynamics of lipid rafts within the plasma membrane of lupus T cells [Jury et al. 2007; Kabouridis and Jury, 2008]. Lipid rafts are cholesterol- and glycosphingolipid-rich membrane microdomains that facilitate and orchestrate close interactions between molecules that are critical in signal transduction [Harder, 2004]. Several investigators have shown that, as compared to T cells derived from control mice or healthy individuals, T cells derived from lupus-prone mice or patients with SLE express increased levels of glycosphingolipid GM1 and possess higher amounts of clustered lipid rafts that are associated with unusual components such as FcRγ activated Syk kinase, and CD45 [Dong et al. 2010; Jury et al. 2004; Krishnan et al. 2004]. Furthermore, it was recently shown that injection of lupus-prone mice with Cholera Toxin B, a component of Vibrio cholerae capable of binding to GM1, induced aggregation of lipid rafts in T cells and consequently accelerated lupus development in those mice [Deng and Tsokos, 2008]. Conversely, statins (inhibitors of HMG-CoA reductase), which decrease cholesterol synthesis and thus can modify lipid raft components, have been shown to restore signaling defects characteristic of SLE T cells [Jury et al. 2006]. Taken together, these studies support a pivotal role for altered lipid rafts in dysregulated T cell signaling in SLE and imply potential utility of levels of T-cell-associated altered lipid rafts as surrogate biomarkers for SLE disease activity.

Biomarkers for lupus nephritis

SLE can affect virtually any tissue or organ. However, not all organs will be affected simultaneously, and the involvement of a specific organ will not necessarily be manifested in the same manner in different patients. The manifestations of specific organ involvement of a given patient may also vary over time. This clinical heterogeneity of SLE poses a great demand for biomarkers that can differentiate, determine, monitor, stratify and/or predict organ-specific involvement in patients with SLE.

Of the myriad manifestations of SLE, renal involvement is one of the most common and it continues to cause significant morbidity and mortality. Lupus nephritis occurs in 25–50% of adult patients with SLE [Cameron, 1999] and 80% of pediatric patients with SLE [Watson and Beresford, 2012]. Among those patients, 25% and 40% of adult and pediatric patients, respectively, may progress to end-stage renal disease. Improved methods for detecting lupus nephritis would allow earlier treatment, which may prevent irreversible kidney damage and thwart declines in renal function.

In lieu of invasive and impractical serial renal biopsies, creatinine clearance, urine protein level, urine sediment, serum C3 and C4 levels, serum creatinine level, and anti-dsDNA titers have for decades been used to follow the onset, course, and severity of lupus nephritis, yet it is generally recognized that these measurements are inadequate. Current efforts are focused on identification of more sensitive and specific biomarkers to diagnose and monitor renal disease in both adult and pediatric patients with SLE, with the hope to optimize synchronization of treatment with active disease, distinguish active inflammation from irreversible damage, and to facilitate development of new therapeutics through clinical trials [Adhya et al. 2011; Mok, 2010; Rovin and Zhang, 2009] (Table 4).

Table 4.

Summary of biomarkers for lupus nephritis.

Traditional Biomarkers

Serum Creatinine levels

Serum C3/C4 levels

Anti-dsDNA levels

Urine protein levels

Urine sediments

Kidney biopsy

Candidate Biomarkers (Serum, peripheral blood, and kidney)

Anti-nucleosome antibodies (serum)

Anti-C1q antibodies (serum)

Complement C4d (kidney biopsy)

Complement C4d (erythrocyte-bound; peripheral blood)

Candidate Biomarkers (Urinary proteins)

Monocyte chemoattractant protein-1 (MCP-1)

Neutrophil gelatinase-associated lipocalin (NGAL)

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)

Transferrin (TF)

α1-acid glycoprotein (AGP; AAG)

Ceruloplasmin (CP)

Lipocalin-type prostaglandin D-synthetase (L-PGDS)

Hepcidin

Tumor growth factor β (TGFβ)

Antinucleosome antibodies

The use of autoantibodies other than anti-dsDNA in monitoring and preferably predicting renal disease in patients with SLE has been explored extensively during the past several decades. Among those autoantibodies, antichromatin/antinucleosome antibodies [Bruns et al. 2000; Gomez-Puerta et al. 2008; Koutouzov et al. 2004], along with anti-C1q antibodies [Pickering and Botto, 2010; Potlukova and Kralikova, 2008] (see the next section), have shown some promise as biomarkers of renal involvement. Chromatin, the DNA-histone complex found in the nucleus of eukaryotic cells, is organized into a repeating series of nucleosomes. Previous studies have demonstrated that the nucleosome is a major autoantigen targeted by T and B cells in SLE [Bruns et al. 2000; Mohan et al. 1993]. Antinucleosome antibodies are reportedly present in 70–100% of patients with SLE, and are fairly sensitive (48–100%) and highly specific (90–99%) for SLE [Cervera et al. 2003; Chabre et al. 1995; Gomez-Puerta et al. 2008]. Among SLE patients, antinucleosome antibodies are more likely to be detected in patients with nephritis and may serve as a useful biomarker in the diagnosis of active lupus nephritis [Cervera et al. 2003; Grootscholten et al. 2007; Gutierrez-Adrianzen et al. 2006].

Moreover, some investigators reported that antinucleosome antibodies could be found in patients who consistently tested negative for anti-dsDNA antibodies and that a significant fraction of those patients indeed had renal disease [Min et al. 2002], suggesting that antinucleosome antibodies may serve as a sensitive marker for renal involvement in the absence of anti-dsDNA. A prospective controlled study was recently conducted with 52 patients with active proliferative lupus nephritis [Grootscholten et al. 2007]. It was found that patients with high titers of antinucleosome antibodies had significantly higher disease activity; the levels of antinucleosome antibodies rapidly declined after treatment. Similarly, another study followed a group of SLE patients with new-onset lupus nephritis for up to 2 years [Manson et al. 2009]. Those investigators showed that antinucleosome levels associated positively with decreasing renal function (measured by urine protein/creatinine ratio and serum albumin level), and the levels of antinucleosome antibodies significantly decreased during remission of lupus nephritis.

Although many studies including those discussed here have reported the promise of antinucleosome antibodies, it should be cautioned that another study showed that antinucleosome antibodies are highly prevalent in both SLE patients with (89%) or without (80%) active proliferative nephritis and have limited value in distinguishing these two subgroups of patients [Bigler et al. 2008]. Nevertheless, these studies collectively provide substantial, but not definitive, evidence that antinucleosome antibodies may be more sensitive and have greater diagnostic efficacy than anti-dsDNA for active disease, especially nephritis, in SLE patients.

Anti-C1q antibodies

Anti-C1q antibodies are mostly of the IgG subtype that react with epitopes present within the collagen-like tail of C1q. Although anti-C1q antibodies can be detected in a small proportion of healthy individuals (3-5%), they are more common in patients with autoimmune disorders such as hypocomplementemic urticarial vasculitis (100%) and SLE (17-63%) [Sinico et al. 2009]. A significant correlation between the presence of anti-C1q antibodies and renal disease in SLE has been reported [Horak et al. 2006; Horvath et al. 2001; Marto et al. 2005; Moroni et al. 2001], with positive predictive value and negative predictive to be 58% and 100%, respectively [Moroni et al. 2001]. Therefore, the absence of anti-C1q antibodies has been suggested as an indicator for excluding a diagnosis of lupus nephritis [Trendelenburg et al. 1999, 2006]. Significant correlations between anti-C1q titers and active lupus nephritis have also been reported [Akhter et al. 2011; Moroni et al. 2001; Trendelenburg et al. 2006], with a sensitivity of 44-100% and a specificity of 70-92% reported by Moroni and colleagues [Moroni et al. 2001]. Another study reported the detection of anti-C1q antibodies in SLE patients who did not have a history of renal disease but eventually developed lupus nephritis [Marto et al. 2005]. The detection of anti-C1q and an increase in anti-C1q antibodies have been suggested to predict renal flares [Meyer et al. 2009; Moroni et al. 2001, 2009]. For example, Moroni and colleagues followed 228 patients with lupus nephritis for 6 years and found that elevation of anti-C1q levels predicted renal flares in patients with proliferative lupus nephritis, with a sensitivity of 81% and a specificity of 71% [Moroni et al. 2009]. Some investigators suggested that the prediction value would be further enhanced when both anti-C1q and anti-dsDNA were measured [Matrat et al. 2011]. Moreover, it has been reported that levels of anti-C1q antibodies decreased after successful treatment of lupus nephritis [Trendelenburg et al. 2006].

Taken together, these recent studies demonstrate a strong correlation between the presence of anti-C1q antibodies and lupus nephritis, and suggest that anti-C1q determination may serve as a noninvasive biomarker (versus the invasive renal biopsy) to monitor renal involvement and/or predict renal flares [Pickering and Botto, 2010; Sinico et al. 2009].

Complement C4d

An expanding literature has demonstrated the utility of histologic identification of complement C4d as an informative biomarker of transplant allograft rejection. Similarly, investigators from the Netherlands have demonstrated a strong relationship between the intensity of glomerular C4d staining and the presence of microthrombi in patients with lupus nephritis (present in seven of eight patients) [Cohen et al. 2008]. Most recently, a prospective assessment was performed to determine the potential value of cell-bound C4d as biomarkers of lupus nephritis [Batal et al. 2012]. Histologic identification of C4d on renal biopsies was compared with the results of a cell-bound complement activation product (CB-CAP) assay panel in 15 patients with lupus nephritis, 239 lupus patients without nephritis and 13 patients with nonlupus nephritis. Patients with lupus nephritis had higher glomerular C4d scores, and also had significantly higher levels of erythrocyte-bound C4s and reticulocyte-bound C4d than the two control groups. SLE patients with lupus nephritis were also more likely to have C4d bound on platelets, as compared with SLE patients without lupus nephritis. Erythrocyte-bound C4d levels correlated with the National Institutes of Health (NIH) activity index. These findings suggest a potential role of circulating cell-bound C4d as a noninvasive biomarker for lupus nephritis.

Urinary chemokine/cytokine biomarkers

Serum and plasma are easy to prepare from patients and have been widely used in developing novel biomarkers for lupus nephritis. However, biological changes in the serum or plasma may not reflect fully and timely the local situation within a specific organ such as the kidneys. Urine, in contrast, may serve as a window looking into the pathological changes in inflamed kidneys in real-time. Significant progress has been made in the discovery of novel urinary biomarkers for lupus nephritis.

Monocyte chemoattractant protein-1

Monocyte chemoattractant protein-1 (MCP-1) participates in the pathogenesis of lupus nephritis. A number of earlier studies showed that urinary MCP-1 (uMCP-1) levels were elevated in patients with active nephritis and declined after immunosuppressive treatment [Noris et al. 1995; Rovin et al. 1996; Wada et al. 1996]. Several more recent studies, both cross-sectional and longitudinal, have confirmed those findings [Kiani et al. 2009; Rovin et al. 2005; Tian et al. 2007; Tucci et al. 2004]. For example, in a study conducted by Rovin and colleagues [Rovin et al. 2005], it was shown that uMCP-1 level was a sensitive indicator for renal flare, with 73% of the uMCP-1 levels measured at renal flares higher than the 95th percentile of the disease controls. uMCP-1 levels did not correlate with extrarenal disease activity. Moreover, uMCP-1 levels increased as early as 2–4 months before renal flares, suggesting that uMCP-1 may serve as a biomarker for predicting impending renal flares. Similarly, uMCP-1 levels have been shown to elevate in pediatric patients with lupus nephritis and to be able to differentiate patients with active or inactive renal disease [Watson et al. 2012]. These results, collectively, indicate that urinary levels of MCP-1 protein and urinary MCP-1 mRNA levels are promising biomarker candidates due to specificity for renal activity, sensitivity in predicting renal flares and the capacity to reflect both the severity of flares, and the proliferative nature of the histology [Chan et al. 2006; Kiani et al. 2009; Rovin et al. 2005].

Neutrophil gelatinase-associated lipocalin

Neutrophil gelatinase-associated lipocalin (NGAL; lipocalin-2) is a small glycoprotein that functions as a carrier for cellular iron transport, apoptosis, and tissue differentiation. NGAL is constitutively produced at low levels in the kidneys, but is upregulated following inflammation, ischemia, and infection [Bolignano et al. 2008; Mishra et al. 2003; Schwartz et al. 2007; Trachtman et al. 2006]. Associations of rising serum and urinary NGAL levels (uNGAL) with acute renal injury have been shown in both adult and pediatric patients [Bolignano et al. 2008; Mishra et al. 2003; Schwartz et al. 2007; Trachtman et al. 2006]. In lupus nephritis, it was postulated that injured tubular cells, infiltrating neutrophils, or inflamed vasculature are the sources of uNGAL [Brunner et al. 2006]. Cross-sectional as well as longitudinal studies have demonstrated the promise of uNAGL as a biomarker of lupus nephritis in both pediatric and adult patients with SLE [Brunner et al. 2006; Hinze et al. 2009; Pitashny et al. 2007; Rubinstein et al. 2010; Suzuki et al. 2008; Yang et al. 2012]. In a cross-sectional study of pediatric patients by Brunner and colleagues, uNGAL levels were significantly elevated in patients with lupus nephritis; increased uNGAL levels were associated with renal SLE disease activity scores and not with global damage or extrarenal disease activity [Brunner et al. 2006]. A cross-sectional study of adult patients with lupus nephritis generated similar findings [Pitashny et al. 2007]. However, it should be pointed out that conflicting reports regarding the association of uNGAL with lupus nephritis in adult patients have been published recently [Kiani et al. 2012; Yang et al. 2012]. Nevertheless, the potential of both serum and urinary NGAL as biomarkers for lupus nephritis is best reflected by longitudinal studies demonstrating the capacity to predict exacerbation of renal disease and flares [Rubinstein et al. 2010; Suzuki et al. 2008].

Tumor necrosis factor-like weak inducer of apoptosis

Tumor necrosis factor like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that belongs to the tumor necrosis factor (TNF) superfamily. The expression of TWEAK has been shown to increase dramatically during inflammation and injury [Winkles, 2008]. In lupus nephritis, it is possible that TWEAK may induce the production of other proinflammatory cytokines and chemokines and thus propagate tissue inflammatory injury [Gao et al. 2009; Sanz et al. 2008]. Urinary TWEAK (uTWEAK) levels were shown by Schwartz and colleagues in a cross-sectional study to be significantly higher in SLE patients with active nephritis as compared with those with inactive or no nephritis [Schwartz et al. 2006]. In addition, uTWEAK levels correlated with anti-dsDNA, serum C3 and C4, uMCP-1 and renal disease activity scores. A subsequent multicenter longitudinal study by this same group further demonstrated the potential value of uTWEAK which was superior to the current standards anti-dsDNA and serum complement levels in differentiating lupus nephritis from nonrenal lupus activity [Schwartz et al. 2009].

Biomarkers predicting histopathologic features of lupus nephritis

Kidney biopsy is currently the gold standard for diagnosing, classifying, and guiding the treatment of lupus nephritis. It standardizes histological classification of lupus nephritis, enables direct assessment of the presence and severity of acute changes due to active lupus nephritis, and provides information of the chronicity of lupus nephritis. However, the invasive nature of kidney biopsy limits its frequent use in monitoring the disease progression and treatment response. Therefore, noninvasive and sensitive biomarkers that might reveal specific histopathologic features of lupus nephritis are in great need.

The associations of serum antinucleosome and anti-C1q levels with renal histopathologic features have recently been reported [Chen et al. 2012; Hung et al. 2011]. Hung and colleagues conducted a cross-sectional study with 36 SLE patients with biopsy-proven proliferative lupus nephritis and 14 patients with non-renal SLE [Hung et al. 2011]. These investigators found that serum levels of antinucleosome antibodies were significantly elevated in patients with lupus nephritis and correlated with SLE disease activity measured by the British Isles Lupus Assessment Group (BILAG) index. The serum antinucleosome antibody levels correlated positively with the histological activity index of lupus nephritis (r(s) = 0.368, p < 0.05), but not with the chronicity index. In another cross-sectional study involving 52 patients who had biopsy-proven lupus nephritis, serum anti-C1q levels were found to correlate positively with the scores of the SLEDAI, anti-dsDNA antibody, and antinucleosome antibody [Chen et al. 2012]. The prevalence of anti-C1q was found to be higher in patients with proliferative lupus nephritis [World Health Organization (WHO) class III and class IV] than those with mesangial lupus nephritis (class II), although the difference did not reach statistical significance. The serum anti-C1q levels were highest in patients with class IV lupus nephritis. Furthermore, the anti-C1q levels correlated positively with renal activity indices but negatively with chronicity indices. These findings suggest that serum anti-C1q antibody may be a valuable noninvasive biomarker for prediction of histopathologic characteristics of lupus nephritis.

Following the identification of several promising urinary biomarkers for diagnosing and differentiating active versus inactive lupus nephritis, investigators examined the relationship between those urinary biomarkers and histological features of lupus nephritis. In a recent study, Brunner and colleagues assayed several established markers (anti-dsDNA, serum C3, C4, creatinine, urinary protein:creatinine ratio, etc.) and urinary biomarkers [MCP-1, NGAL, lipocalin-type prostaglandin D-synthetase (L-PGDS), α1-acid-blycoprotein (AAG/AGP), transferrin (TF), and ceruloplasmin (CP)] in urine samples from 76 SLE patients collected within 2 months of kidney biopsy [Brunner et al. 2012]. These urinary biomarkers were compared with histopathologic features of the kidney biopsy (mesangial expansion, capillary proliferation, crescent formation, wire loops, fibrosis, etc.). Statistical analyses included nonparametric analysis and area under the receiver operating characteristic curve (AUC) calculation. The results revealed that differential increases in levels of urinary biomarkers in patients with active lupus nephritis appeared to reflect specific histologic features in the kidneys. Specifically, the combination of urinary MCP-1, AAG, and CP levels and protein:creatinine ratio performed well in predicting lupus nephritis activity (AUC = 0.85). On the other hand, NGAL together with MCP-1 and creatinine clearance appeared to be a valuable indicator of lupus nephritis chronicity (AUC = 0.83). The combined panel of MCP-1, AAG, TF, creatinine clearance, and serum C4 was shown to be a potential biomarker for membranous nephritis (AUC = 0.75).

Collectively, these studies suggest that panels of serum and urinary assays hold promise as potential noninvasive biomarkers for monitoring the activity, chronicity, and type of lupus nephritis.

SLE biomarker panels

Undoubtedly, no single biomarker will be sufficient ultimately to diagnose, monitor and stratify all patients with SLE. Thus, investigations have recently evolved toward discovery and validation of lupus biomarker ‘panels’. One approach in this regard has been technology-driven with the use of microarrays or proteomics for identifying novel gene or protein ‘biosignatures’ of SLE such as that discussed above for lupus nephritis [Brunner et al. 2012]. Additional ongoing developments on this front are discussed briefly here.

Biomarker panels for diagnosis and disease activity monitoring of SLE

Recognizing the importance of aberrant T-cell function in SLE, a gene expression array consisting of 30 genes thought to contribute to the pathogenesis of SLE has recently been developed. To examine the utility of this T cell gene expression array, an initial study was conducted with 10 patients with SLE, 6 patients with rheumatoid arthritis, and 19 healthy individuals [Juang et al. 2011]. The results showed that the gene expression array could faithfully represent the expression levels of different genes. Moreover, principal component analysis (PCA) was used to evaluate the contribution of the arrayed genes to diagnosis and stratification. PCA of gene expression levels showed that SLE samples were distinguished from samples of RA and healthy individual. Notably, individual principal components appeared to define specific disease parameters such as arthritis and proteinuria. These results suggest potential for this T-cell gene expression as lupus biomarkers for diagnosis and stratification. Further validation, using larger cohorts of patients with lupus compared with patients with other lupus-like diseases is warranted.

Another gene expression-based search for biomarkers of autoimmune disease involved profiling the transcriptomes of purified CD8 T cells [McKinney et al. 2010]. McKinney and colleagues identified a subset of genes associated with a poor prognosis in patients with SLE. This subset of genes is enriched for genes involved in TCR signaling, IL-7 receptor signaling, and memory T-cell phenotype. They further showed that these subgroups of genes can be identified by measuring the expression of only three representative genes. These findings suggest the prospect of a streamlined version of gene expression-based biomarkers that might be used for identifying patients with poor prognosis and thus for personalized treatment of those patients.

A more recent study investigated the value of a biomarker panel consisting of traditional markers (e.g. ANA and anti-dsDNA) and the newly developed CB-CAP biomarkers [Liu et al. 2010]. In this multicenter, cross-sectional study, a total of 593 subjects (210 SLE patients, 178 patients with other rheumatic diseases, and 205 healthy individuals) were enrolled [Kalunian et al. 2012]. Erythrocyte-bound C4d (E-C4d), B-cell-bound C4d (B-C4d), ANA, and anti-mutated citrullinated vimentin antibody (anti-MCV) were measured and an index score system corresponding to the weighted sum of these four markers was developed. It was reported that a positive index score correctively categorized 72% of SLE patients. Furthermore, the sensitivity and specificity of the combination of anti-dsDNA and index score positivity in differentiating SLE from other rheumatic disease was 80% and 87%, respectively. These results support the use a biomarker panel combining anti-dsDNA, ANA, anti-MCV, E-C4d, and B-C4d for the diagnosis of SLE. This is the only biomarker panel validated to date for lupus diagnosis.

Composite urinary biomarkers for lupus nephritis

The proteomics approach has recently been taken to identify protein markers/profiles that can differentiate SLE with or without nephritis or differentiate active lupus nephritis from inactive lupus [Mosley et al. 2006; Suzuki et al. 2007]. In one of the first urinary proteomics studies, Mosley and colleagues utilized the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SLEDI-TOF MS) technology and identified proteins with masses of 3340 and 3980 that best distinguished active from inactive lupus nephritis [Mosley et al. 2006]. Further analysis with serial urine samples of six patients with biopsy-confirmed lupus nephritis showed that these proteins could predict renal flares and remission earlier than conventional markers for nephritis. Subsequently, Suzuki and colleagues used a similar technique to investigate the urinary protein profile (‘signature’) in pediatric patients with lupus nephritis [Suzuki et al. 2007]. They identified 8 candidate peptides/proteins with molecular masses from 2700 to 133,000, including TF, AGP, CP, and L-PGDS. The peak intensities of these proteins were significantly stronger in patients with nephritis than in patients without nephritis and healthy controls. Moreover, these proteins significantly correlated with renal disease activity.

In a subsequent validation study, these investigators measured the concentrations of urinary TF, AGP, CP, and L-PGDS in serial samples from 98 pediatric SLE patients and 30 patients with juvenile idiopathic arthritis [Suzuki et al. 2009]. All four urinary proteins were present at significantly higher concentrations in SLE patients with active nephritis, compared to those with inactive nephritis or juvenile idiopathic arthritis. By analyzing the AUC, these urinary proteins performed better than traditional renal markers in identifying the presence of active nephritis. Furthermore, prospective analysis showed that significant increases of urinary TF, L-PGDS, and AGP, occurred as early as 3 months before clinical worsening of lupus nephritis. This latter result suggests that these urinary proteins, particularly TF, are sensitive biomarkers for changes in renal disease activity.

Most recently, Zhang and colleagues demonstrated the potential of a composite biomarker panel for lupus nephritis in adult SLE patients [Zhang et al. 2012]. In a study that included evaluation of 64 renal biopsies and simultaneous (or close-in-time) urine samples from 61 patients with lupus nephritis, they identified a composite panel including MCP-1 and serum creatinine that had sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 81%, 67%, and 100%, respectively with only 14% of biopsies being misclassified.

Future prospects

Although many SLE biomarker reports have been published during the past decade, the majority of these have studied small numbers of patients and/or have been limited to cross-sectional observations. Owing to the heterogeneous nature of SLE with mounting evidence of the influence of geoepidemiology and epigenetics, it is perhaps not surprising that some of the biomarkers have yielded conflicting results in different studies or have failed to fulfill the early promising potential. The few longitudinal investigations that have been published have followed promising initial cross-sectional studies and more such long term prospective analyses are ongoing. Ultimately, validation of lupus biomarkers will require multicenter studies.

Until recently, the development of SLE biomarkers has primarily been focused on biomarkers that may assist in making a precise diagnosis or monitoring disease activity. Attempts have begun toward discovering biomarkers that might aid in predicting the onset of SLE in susceptible individuals and/or development of flares (systemic or organ-specific) in patients with established SLE, predicting disease outcome, and assessing the effectiveness of therapeutic interventions. These ‘next-generation’ SLE biomarkers will be particularly important because more sensitive and specific markers for the onset or flare of SLE disease activity may allow proactive institution of therapeutic and even preventive strategies so that the therapeutic efficacy can be enhanced while treatment-related side effects can be minimized. In the burgeoning era of biologic therapeutics, some of which have resulted from recent biomarker studies, a new class of pharmacodynamic biomarkers is needed to aid identification of patients who might respond favorably to a particular biologic, selection of the type and dose of biologics used, and evaluation of therapeutic efficacy. An illustrative example is sifalimumab and rontalizumab, anti-IFNα monoclonal antibodies under evaluation for the treatment of SLE, which were developed based on the seminal discovery of the IFN signature and related biomarkers. Specific and dose-dependent inhibition of type 1 IFN-inducible gene expression and improvement of skin lesions has been observed in the blood of treated SLE patients [Merrill et al. 2011]. It has been shown that, using a scoring or a metric system based on the expression of type 1 IFN-inducible genes, SLE patients could be divided into two distinct subpopulations [Yao et al. 2010]. This latter finding supports the possibility of using the IFN signature metric as predictive biomarkers to identify SLE patients who might respond more favorably to these biologics and as monitoring biomarkers to stratify patients’ responses.

Owing to the extreme complexity of the disease in its diagnosis, course and organ-specific manifestations as described above, we will most likely depend upon a panel of SLE biomarkers that will be used by physicians, scientists and industry for patient care, research, and drug discovery. This potentially daunting task will require collaborative efforts and novel approaches moving forward.

Footnotes

Funding

The authors’ studies mentioned in this article were supported by grants from the National Institutes of Health (RO1 HL074335 and RO1 AI077591), the Lupus Research Institute, and the Department of Defense.

Conflict of interest statement

The authors are co-inventors of patents related to the CB-CAP biomarkers that were mentioned in this article. Dr Ahearn and Dr Manzi are consultants of the Exagen Diagnostics, Vista, CA.

References

Adhya

Borozdenkova

Karim

(2011) The role of cytokines as biomarkers in systemic lupus erythematosus and lupus nephritis. Nephrol Dial Transplant 26: 3273–3280.

Ahearn

Liu

Kao

Manzi

(2012) Biomarkers for systemic lupus erythematosus. Transl Res 159: 326–342.

Akhter

Burlingame

Seaman

Magder

Petri

(2011) Anti-C1q antibodies have higher correlation with flares of lupus nephritis than other serum markers. Lupus 20: 1267–1274.

Allan

Zueva

Cammas

Schreiber

Masson

Belz

. (2012) An epigenetic silencing pathway controlling T helper 2 cell lineage commitment. Nature 487: 249–253.

Apostolidis

Lieberman

Kis-Toth

Crispin

Tsokos

(2011) The dysregulation of cytokine networks in systemic lupus erythematosus. J Interferon Cytokine Res 31: 769–779.

Aringer

Stummvoll

Steiner

Koller

Steiner

Hofler

. (2001) Serum interleukin-15 is elevated in systemic lupus erythematosus. Rheumatology (Oxford) 40: 876–881.

Awasthi

Carrier

Peron

Bettelli

Kamanaka

Flavell

. (2007) A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. Nat Immunol 8: 1380–1389.

Baechler

Batliwalla

Karypis

Gaffney

Ortmann

Espe

. (2003) Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci USA 100: 2610–2615.

Baltimore

Boldin

O’Connell

Rao

Taganov

(2008) MicroRNAs: new regulators of immune cell development and function. Nat Immunol 9: 839–845.

10.

Bartel

(2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.

11.

Basu

Liu

Yarlagadda

Gorelik

Kaplan

. (2009) Stimulatory and inhibitory killer Ig-like receptor molecules are expressed and functional on lupus T cells. J Immunol 183: 3481–3487.

12.

Batal

Liang

Bastacky

Kiss

McHale

Wilson

. (2012) Prospective assessment of C4d deposits on circulating cells and renal tissues in lupus nephritis: a pilot study. Lupus 21: 13–26.

13.

Bauer

Baechler

Petri

Batliwalla

Crawford

Ortmann

. (2006) Elevated serum levels of interferon-regulated chemokines are biomarkers for active human systemic lupus erythematosus. PLoS Med 3(12): e491.

14.

Bauer

Petri

Batliwalla

Koeuth

Wilson

Slattery

. (2009) Interferon-regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: a validation study. Arthritis Rheum 60: 3098–3107.

15.

Becker-Merok

Nikolaisen

Nossent

(2006) B-lymphocyte activating factor in systemic lupus erythematosus and rheumatoid arthritis in relation to autoantibody levels, disease measures and time. Lupus 15: 570–576.

16.

Bennett

Palucka

Arce

Cantrell

Borvak

Banchereau

. (2003) Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 197: 711–723.

17.

Biesen

Demir

Barkhudarova

Grun

Steinbrich-Zollner

Backhaus

. (2008) Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 58: 1136–1145.

18.

Bigler

Lopez-Trascasa

Potlukova

Moll

Danner

Schaller

. (2008) Antinucleosome antibodies as a marker of active proliferative lupus nephritis. Am J Kidney Dis 51: 624–629.

19.

Bird

(2007) Perceptions of epigenetics. Nature 447: 396–398.

20.

Bolignano

Donato

Coppolino

Campo

Buemi

Lacquaniti

. (2008) Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. Am J Kidney Dis 52: 595–605.

21.

Brunner

Bennett

Mina

Suzuki

Petri

Kiani

. (2012) Association of noninvasively measured renal protein biomarkers with histologic features of lupus nephritis. Arthritis Rheum 64: 2687–2697.

22.

Brunner

Mueller

Rutherford

Passo

Witte

Grom

. (2006) Urinary neutrophil gelatinase-associated lipocalin as a biomarker of nephritis in childhood-onset systemic lupus erythematosus. Arthritis Rheum 54: 2577–2584.

23.

Bruns

Blass

Hausdorf

Burmester

Hiepe

(2000) Nucleosomes are major T and B cell autoantigens in systemic lupus erythematosus. Arthritis Rheum 43: 2307–2315.

24.

Cameron

(1999) Lupus nephritis. J Am Soc Nephrol 10: 413–424.

25.

Carthew

R.W.

Sontheimer

E.J.

(2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136: 642–655.

26.

Cervera

Vinas

Ramos-Casals

Font

Garcia-Carrasco

Siso

. (2003) Anti-chromatin antibodies in systemic lupus erythematosus: a useful marker for lupus nephropathy. Ann Rheum Dis 62: 431–434.

27.

Chabre

Amoura

Piette

Godeau

Bach

Koutouzov

(1995) Presence of nucleosome-restricted antibodies in patients with systemic lupus erythematosus. Arthritis Rheum 38: 1485–1491.

28.

Chan

Lai

Tam

Chow

. (2006) The effect of immunosuppressive therapy on the messenger RNA expression of target genes in the urinary sediment of patients with active lupus nephritis. Nephrol Dial Transplant 21: 1534–1540.

29.

Cheema

Roschke

Hilbert

Stohl

(2001) Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum 44: 1313–1319.

30.

Chen

Wang

(2012) Anti-C1q antibody is a valuable biological marker for prediction of renal pathological characteristics in lupus nephritis. Clin Rheumatol 31: 1323–1329.

31.

Chun

Chung

Kim

Yun

Jeon

. (2007) Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus. J Clin Immunol 27: 461–466.

32.

Cohen

Koopmans

Kremer Hovinga

Berger

Roos van Groningen

Steup-Beekman

. (2008) Potential for glomerular C4d as an indicator of thrombotic microangiopathy in lupus nephritis. Arthritis Rheum 58: 2460–2469.

33.

Cornacchia

Golbus

Maybaum

Strahler

Hanash

Richardson

(1988) Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 140: 2197–2200.

34.

Crispin

Keenan

Finnell

Bermas

Schur

Massarotti

. (2010a) Expression of CD44 variant isoforms CD44v3 and CD44v6 is increased on T cells from patients with systemic lupus erythematosus and is correlated with disease activity. Arthritis Rheum 62: 1431–1437.

35.

Crispin

Liossis

Kis-Toth

Lieberman

Kyttaris

Juang

. (2010b) Pathogenesis of human systemic lupus erythematosus: recent advances. Trends Mol Med 16(2): 47–57.

36.

Crispin

Oukka

Bayliss

Cohen

Van Beek

Stillman

. (2008) Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys. J Immunol 181: 8761–8766.

37.

Crispin

Tsokos

(2010a) IL-17 in systemic lupus erythematosus. J Biomed Biotechnol 2010: 943254.

38.

Crispin

Tsokos

(2010b) Interleukin-17-producing T cells in lupus. Curr Opin Rheumatol 22: 499–503.

39.

Crow

(2007) Type I interferon in systemic lupus erythematosus. Curr Top Microbiol Immunol 316: 359–386.

40.

Dai

Ahmed

(2011) MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl Res 157: 163–179.

41.

Dai

Huang

Tang

Tan

. (2007) Microarray analysis of microRNA expression in peripheral blood cells of systemic lupus erythematosus patients. Lupus 16: 939–946.

42.

Dai

Sui

Lan

Yan

Huang

(2009a) Comprehensive analysis of microRNA expression patterns in renal biopsies of lupus nephritis patients. Rheumatol Int 29: 749–754.

43.

Dai

Turtle

Booth

Riddell

Gooley

Stevens

. (2009b) Normally occurring NKG2D+CD4+ T cells are immunosuppressive and inversely correlated with disease activity in juvenile-onset lupus. J Exp Med 206: 793–805.

44.

Davis

Hutcheson

Mohan

(2011) The role of cytokines in the pathogenesis and treatment of systemic lupus erythematosus. J Interferon Cytokine Res 31: 781–789.

45.

Deapen

Escalante

Weinrib

Horwitz

Bachman

Roy-Burman

. (1992) A revised estimate of twin concordance in systemic lupus erythematosus. Arthritis Rheum 35: 311–318.

46.

Deng

Tsokos

(2008) Cholera toxin B accelerates disease progression in lupus-prone mice by promoting lipid raft aggregation. J Immunol 181: 4019–4026.

47.

Deng

Tsao

(2010) Genetic susceptibility to systemic lupus erythematosus in the genomic era. Nat Rev Rheumatol 6: 683–692.

48.

Dong

Chen

Lei

. (2010) Increased expression of ganglioside GM1 in peripheral CD4+ T cells correlates soluble form of CD30 in systemic lupus erythematosus patients. J Biomed Biotechnol 2010: 569053.

49.

Doreau

Belot

Bastid

Riche

Trescol-Biemont

Ranchin

. (2009) Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus. Nat Immunol 10: 778–785.

50.

Elkon

Wiedeman

(2012) Type I IFN system in the development and manifestations of SLE. Curr Opin Rheumatol 24: 499–505.

51.

Ettinger

Kuchen

Lipsky

(2008) The role of IL-21 in regulating B-cell function in health and disease. Immunol Rev 223: 60–86.

52.

Fabian

Sonenberg

Filipowicz

(2010) Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79: 351–379.

53.

Feng

Grossman

Hanvivadhanakul

FitzGerald

Park

. (2006) Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus. Arthritis Rheum 54: 2951–2962.

54.

Fields

Kim

Flavell

(2002) Cutting edge: changes in histone acetylation at the IL-4 and IFN-gamma loci accompany Th1/Th2 differentiation. J Immunol 169: 647–650.

55.

Flesher

Sun

Behrens

Graham

Criswell

(2010) Recent advances in the genetics of systemic lupus erythematosus. Expert Rev Clin Immunol 6: 461–479.

56.

Fraga

Ballestar

Paz

Ropero

Setien

Ballestar

. (2005) Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci U S A 102: 10604–10609.

57.

Gabay

McInnes

(2009) The biological and clinical importance of the ‘new generation’ cytokines in rheumatic diseases. Arthritis Res Ther 11: 230.

58.

Gao

Campbell

Burkly

Jakubowski

Jarchum

Banas

. (2009) TNF-like weak inducer of apoptosis (TWEAK) induces inflammatory and proliferative effects in human kidney cells. Cytokine 46: 24–35.

59.

Gomez-Puerta

Burlingame

Cervera

(2008) Anti-chromatin (anti-nucleosome) antibodies: diagnostic and clinical value. Autoimmun Rev 7: 606–611.

60.

Grootscholten

Dieker

McGrath

Roos

Derksen

van der Vlag

. (2007) A prospective study of anti-chromatin and anti-C1q autoantibodies in patients with proliferative lupus nephritis treated with cyclophosphamide pulses or azathioprine/methylprednisolone. Ann Rheum Dis 66: 693–696.

61.

Gutierrez-Adrianzen

Koutouzov

Mota

das Chagas Medeiros

Bach

de Holanda Campos

(2006) Diagnostic value of anti-nucleosome antibodies in the assessment of disease activity of systemic lupus erythematosus: a prospective study comparing anti-nucleosome with anti-dsDNA antibodies. J Rheumatol 33: 1538–1544.

62.

Harder

(2004) Lipid raft domains and protein networks in T-cell receptor signal transduction. Curr Opin Immunol 16: 353–359.

63.

Harigai

Kawamoto

Hara

Kubota

Kamatani

Miyasaka

(2008) Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B. J Immunol 181: 2211–2219.

64.

Herbst

Liu

Jallal

Yao

(2012) Biomarkers for systemic lupus erythematosus. Int J Rheum Dis 15: 433–444.

65.

Hinze

Suzuki

Klein-Gitelman

Passo

Olson

Singer

. (2009) Neutrophil gelatinase-associated lipocalin is a predictor of the course of global and renal childhood-onset systemic lupus erythematosus disease activity. Arthritis Rheum 60: 2772–2781.

66.

Hoffman

(2004) T cells in the pathogenesis of systemic lupus erythematosus. Clin Immunol 113: 4–13.

67.

Hooks

Moutsopoulos

Geis

Stahl

Decker

Notkins

(1979) Immune interferon in the circulation of patients with autoimmune disease. N Engl J Med 301: 5–8.

68.

Horak

Hermanova

Zadrazil

Ciferska

Ordeltova

Kusa

. (2006) C1q complement component and -antibodies reflect SLE activity and kidney involvement. Clin Rheumatol 25: 532–536.

69.

Horvath

Czirjak

Fekete

Jakab

Pozsonyi

Kalabay

. (2001) High levels of antibodies against Clq are associated with disease activity and nephritis but not with other organ manifestations in SLE patients. Clin Exp Rheumatol 19: 667–672.

70.

Qiu

Luo

Yuan

Lei

. (2008) Abnormal histone modification patterns in lupus CD4+ T cells. J Rheumatol 35: 804–810.

71.

Hughes

Sawalha

(2011) The role of epigenetic variation in the pathogenesis of systemic lupus erythematosus. Arthritis Res Ther 13: 245.

72.

Hughes

Webb

Fei

Wren

Sawalha

(2010) DNA methylome in human CD4+ T cells identifies transcriptionally repressive and non-repressive methylation peaks. Genes Immun 11: 554–560.

73.

Hung

Chen

Lan

Chen

. (2011) Antinucleosome antibodies as a potential biomarker for the evaluation of renal pathological activity in patients with proliferative lupus nephritis. Lupus 20: 1404–1410.

74.

Illei

Tackey

Lapteva

Lipsky

(2004a) Biomarkers in systemic lupus erythematosus. I. General overview of biomarkers and their applicability. Arthritis Rheum 50: 1709–1720.

75.

Illei

Tackey

Lapteva

Lipsky

(2004b) Biomarkers in systemic lupus erythematosus: II. Markers of disease activity. Arthritis Rheum 50: 2048–2065.

76.

Javierre

Fernandez

Richter

Al-Shahrour

Martin-Subero

Rodriguez-Ubreva

. (2010) Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res 20: 170–179.

77.

Jeffries

Dozmorov

Tang

Merrill

Wren

Sawalha

(2011) Genome-wide DNA methylation patterns in CD4+ T cells from patients with systemic lupus erythematosus. Epigenetics 6: 593–601.

78.

Jeffries

Sawalha

(2011) Epigenetics in systemic lupus erythematosus: leading the way for specific therapeutic agents. Int J Clin Rheumatol 6: 423–439.

79.

Juang

Peoples

Kafri

Kyttaris

Sunahori

Kis-Toth

. (2011) A systemic lupus erythematosus gene expression array in disease diagnosis and classification: a preliminary report. Lupus 20: 243–249.

80.

Jury

Flores-Borja

Kabouridis

(2007) Lipid rafts in T cell signalling and disease. Semin Cell Dev Biol 18: 608–615.

81.

Jury

Isenberg

Mauri

Ehrenstein

(2006) Atorvastatin restores Lck expression and lipid raft-associated signaling in T cells from patients with systemic lupus erythematosus. J Immunol 177: 7416–7422.

82.

Jury

Kabouridis

Flores-Borja

Mageed

Isenberg

(2004) Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus. J Clin Invest 113: 1176–1187.

83.

Kabouridis

Jury

(2008) Lipid rafts and T-lymphocyte function: implications for autoimmunity. FEBS Lett 582: 3711–3718.

84.

Kalunian

Chatham

Massarotti

Reyes-Thomas

Harris

Furie

. (2012) Measurement of cell-bound complement activation products enhances diagnostic performance in systemic lupus erythematosus. Arthritis Rheum 64: 4040–4047.

85.

Kaplan

Attwood

Richardson

(2004) Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. J Immunol 172: 3652–3661.

86.

Karageorgas

Tseronis

Mavragani

(2011) Activation of type I interferon pathway in systemic lupus erythematosus: association with distinct clinical phenotypes. J Biomed Biotechnol 2011: 273907.

87.

Kiani

Johnson

Chen

Diehl

Vasudevan

. (2009) Urine osteoprotegerin and monocyte chemoattractant protein-1 in lupus nephritis. J Rheumatol 36: 2224–2230.

88.

Kiani

Fang

Zhou

Ahn

Magder

. (2012) Urinary vascular cell adhesion molecule, but not neutrophil gelatinase-associated lipocalin, is associated with lupus nephritis. J Rheumatol 39: 1231–1237.

89.

Kirou

Lee

George

Louca

Peterson

Crow

(2005) Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum 52: 1491–1503.

90.

Korn

Bettelli

Oukka

Kuchroo

(2009) IL-17 and Th17 Cells. Annu Rev Immunol 27: 485–517.

91.

Koutouzov

Jeronimo

Campos

Amoura

(2004) Nucleosomes in the pathogenesis of systemic lupus erythematosus. Rheum Dis Clin North Am 30: 529–558.

92.

Krishnan

Nambiar

Warke

Fisher

Mitchell

Delaney

. (2004) Alterations in lipid raft composition and dynamics contribute to abnormal T cell responses in systemic lupus erythematosus. J Immunol 172: 7821–7831.

93.

Landolt-Marticorena

Bonventi

Lubovich

Ferguson

Unnithan

. (2009) Lack of association between the interferon-alpha signature and longitudinal changes in disease activity in systemic lupus erythematosus. Ann Rheum Dis 68: 1440–1446.

94.

Laurent

Wong

Huynh

Tsirigos

Ong

. (2010) Dynamic changes in the human methylome during differentiation. Genome Res 20: 320–331.

95.

Zhang

Chen

Zhu

Tao

Pan

. (2010) Low level of serum interleukin 27 in patients with systemic lupus erythematosus. J Investig Med 58: 737–739.

96.

Lin

Hsieh

Lin

Lee

Tsai

Lai

. (2012) A whole genome methylation analysis of systemic lupus erythematosus: hypomethylation of the IL10 and IL1R2 promoters is associated with disease activity. Genes Immun 13: 214–220.

97.

Liu

Manzi

Kao

Navratil

Ahearn

(2010) Cell-bound complement biomarkers for systemic lupus erythematosus: from benchtop to bedside. Rheum Dis Clin North Am 36: 161–172.

98.

Liu

Kuick

Hanash

Richardson

(2009) DNA methylation inhibition increases T cell KIR expression through effects on both promoter methylation and transcription factors. Clin Immunol 130: 213–224.

99.

Kaplan

Ray

Zacharek

Gutsch

Richardson

(2002) Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 46: 1282–1291.

100.

Richardson

(2005) Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs. J Immunol 174: 6212–6219.

101.

Tesmer

Ray

Yousif

Richardson

(2007) Demethylation of CD40LG on the inactive X in T cells from women with lupus. J Immunol 179: 6352–6358.

102.

Luger

Dechassa

Tremethick

(2012) New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell Biol 13: 436–447.

103.

Mackay

Schneider

(2009) Cracking the BAFF code. Nat Rev Immunol 9: 491–502.

104.

Manderson

Botto

Walport

(2004) The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 22: 431–456.

105.

Manolio

Collins

Cox

Goldstein

Hindorff

Hunter

. (2009) Finding the missing heritability of complex diseases. Nature 461: 747–753.

106.

Manson

Rogers

Mason

Berden

van der Vlag

. (2009) Relationship between anti-dsDNA, anti-nucleosome and anti-alpha-actinin antibodies and markers of renal disease in patients with lupus nephritis: a prospective longitudinal study. Arthritis Res Ther 11(5): R154.

107.

Marto

Bertolaccini

Calabuig

Hughes

Khamashta

(2005) Anti-C1q antibodies in nephritis: correlation between titres and renal disease activity and positive predictive value in systemic lupus erythematosus. Ann Rheum Dis 64: 444–448.

108.

Matrat

Veysseyre-Balter

Trolliet

Villar

Dijoud

Bienvenu

. (2011) Simultaneous detection of anti-C1q and anti-double stranded DNA autoantibodies in lupus nephritis: predictive value for renal flares. Lupus 20: 28–34.

109.

McKinney

Lyons

Carr

Hollis

Jayne

Willcocks

. (2010) A CD8+ T cell transcription signature predicts prognosis in autoimmune disease. Nat Med 16: 586–591, 581p following 591.

110.

Merrill

Wallace

Petri

Kirou

Yao

White

. (2011) Safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase I, multicentre, double-blind randomised study. Ann Rheum Dis 70: 1905–1913.

111.

Meyer

Nicaise-Roland

Cadoudal

Grootenboer-Mignot

Palazzo

Hayem

. (2009) Anti-C1q antibodies antedate patent active glomerulonephritis in patients with systemic lupus erythematosus. Arthritis Res Ther 11(3): R87.

112.

Min

Kim

Park

Seo

Kang

Kim

. (2002) Anti-nucleosome antibody: significance in lupus patients lacking anti-double-stranded DNA antibody. Clin Exp Rheumatol 20: 13–18.

113.

Mishra

Prada

Mitsnefes

Zahedi

Yang

. (2003) Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14: 2534–2543.

114.

Mitoma

Horiuchi

Kimoto

Tsukamoto

Uchino

Tamimoto

. (2005) Decreased expression of interleukin-21 receptor on peripheral B lymphocytes in systemic lupus erythematosus. Int J Mol Med 16: 609–615.

115.

Mohan

Adams

Stanik

Datta

(1993) Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus. J Exp Med 177: 1367–1381.

116.

Mok

(2010) Biomarkers for lupus nephritis: a critical appraisal. J Biomed Biotechnol 2010: 638413.

117.

Mok

Lau

(2003) Pathogenesis of systemic lupus erythematosus. J Clin Pathol 56(7): 481–490.

118.

Moore

Belvedere

Orr

Pieri

LaFleur

Feng

. (1999) BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285: 260–263.

119.

Moroni

Radice

Giammarresi

Quaglini

Gallelli

Leoni

. (2009) Are laboratory tests useful for monitoring the activity of lupus nephritis? A 6-year prospective study in a cohort of 228 patients with lupus nephritis. Ann Rheum Dis 68: 234–237.

120.

Moroni

Trendelenburg

Papa

Quaglini

Raschi

Panzeri

. (2001) Anti-C1q antibodies may help in diagnosing a renal flare in lupus nephritis. Am J Kidney Dis 37: 490–498.

121.

Moser

Kelly

Lessard

Harley

(2009) Recent insights into the genetic basis of systemic lupus erythematosus. Genes Immun 10: 373–379.

122.

Mosley

Tam

Edwards

Crozier

Pusey

Lightstone

(2006) Urinary proteomic profiles distinguish between active and inactive lupus nephritis. Rheumatology (Oxford) 45: 1497–1504.

123.

Moulton

Tsokos

(2011) Abnormalities of T cell signaling in systemic lupus erythematosus. Arthritis Res Ther 13: 207.

124.

Nardelli

Belvedere

Roschke

Moore

Olsen

Migone

. (2001) Synthesis and release of B-lymphocyte stimulator from myeloid cells. Blood 97: 198–204.

125.

Nath

Kilpatrick

Harley

(2004) Genetics of human systemic lupus erythematosus: the emerging picture. Curr Opin Immunol 16: 794–800.

126.

Nikpour

Dempsey

Urowitz

Gladman

Barnes

(2008) Association of a gene expression profile from whole blood with disease activity in systemic lupus erythematosus. Ann Rheum Dis 67: 1069–1075.

127.

Noris

Bernasconi

Casiraghi

Sozzani

Gotti

Remuzzi

. (1995) Monocyte chemoattractant protein-1 is excreted in excessive amounts in the urine of patients with lupus nephritis. Lab Invest 73: 804–809.

128.

O’Brien

Roark

Born

. (2009) IL-17-producing gammadelta T cells. Eur J Immunol 39: 662–666.

129.

Obermoser

Pascual

(2010) The interferon-alpha signature of systemic lupus erythematosus. Lupus 19: 1012–1019.

130.

Oelke

Richardson

Deng

Hanash

. (2004) Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum 50: 1850–1860.

131.

Ouyang

Kolls

Zheng

(2008) The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28: 454–467.

132.

Pan

Zhu

Yuan

Cui

Wang

Luo

. (2010) MicroRNA-21 and microRNA-148a contribute to DNA hypomethylation in lupus CD4+ T cells by directly and indirectly targeting DNA methyltransferase 1. J Immunol 184: 6773–6781.

133.

Pers

Daridon

Devauchelle

Jousse

Saraux

Jamin

. (2005) BAFF overexpression is associated with autoantibody production in autoimmune diseases. Ann N Y Acad Sci 1050: 34–39.

134.

Petri

Singh

Tesfasyone

Dedrick

Fry

Lal

. (2009) Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus. Lupus 18: 980–989.

135.

Petri

Stohl

Chatham

McCune

Chevrier

Ryel

. (2008) Association of plasma B lymphocyte stimulator levels and disease activity in systemic lupus erythematosus. Arthritis Rheum 58: 2453–2459.

136.

Pickering

Botto

(2010) Are anti-C1q antibodies different from other SLE autoantibodies? Nat Rev Rheumatol 6: 490–493.

137.

Pickering

Botto

Taylor

Lachmann

Walport

(2000) Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol 76: 227–324.

138.

Pitashny

Schwartz

Qing

Hojaili

Aranow

Mackay

. (2007) Urinary lipocalin-2 is associated with renal disease activity in human lupus nephritis. Arthritis Rheum 56: 1894–1903.

139.

Potlukova

Kralikova

(2008) Complement component c1q and anti-c1q antibodies in theory and in clinical practice. Scand J Immunol 67: 423–430.

140.

Rahman

Isenberg

(2008) Systemic lupus erythematosus. N Engl J Med 358: 929–939.

141.

Rauch

Zhong

Riggs

Pfeifer

(2009) A human B cell methylome at 100-base pair resolution. Proc Natl Acad Sci U S A 106: 671–678.

142.

Renaudineau

Youinou

(2011) Epigenetics and autoimmunity, with special emphasis on methylation. Keio J Med 60: 10–16.

143.

Richardson

Scheinbart

Strahler

Gross

Hanash

Johnson

(1990) Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum 33: 1665–1673.

144.

Ritterhouse

Crowe

Niewold

Merrill

Roberts

Dedeke

. (2011) B lymphocyte stimulator levels in systemic lupus erythematosus: higher circulating levels in African American patients and increased production after influenza vaccination in patients with low baseline levels. Arthritis Rheum 63: 3931–3941.

145.

Roh

Cuddapah

Zhao

(2005) Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping. Genes Dev 19: 542–552.

146.

Rose

Grutzkau

Hirseland

Huscher

Dahnrich

Dzionek

. (2012) IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus. Ann Rheum Dis, in press.

147.

Rovin

Doe

Tan

(1996) Monocyte chemoattractant protein-1 levels in patients with glomerular disease. Am J Kidney Dis 27: 640–646.

148.

Rovin

Song

Birmingham

Hebert

Nagaraja

(2005) Urine chemokines as biomarkers of human systemic lupus erythematosus activity. J Am Soc Nephrol 16: 467–473.

149.

Rovin

Zhang

(2009) Biomarkers for lupus nephritis: the quest continues. Clin J Am Soc Nephrol 4: 1858–1865.

150.

Rubinstein

Pitashny

Levine

Schwartz

Schwartzman

Weinstein

. (2010) Urinary neutrophil gelatinase-associated lipocalin as a novel biomarker for disease activity in lupus nephritis. Rheumatology (Oxford) 49: 960–971.

151.

Santer

Yoshio

Minota

Moller

Elkon

(2009) Potent induction of IFN-alpha and chemokines by autoantibodies in the cerebrospinal fluid of patients with neuropsychiatric lupus. J Immunol 182: 1192–1201.

152.

Sanz

Justo

Sanchez-Nino

Blanco-Colio

Winkles

Kreztler

. (2008) The cytokine TWEAK modulates renal tubulointerstitial inflammation. J Am Soc Nephrol 19: 695–703.

153.

Sarzi-Puttini

Atzeni

Iaccarino

Doria

(2005) Environment and systemic lupus erythematosus: an overview. Autoimmunity 38: 465–472.

154.

Schwartz

Michaelson

Putterman

(2007) Lipocalin-2, TWEAK, and other cytokines as urinary biomarkers for lupus nephritis. Ann N Y Acad Sci 1109: 265–274.

155.

Schwartz

Rubinstein

Burkly

Collins

Blanco

. (2009) Urinary TWEAK as a biomarker of lupus nephritis: a multicenter cohort study. Arthritis Res Ther 11(5): R143.

156.

Schwartz

Burkly

Mackay

Aranow

Kollaros

. (2006) Urinary TWEAK and the activity of lupus nephritis. J Autoimmun 27: 242–250.

157.

Shah

Lee

Kim

Kang

Craft

. (2010) Dysregulated balance of Th17 and Th1 cells in systemic lupus erythematosus. Arthritis Res Ther 12(2): R53.

158.

Shen

Liang

Tang

de Vries

Tak

(2012) MicroRNAs-novel regulators of systemic lupus erythematosus pathogenesis. Nat Rev Rheumatol 8: 701–709.

159.

Sinico

Rimoldi

Radice

Bianchi

Gallelli

Moroni

(2009) Anti-C1q autoantibodies in lupus nephritis. Ann N Y Acad Sci 1173: 47–51.

160.

Snowden

Gregory

Case

Pabo

(2002) Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr Biol 12: 2159–2166.

161.

Stagakis

Bertsias

Verginis

Nakou

Hatziapostolou

Kritikos

. (2011) Identification of novel microRNA signatures linked to human lupus disease activity and pathogenesis: miR-21 regulates aberrant T cell responses through regulation of PDCD4 expression. Ann Rheum Dis 70: 1496–1506.

162.

Stohl

Metyas

Tan

Cheema

Oamar

. (2003) B lymphocyte stimulator overexpression in patients with systemic lupus erythematosus: longitudinal observations. Arthritis Rheum 48: 3475–3486.

163.

Suzuki

Ross

Wiers

Nelson

Bennett

Passo

. (2007) Identification of a urinary proteomic signature for lupus nephritis in children. Pediatr Nephrol 22: 2047–2057.

164.

Suzuki

Wiers

Brooks

Greis

Haines

Klein-Gitelman

. (2009) Initial validation of a novel protein biomarker panel for active pediatric lupus nephritis. Pediatr Res 65: 530–536.

165.

Suzuki

Wiers

Klein-Gitelman

Haines

Olson

Onel

. (2008) Neutrophil gelatinase-associated lipocalin as a biomarker of disease activity in pediatric lupus nephritis. Pediatr Nephrol 23: 403–412.

166.

Tang

Luo

Cui

Yuan

Guo

. (2009) MicroRNA-146A contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins. Arthritis Rheum 60: 1065–1075.

167.

Dozmorov

Guthridge

Nguyen

Cavett

Kelly

. (2010) Identification of unique microRNA signature associated with lupus nephritis. PLoS One 5(5): e10344.

168.

Tian

Wang

Liu

Cheng

. (2007) Urinary levels of RANTES and M-CSF are predictors of lupus nephritis flare. Inflamm Res 56: 304–310.

169.

Trachtman

Christen

Cnaan

Patrick

Mai

Mishra

. (2006) Urinary neutrophil gelatinase-associated lipocalcin in D+HUS: a novel marker of renal injury. Pediatr Nephrol 21: 989–994.

170.

Trendelenburg

Lopez-Trascasa

Potlukova

Moll

Regenass

Fremeaux-Bacchi

. (2006) High prevalence of anti-C1q antibodies in biopsy-proven active lupus nephritis. Nephrol Dialysis Transplant 21: 3115–3121.

171.

Trendelenburg

Marfurt

Gerber

Tyndall

Schifferli

(1999) Lack of occurrence of severe lupus nephritis among anti-C1q autoantibody-negative patients. Arthritis Rheum 42: 187–188.

172.

Tsokos

(2011) Systemic lupus erythematosus. N Engl J Med 365: 2110–2121.

173.

Tucci

Barnes

Sobel

Croker

Segal

Reeves

. (2004) Strong association of a functional polymorphism in the monocyte chemoattractant protein 1 promoter gene with lupus nephritis. Arthritis Rheum 50: 1842–1849.

174.

Vila

Molina

Mayor

Cruz

Rios-Olivares

Rios

(2007) Association of serum MIP-1alpha, MIP-1beta, and RANTES with clinical manifestations, disease activity, and damage accrual in systemic lupus erythematosus. Clin Rheumatol 26: 718–722.

175.

Wada

Yokoyama

Mukaida

Iwano

Dohi

. (1996) Monitoring urinary levels of monocyte chemotactic and activating factor reflects disease activity of lupus nephritis. Kidney Int 49: 761–767.

176.

Wang

Tam

Kwan

Chow

Luk

. (2011) Serum and urinary cell-free MiR-146a and MiR-155 in patients with systemic lupus erythematosus. J Rheumatol 37: 2516–2522.

177.

Wang

Peng

Ouyang

Dai

(2012) Circulating microRNAs as candidate biomarkers in patients with systemic lupus erythematosus. Transl Res 160: 198–206.

178.

Watson

Beresford

(2012) Urine biomarkers in juvenile-onset SLE nephritis. Pediatr Nephrol, in press.

179.

Watson

Midgley

Pilkington

Tullus

Marks

Holt

. (2012) Urinary monocyte chemoattractant protein 1 and alpha 1 acid glycoprotein as biomarkers of renal disease activity in juvenile-onset systemic lupus erythematosus. Lupus 21: 496–501.

180.

Weckerle

Niewold

(2011) The unexplained female predominance of systemic lupus erythematosus: clues from genetic and cytokine studies. Clin Rev Allergy Immunol 40: 42–49.

181.

Williamson

Pinto

(2012) Histones and genome integrity. Front Biosci 17: 984–995.

182.

Wilson

Rowell

Sekimata

(2009) Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol 9: 91–105.

183.

Winfield

Shaw

Silverman

Eisenberg

Wilson

III Koffler

(1983) Intrathecal IgG synthesis and blood–brain barrier impairment in patients with systemic lupus erythematosus and central nervous system dysfunction. Am J Med 74: 837–844.

184.

Winkles

(2008) The TWEAK-Fn14 cytokine-receptor axis: discovery, biology and therapeutic targeting. Nat Rev Drug Discov 7: 411–425.

185.

Wong

Lam

(2000) Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 9: 589–593.

186.

Xiao

Rajewsky

(2009) MicroRNA control in the immune system: basic principles. Cell 136: 26–36.

187.

Yang

Hsieh

Tsai

. (2012) Urinary neutrophil gelatinase-associated lipocalin is a potential biomarker for renal damage in patients with systemic lupus erythematosus. J Biomed Biotechnol 2012: 759313.

188.

Yang

Chu

Yang

Gao

Zhu

Wan

. (2009) Th17 and natural Treg cell population dynamics in systemic lupus erythematosus. Arthritis Rheum 60: 1472–1483.

189.

Yao

Higgs

B.W.

Richman

White

Jallal

(2010) Use of type I interferon-inducible mRNAs as pharmacodynamic markers and potential diagnostic markers in trials with sifalimumab, an anti-IFNalpha antibody, in systemic lupus erythematosus. Arthritis Res Ther 12(Suppl. 1): S6.

190.

Ytterberg

Schnitzer

(1982) Serum interferon levels in patients with systemic lupus erythematosus. Arthritis Rheum 25: 401–406.

191.

Shao

Yue

Zhang

Guan

Wan

. (2011) Copy number variations of Interleukin-12B and T-bet are associated with systemic lupus erythematosus. Rheumatology (Oxford) 50: 1201–1205.

192.

Zhang

Roschke

Baker

Wang

Alarcon

Fessler

. (2001) Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J Immunol 166: 6–10.

193.

Zhang

Nagaraja

Nadasdy

Song

McKinley

Prosek

. (2012) A composite urine biomarker reflects interstitial inflammation in lupus nephritis kidney biopsies. Kidney Int 81: 401–406.

194.

Zhang

Kyttaris

Tsokos

(2009) The role of IL-23/IL-17 axis in lupus nephritis. J Immunol 183: 3160–3169.

195.

Zhang

Song

Maurer

Petri

Sullivan

(2010) Global H4 acetylation analysis by ChIP-chip in systemic lupus erythematosus monocytes. Genes Immun 11: 124–133.

196.

Zhao

Tang

Gao

Liang

Yin

. (2010a) Hypomethylation of IL10 and IL13 promoters in CD4+ T cells of patients with systemic lupus erythematosus. J Biomed Biotechnol 2010: 931018.

197.

Zhao

Wang

Liang

Zhao

Long

Ding

. (2011) MicroRNA-126 regulates DNA methylation in CD4+ T cells and contributes to systemic lupus erythematosus by targeting DNA methyltransferase 1. Arthritis Rheum 63: 1376–1386.

198.

Zhao

Tang

Cui

Wang

. (2010b) MicroRNA-125a contributes to elevated inflammatory chemokine RANTES levels via targeting KLF13 in systemic lupus erythematosus. Arthritis Rheum 62: 3425–3435.