Identification of intra-group, inter-individual, and gene-specific variances in mRNA expression profiles in the rheumatoid arthritis synovial membrane

Human rheumatoid arthritis (RA) is characterized by chronic inflammation and destruction of multiple joints, perpetuated by an abnormally transformed and invasive synovial membrane (SM), forming the so-called pannus tissue [1]. Many activated cell types contribute to the development and progression of RA. Monocytes/macrophages, dendritic cells, T and B cells, endothelial cells, and synovial fibroblasts are major components of the pannus [2‐8] and participate in maintaining joint inflammation, degradation of extracellular matrix (ECM) components, and invasion of cartilage and bone [2, 4] as well as fibrosis of the affected joints [9].

The extended analysis of gene expression profiles in RA SM during the last decades has revealed several relevant gene groups affecting development and progression of the disease. Central transcription factors involved as key players in RA pathogenesis are AP-1, NF-κB, Ets-1, and SMADs [10‐12]. These factors show binding activity for their cognate recognition sites in the promoters of inflammation-related cytokines (for example, tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6 [3]) and matrix-degrading enzymes (for example, matrix metalloproteinase [MMP]-1 and MMP-3 [13, 14]). The latter contribute to tissue degradation by destruction of ECM components, including aggrecan or collagen type I-IV, X, and XI [15].

The analysis of those comprehensive expression data has become feasible due to the implementation of microarray-based methods [16]. Therefore, a variety of comparisons can be performed, including differences in gene expression among different groups and/or individuals. In contrast to conventional differential gene expression analyses, the determination of inter-individual gene expression variances, often affecting gene expression of members of the same patient/donor group, is generally not considered in rheumatology, although those variances are known to be a characteristic of many diseases. In trisomy 21, for instance, inter-individual expression variances affect a number of tightly regulated genes. In addition, the variances are independent of the respective level of gene expression, and although only a minority of genes are affected, these genes are thought to be involved in the symptoms of trisomy 21 with the highest phenotypical differences [17]. Significant inter-individual expression variances have also been reported to affect the expression of telomerase subunits in malignant glioma [18] as well as protein tyrosine kinases and phosphatases in human basophils in asthma and inflammatory allergy [19]. The latter implies that such alterations may also play an important role within inflammatory diseases, reflected in either synchronization (that is, a loss of inter-individual gene expression variances) or desynchronization (that is, increased inter-individual gene expression variances) of gene expression within a group of different individuals/patients.

In RA, differences in gene expression profiles for specific genes among two subgroups of RA patients have been reported, but within these subgroups, the differences are limited to distinct expression levels without significant intra-subgroup expression variances [12]. To the best of our knowledge, there are as yet no reports on broad intra-group inter-individual gene expression variations among RA patients.

Interestingly, although the majority of reports show expression variances in tissues from patients with different diseases, variances have also been reported in normal tissues (for example, the human retina [20] or human B-lymphoblastoid cells [21]). In contrast to expression variations in diseases, the variations in normal donors are generally limited to a small number of genes (for example, 2.6% in the human retina [20]). To analyze inter-individual mRNA expression variances in RA, the occurrence of gene-specific expression differences in the SM was analyzed using the Bonferroni/Holm corrected Brown-Forsythe version of the Levene test for variance analysis [22‐24] on the basis of genome-wide mRNA expression data in RA (n = 12), osteoarthritis (OA) (n = 10), and normal control (NC) (n = 9) synovial tissue.

The present microarray-based and real-time RT-PCR-validated, genome-wide mRNA expression analysis in RA, OA, and NC SM by KEGG mapping shows that gene-specific, significant, intra-group/inter-individual variances in gene expression profiles occur in RA. These variances affect a variety of genes involved in numerous pathways/complexes potentially relevant for RA pathogenesis. Since significant variance-fold values are observed for many genes with comparable mean expression levels among different patient/donor groups (data not shown), the manifestation of gene expression variances does not necessarily depend on the respective mean mRNA expression level.

To our knowledge, gene expression variances in RA samples have been reported only for distinct subgroup-specific differences in gene expression profiles of RA patients [12]. Consequently, the present data demonstrate for the first time broad intra-group/inter-individual gene expression variances in RA SM samples, previously observed in other severe diseases such as trisomy 21, malignant glioma, and inflammatory allergy [17‐19]. It has been hypothesized that expression variances of regulatory key genes contribute to the individual phenotype of the given disease [17], whether independent of or depending on the expression level.

Currently, the causes for gene expression variances among RA patients are unknown. Possible external reasons may include the higher average age of the individuals in the RA group as well as medication influencing immunological processes and the expression of immunologically relevant genes (for example, methotrexate, prednisolone, sulfasalazine, and/or nonsteroidal anti-inflammatory drugs [35, 36]) or differences in nutrition, with general effects on individual gene expression [37]. The inflammatory status of the respective joint at the time of surgical intervention may also substantially influence gene expression in the RA SM [38]. However, an analysis of the differential gene expression shows that the present RA group is generally characterized by an expression profile highly compatible with previous gene expression studies [39], including the overexpression of several transcription factors (for example, FOS, FOSB, JUN, and STAT1 [10‐12]), cytokines/chemokines (for example, IL2, IL4, CCL23, and CCL25 [40]), signal transduction molecules (for example, MAPK9, MAP3K2, PTPN7, and AKT2 [41, 42]), cell cycle regulators (for example, CDC12, CCNB2, and CCNE2 [43]), and heat shock proteins (DNAJ molecules; [44]; data not shown), indicating that the present RA cohort is representative for RA patients in general.

Regarding internal molecular changes in the individuals, a participation of mutations or single nucleotide polymorphisms in different genes is plausible, either directly [45, 46] or via mutated regulators (for example, transcription factors, mRNA stability modifiers, and so on [47]). This also includes broader genomic rearrangements (for example, chromosomal translocations or polysomies [48, 49]) as well as epigenomic modifications (for example, gene/promoter methylation [50]). In addition, the individual composition of cell types in the analyzed SM samples may influence the mRNA expression profile, depending on the inflammatory status and/or cell proliferation, potentially resulting in enhanced immigration/proliferation of T cells, B cells, or synovial fibroblasts [51].

In RA compared with NC, 10 KEGG pathways/complexes are specifically and significantly affected by gene expression variances. As expected, the importance of immunological processes for RA progression [8] is reflected in several pathways directly involved in such networks (Toll-like, T cell, and Fc ε receptor signaling [52‐54]). In the SM, alterations in immunological pathways/complexes may contribute to the development of local (and systemic) inflammation, reflecting the highly inflamed status of the joint as one of the major characteristics of RA [2, 55].

RA-specific gene expression variances also occur in cytokine–cytokine receptor interactions. Within this complex, a striking involvement of sub-pathways can be observed, with relevance for chemotaxis (CXC family chemokines [56]), angiogenesis, proliferation, and cell survival (TGF-β family [57, 58]) as well as inflammation, joint destruction, and fibrosis (TNF family [59, 60] and IL2RG shared pathway [9, 61]; Figure 2). Sub-pathways influencing tissue protection (interferon family [62]) or anti-inflammation and anti-angiogenesis (IL13RA1 [interleukin-13 receptor alpha-1] shared pathway [63]) are scarcely affected. Therefore, a specific influence of gene expression variances on cytokine-mediated aspects of the RA can be assumed [64].

Although the following pathways/complexes are not significantly affected by gene expression variances in total, embedded sub-pathways include the majority of affected genes, thus reaching statistical significance. In the TGF-β pathway, only members of the classical TGF-β sub-pathway are significantly affected, thus potentially influencing angiogenesis [58], cell survival [65], and cell proliferation [66] amongst others (Figure 3). Indeed, this (sub-) pathway appears to occupy a central position for the RA pathogenesis, due to the integration of various RA-relevant cellular functions. This is further underlined by its prominent role within the framework of cytokine–cytokine receptor interactions (Figure 2) and its influence on pro-inflammatory/pro-destructive features, either independent of or via MAPK (Figures 3 and 4). Within the MAPK signaling pathway, the 'classical' and the JNK/p38 MAPK sub-pathways – regulating proliferation, anti-apoptosis, and inflammation – are significantly affected by gene expression variances (Figure 4). This may be an indication of a participation of variable gene expression in inflammatory processes via MAPK variants (especially via JNK/MAPK8 [67]) and proliferation of activated cells (for example, synovial fibroblasts and T cells) in RA [68, 69] and MAPK-mediated anti-apoptosis (Figure 4).

Regarding apoptosis, genes particularly involved in the regulation of cell survival and anti-apoptosis are significantly affected by expression variances (Figure 5) [70]. Interestingly, the respective genes in this particular pathway also show increased expression levels in RA SM (data not shown). Pro-apoptotic genes are not affected in this pathway, corresponding to the absence of gene expression variances within the complex of p53-induced apoptosis (data not shown).

Depending on the individual gene expression level in each patient, gene expression variances in regulatory pathways may lead to enhanced inflammation [53, 54], angiogenesis [71, 72], enhanced collagen synthesis and secretion [9], and/or a reduced rate of apoptosis [73], thus potentially contributing to hyperplasia of the SM [74], collagen-dependent fibrosis of the joints [64], and a prolonged life span of activated synovial cells in RA [73, 75].

Since RA and OA samples share many aspects of their respective mRNA expression profiles [76, 77], genes in a number of pathways show comparable variance-fold values in both RA and OA (for example, apoptosis; Tables 2 and 4), thus reflecting basic similarities of joint diseases. However, RA and OA SM samples can be clearly differentiated regarding gene expression variances in other pathways/complexes. In OA, the pathways/complexes affected by higher expression variances than in NC indicate an OA-specific desynchronization of metabolic processes (Table 7). In contrast, RA-specific pathways/complexes are involved in the regulation of VEGF-mediated angiogenesis [74, 75] and vascular permeability [78], as well as B cell-dependent auto-immunity and inflammation [79]. The latter represents the elevated activity status of B cells (including cytokine production and T-cell activation) and – in connection with the affection of the anti-apoptotic sub-pathway – the enhanced survival of self-reactive B cells [5, 6, 80]. This may result in a pronounced role of B cells for disease development in RA compared with OA, which is also reflected in the increasing impact of B cell-directed treatment in RA [81].

In summary, these pathways indicate limited but distinct molecular/cellular differences between RA and OA and demonstrate a major contribution of inflammation and angiogenesis in RA. It is reasonable to assume that the RA pathogenesis is influenced by broad alterations of gene expression in general. For years, only differential gene expression analyses have been performed, resulting in the identification of some key genes but leading to the disregard of several genes with a more limited influence on RA, whose collective influence may still be as large as that of the already-known key players. Therefore, besides ubiquitous elevated expression levels of exceptional pro-inflammatory/pro-destructive key regulators/mediators like TNF-α, IL-1β [82], or MMP-1 [83], elevated or reduced expression levels of many different genes in various pathways/complexes may also influence RA development and progression. In this process, the affection of pathologically relevant pathways with differentially expressed genes may be more important than the character of the respective genes, resulting in different gene expression profiles among individual RA patients as reflected in the gene expression variances of the present study. As a consequence, synchronized or desynchronized gene expression in RA potentially shifts cellular activity from the normal to an activated status.

Regarding diagnosis and therapy of RA, the present results indicate that a more individualized approach for different patients may represent the future of RA treatment. Thus, the determination of individual gene expression patterns may facilitate the selection of the best medication or, more ambitiously, may allow directed modulation of (individually) selected pathways/complexes instead of broad suppression of inflammation by anti-inflammatory/anti-rheumatic drugs [84]. In addition, the present study helped to identify the TGF-β pathway as an accessory key player in RA, due to its central position within the regulatory networks. This suggestion is strongly supported by an emerging number of publications reporting a decisive impact of TGF-β on RA development/progression [57, 58, 85, 86]. The affected pathways (and the respective genes) reported here may provide the basis for further analyses of the RA pathogenesis and the differences between RA and OA on a cellular and molecular level.

In RA, a number of disease-relevant or even disease-specific KEGG pathways/complexes (for example, TGF-β signaling and anti-apoptosis) are characterized by broad intra-group inter-individual expression variances. This indicates that RA pathogenesis in different individuals may depend to a lesser extent on common alterations of the expression of specific key genes, and rather on individual-specific alterations of different genes resulting in common disturbances of key pathways. Numerous affected pathways, including TGF-β signaling in a central position, are involved in inflammation, angiogenesis, proliferation, and cell survival, thus potentially influencing characteristic features of RA pathology.