Introduction
Rheumatoid arthritis (RA) is a chronic, autoimmune, and inflammatory polyarthritis that induces joint damage and disability. It is a heterogeneous disease with different clinical presentations and courses, ranging from mild to severe. Histological and molecular variations in synovial tissues were previously described among RA patients [
1,
2]. However, the few microarray studies available were conducted on human RA synovia from patients with long-standing (LS) disease and/or treated with disease-modifying antirheumatic drugs (DMARDs) and/or glucocorticoids [
2‐
4]. Moreover, many studies used osteoarthritis synovia as the control because of the difficulties in obtaining healthy synovial samples [
5‐
8]. The molecular differences observed in those comparisons did not precisely indicate the pathological processes involved in RA, especially if we consider that different biological processes are at work throughout the course of RA. The tight link between the molecular pattern and the disease stage was previously described in murine autoimmune arthritis but no data are available on human RA [
9]. Therefore, we applied gene-expression profiling to synovial biopsies from patients with early untreated or treated LS RA and control synovia to try to identify biological processes corresponding to the RA stages.
Discussion
A previous report [
2] already showed that RA is a heterogeneous disease with distinct molecular forms, but to date, few studies have focused on different RA stages [
17]. Herein, we showed, for the first time, that gene-expression profiling in synovia from patients with early untreated RA and those with treated LS RA revealed opposite gene regulations, suggesting the involvement of different pathophysiological mechanisms during the disease course. Moreover, the novel comparison of the gene-expression levels in early or LS RA with those observed in healthy (mechanical or traumatic lesions) synovia brings information that is more appropriate than that obtained with osteoarthritis synovia (used as the reference in most of the published studies). The synovial biopsies, taken from sites of active RA-associated synovitis, enabled us to examine RA molecular processes. In LS RA, because the biopsied joints still harbored active disease, it seems likely that the pathophysiology and molecular processes were not influenced by methotrexate or that, at worst, they were weakly influenced by methotrexate.
The small number of samples and the fact that we did not perform a pangenomic study limit the possibility of making any distinction between RA with and RA without anti-cyclic citrullinated peptide autoantibodies and thus of drawing putative pathophysiological conclusions. Indeed, the 12,000 cDNA probes covering our array were selected on the basis of tissue-preferred expression in liver and corresponded to genes with liver-restricted expression (10% of the probes) and genes with combined hepatic and broad expression (the other 90%). Moreover, preliminary comparison with a pangenomic array indicated that our array is able to detect more than two thirds of the genes from healthy synovia (data not shown). Because this restrictive procedure cannot measure every transcript expressed in the tissue, it is not intended to provide a genome-wide view of the RA-associated gene dysregulations. Nevertheless, our approach seems quite acceptable, as our major task here was a preliminary study of molecular profiles in early and LS RA versus control synovia.
These limitations aside, our observations identified several differences in ontological processes, thereby also suggesting several putative differences in the RA pathophysiological mechanisms as a function of disease progression. In addition, the distinct biological processes identified here according to RA stage are compatible with our knowledge of its pathophysiology. Indeed, the genes linked to the biological processes, referred to as MHC class II-mediated immunity, immunity and defense, and T cell-mediated immunity, which represent the hallmarks of immune system activation, were upregulated in early RA. The antibacterial response, T cell-mediated immunity, and macrophage-mediated immunity are also the main functional and biological processes involved at RA onset. Conversely, genes involved in the cell cycle, apoptosis inhibition, and granulocyte-mediated immunity were upregulated in LS RA, suggesting the involvement of a proliferative process. By identifying dysregulated genes in early and LS RA as compared with healthy synovia, we provide arguments that the biological processes, molecular functions, and pathways differed according to the stage.
However, the results of the comparisons, early or LS RA versus controls, seem to be contradictory for some biological processes (Tables
1 and
2) (Table S3 in Additional data file
3). For example, the genes involved in T cell-mediated immunity were upregulated in early versus LS RA (
HLAB,
HLADRB1,
CLEC4M, and so on) but were downregulated in early versus controls (
IFFG1,
LTB, and so on). These findings are not discordant, because the genes belonging to each class were different and did not overlap. Moreover, some processes/pathways are common to early and LS RA but the expressions of the genes belonging to these classes are subjected to opposite regulations. For instance, genes for protein biosynthesis, for nucleoside, nucleotide, and nucleic acid metabolism, and for ribosomal proteins were upregulated in early RA versus controls but were downregulated in LS RA versus controls. These opposite regulations could reflect disease processes involved during the different stages of disease. On the contrary, genes encoding metalloprotease inhibitor and salvage pyrimidine deoxyribonucleotide ontological classes were regulated similarly in early and LS RA. This overlap could reflect an RA-specific mechanism, regardless of the disease stage. But when we compared the gene sets involved in that mechanism and those dysregulated in RA versus previously published controls [
2,
17,
18], we found only three genes in common:
CEBPβ,
HCLS1, and
TIMP1.
Notably, the age differences among controls and early RA and LS RA patients might influence the results. Indeed, de Magalhães and colleagues [
19] reported that gene expression relative to inflammation, immune response, lysosome, energy metabolism, cell cycle, and cellular senescence could be affected by the aging process. Consequently, it is conceivable that differences observed herein could be explained, at least in part, by age rather than RA. This limitation is very difficult to avoid in clinical practice because restricting selection of RA patients to those of the same age but with different RA stages represents a real, if not impossible, challenge.
Previously, Olsen and colleagues [
20] performed a similar study on early and LS RA but they used peripheral blood mononuclear cells (PBMCs) from patients being treated with DMARDs or corticosteroids. The authors found that early RA was associated with a distinct gene-expression profile in PBMCs, with a signature that reflected an immune response to an unidentified infectious agent. Despite the differences in study designs, some families of genes (cytochrome
P450, zinc-finger protein, chemokine receptors, MHC class II, and
RAS oncogene family) were common to their study and ours. For example, the
S100A10 gene was downregulated in both early RA PBMCs and synovial tissues, whereas
HLA-DPA1,
B2M, and
ARHGDIB were dysregulated in early RA PBMCs and synovial tissues. These latter discrepancies might be explained by the differences between the tissues (PBMCs versus synovia) and the controls (vaccinated versus healthy donors) used in the two studies. Even though these two studies are not completely comparable, some of their findings converge, pointing to the involvement of stress responses and defense mechanisms in early RA, and are therefore complementary, contributing to a better understanding of the different RA stages.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TL had full access to all of the study data and takes responsibility for the integrity of the data and the accuracy of the data analysis. He helped conceive of the study and participated in its design and coordination and helped perform or collect the synovial biopsies and clinical data, analyze and interpret biological data, draft the manuscript, and perform the statistical analyses. OV and JPS helped conceive of the study and participated in its design and coordination and helped analyze and interpret biological data and draft the manuscript. XLL helped conceive of the study and participated in its design and coordination and helped draft the manuscript. MH, XA, NB, IAA, and GC helped perform or collect the synovial biopsies and clinical data. CB and CD helped analyze and interpret biological data, draft the manuscript, and perform the statistical analyses. FT helped analyze and interpret biological data. MD helped draft the manuscript. All authors read and approved the final manuscript.