Rituximab treatment induces the expression of genes involved in healing processes in the rheumatoid arthritis synovium

Objectives: In previous experiments, we performed transcriptomic analyses on synovial biopsies obtained from anti-TNF resistant rheumatoid arthritis (RA) patients prior to (T0) and 12 weeks (T12) after initiation of rituximab therapy. We found that genes down-regulated between T0 and T12 were significantly enriched in immunoglobulin genes, and genes involved in chemotaxis, leucocyte activation and immune responses. By contrast, genes up-regulated between T0 and T12 were significantly enriched in transcripts involved in cell development and wound healing. We wondered whether up-regulation of the latter genes was a true effect of rituximab therapy, or whether the relative decrease in inflammatory cells and relative increase in resident synovial fibroblasts resulted in a biased appreciation of global gene expression profiles at T12. Methods: Paired synovial biopsies were obtained from the affected knee of 20 anti-TNF resistant RA patients at T0 and T12. Fresh synovial biopsy tissue samples were fixed overnight in 10% formalin buffer and embedded in paraffin. Immunolabeling experiments were performed using the following antibodies: BMPR1A (Bone Morphogenetic Protein Receptor 1A), MEOX2 (Mesenchyme Homeobox2), LAMA4 (Laminin alpha 4), phospho-SMAD1/5 and phospho-SMAD3. Quantitative analysis of the antibody immunostained sections was performed using ImageJ software, according to the Digital Image Analysis process. Results: BMPR1A, MEOX2 and LAMA4 are markers of mesenchymal cell differentiation. Immunolabeling experiments and digital quantification of the slides indicated that synovial expression of the three molecules is significantly induced by rituximab therapy in RA synovial tissue. Up-regulation of these genes triggered us to investigate whether administration of rituximab resulted in synovial activation of TGF-β or BMP signaling. To explore this hypothesis, we performed phospho-SMAD3 (a marker of TGF-β activation) and phospho-SMAD1/5 (a marker of BMP activation) immunohistochemistry studies. No significant difference in phospho-SMAD3 staining was observed between T0 and T12. The phospho-SMAD1/5 signal significantly decreased between T0 and T12. These results indicate that synovial up-regulation of genes involved in healing processes is not associated with an increased TGF-β or BMP activity through its canonical pathways at T12. Conclusion: Rituximab induces the expression of molecular markers of mesenchymal cell differentiation in the synovium. We did not find evidence of increased TGF-β and BMP signalling in the synovium at T12. Further studies are needed in order to investigate the possibility of increased BMP or TGF-β signaling at earlier time-points or the presence of alternative mechanisms of induction of mesenchymal cell differentiation.