Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients

Abstract

We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-alpha status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log2 fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-alpha was found and nonresponders, indicating that TNF-alpha could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-alpha was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3 and CXCL14). Subsequent Gene Ontology analysis revealed that several 'themes' were significantly over-represented that are known to be affected by anti-TNF treatment in inflammatory tissue; for example, immune response (GO:0006955), cell communication (GO:0007154), signal transduction (GO:0007165) and chemotaxis (GO:0006935). No genes reached statistical significance in the moderately responding or nonresponding groups. In conclusion, this pilot study suggests that further investigation is warranted on the usefulness of gene expression profiling of synovial tissue to predict and monitor the outcome of rheumatoid arthritis therapies.

Figure 1

Hierarchical cluster dendrogram of the biopsies taken before treatment. A hierarchical cluster dendrogram of the 876 genes with M values (log₂ fold change) with interquartile range > 1 in the first series of hybridizations. The clinical response of each patient is given in the plot (European League Against Rheumatism response criteria). pat, patient; cc, close to cartilage; ncc, not close to cartilage; biopsy 1 or 2 for each biopsy type, technical replicate A or B.

Figure 2

Volcano plots displaying differential expression. The y axis corresponds to the negative false discovery rate on a log₁₀ scale. The x axis displays the M value (log₂ fold change). Differential expression (proportion of false positives < 0.025): (a) between TNF-positive patients and nonresponders (685 differentially expressed (DE) genes) before treatment, (b) before and after treatment in the good responders (115 DE genes), (c) before and after treatment in the moderate responders (0 DE genes), (d) due to treatment in the nonresponders (no DE genes), (e) before and after treatment in all patients (974 DE genes), and (f) before and after treatment in the TNF-positive group of patients (1058 DE genes). TNF-α is highlighted in (e) and (f). FDR, false discovery rate.

Figure 3

Gene Ontology analysis of the genes differentially expressed before and after treatment in the TNF-positive group of patients. The 20 most significant biological processes (Gene Ontology categories) and their parent terms are shown. The color of each node illustrates significance and can be interpreted in the scale bar, which displays the false discovery rate on a log₁₀ scale. The arrows indicate the parent to child direction. The numbers of each arrow demonstrate the amount of unique Entrez Gene IDs [57] among the DE genes that are mapped to the parent term, and the number of unique Entrez Gene IDs among all genes present on the chip that are mapped to the parent term.

Figure 4

Real-time PCR results. (a) Real-time results for four genes in the first series of hybridizations comparing TNF-positive patients and nonresponders. (b) Real-time results for nine genes in the second series of hybridizations in the TNF-positive patients. The whiskers display the standard deviations in both (a) and (b).