Expression of STAT3-regulated genes in circulating CD4+ T cells discriminates rheumatoid arthritis independently of clinical parameters in early arthritis

Abstract

Objectives: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance.

Methods: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis.

Results: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response.

Conclusion: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

Keywords: T lymphocytes; gene expression; rheumatoid arthritis.

Fig. 1

Expression of STAT3-regulated genes in circulating CD4+ lymphocytes of an independent early arthritis cohort (A–C) Normalized gene expression of three STAT3-regulated genes in circulating CD4+ T cells of an independent early arthritis cohort of 161 patients presenting with RA or other arthritides (Non-RA). Mann–Whitney U tests used to determine P-values; FC denotes fold-change. (D–F) Bivariate correlation between the same genes’ normalized expression and pSTAT3 measurements in paired circulating CD4+ T cells of early arthritis clinic attendees. Spearman’s Rho correlation coefficients and associated P-values are depicted. STAT3: signal transduction and activator of transcription-3; pSTAT: phospho-STAT3.

Fig. 2

Discriminatory utility of a proposed CD4+ lymphocyte signature is primarily accounted for by three genes (A) Dendrogram and heat map depicting results of hierarchical clustering of 279 early arthritis patients (columns) according to normalized expression of 12 signature genes in circulating CD4+ T cells (rows). Red dotted line identifies RA-enriched cluster highlighted in text. (B) Analogous result as for (A), based on normalized expression levels of three-gene signature only. (C) Scatterplots overlaid with non-parametric density plots separate RA patients and non-RA patients based on normalized expression alone, such that the two populations in each case preferentially occupy the top right and bottom left quadrants, respectively.

Fig. 3

CD4+ lymphocyte gene signatures add independent diagnostic value to clinical parameters Receiver-operating characteristic curves depicting the extent to which consideration of the 12-gene and three-gene signatures (blue and green lines, respectively) add independent discriminatory value to five clinical parameters (age, swollen and tender joint count, CRP and ESR; red line), with respect to a diagnosis of RA vs non-RA (see text). AUC: area under curve.