Investigation of interleukin-6-driven STAT3 signalling in circulating lymphocytes of patients with early rheumatoid arthritis as a route to biomarker discovery

BackgroundRheumatoid arthritis is a chronic, debilitating, inflammatory, autoimmune disease caused by a breakdown in self tolerance. To treat the disease, rapid and accurate diagnosis is needed, which will be greatly aided by the identification of robust biomarkers. We recently identified a transcriptional signature in circulating CD4 T cells that predicted disease progression among patients attending a clinic for early arthritis. The signature appeared most prominently in a diagnostically challenging subgroup of individuals who were seronegative for anti-citrullinated peptide autoantibodies (ACPAs); in additon, the signature contained an over-representation of genes that regulate signal transducer and activator of transcription 3 (STAT3), whose expression in turn correlated with serum interleukin (IL) 6. We therefore sought an improved understanding of STAT3 signalling among immune cell subsets of an independent cohort of patients with early arthritis.Methods94 newly presenting patients, naive to immunomodulatory treatment (including steroids), were recruited from a clinic for early arthritis, and followed until diagnoses were confirmed. Basal and IL6-induced expression levels of pY705STAT3 (pSTAT3) were determined in T-cell and B-cell subsets using Phosflow, a flow-cytometry-based method for measuring intracellular phosphoproteins. Contemporaneous serum IL6, IL6R, and soluble gp130 concentrations were measured by immunoassay.FindingsBasal pSTAT3 concentrations were high in circulating CD4 T cells, but low in both CD8 T cells and B cells. Basal pSTAT3 expression correlated with serum IL6 most strongly in CD4 T cells (Spearman's r=0·74, p<0·01) and, to a lesser extent, CD8 T cells, but not B cells. The expected pSTAT3 induction after IL6 stimulation, recorded in all subsets, was most pronounced in CD4 T cells, and this reflected significantly higher basal IL6R surface expression in this cell population. When patients were categorised by diagnostic outcome, ACPA-negative patients had significantly higher basal pSTAT3 in CD4 T cells than did ACPA-positive patients, inflammatory non-rheumatoid arthritis patients, and non-inflammatory arthritis patients (p<0·05 for each comparison, Mann-Whitney U test). This pattern, which was not seen in CD8 T cells or B cells, corroborates our previous observations in respect of STAT3 target gene expression.InterpretationOur findings support a particular role for IL6-driven CD4 T-cell activation, primarily via STAT3, during the induction of rheumatoid arthritis. Since CD4 T cells preferentially express surface IL6R, we suggest that classic IL6 signalling (rather than trans signalling, which is not dependent on IL6R surface expression) has a crucial role in CD4 T-cell activation in early rheumatoid arthritis. Expression of pSTAT3 in CD4 T cells might be a biomarker for predicting progression to rheumatoid arthritis among ACPA-negative patients and could also be useful for predicting efficacy of therapies that target IL6 signalling.FundingPfizer, Academy of Medical Sciences.