Quantifying circulating Th17 cells by qPCR: potential as diagnostic biomarker for rheumatoid arthritis.

OBJECTIVE: The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. METHODS: We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. RESULTS: In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P<0.0001). Regression models demonstrated a high predictive value for RA development (75.8% correct prediction), and particularly for the ACPA-negative group (n = 125) where Th17 and swollen joint count (SJC) were the only predictors (73% correct prediction). The chemokine receptor CXCR4 had significantly higher expression on Th17 from early RA patients (n = 11) compared with HC (n = 15). CONCLUSION: The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.

Heterogeneity of the cytokinome in undifferentiated arthritis progressing to rheumatoid arthritis and its change in the course of therapy. Move toward personalized medicine.

OBJECTIVES: To conduct a comprehensive analysis of cytokine concentrations in sera and mononuclear cell supernatants in order to examine inter- and intra-individual cytokine variations in undifferentiated arthritis progressing to rheumatoid arthritis and healthy control groups. METHODS: Patients with UA (undifferentiated arthritis) developing RA (rheumatoid arthritis) (UA→RA) (n=16) and healthy controls (n=16) were enrolled into the study. UA→RA patients were followed up for six months since the final RA diagnosis. Cytokines IFN-γ, IL-10, TNF, IL-17A, IL-6, IL-1ß, IL-2 in sera and mononuclear cell supernatants in 72h and 120h culture variants with- and without anti-CD3 stimulations were assayed using flow cytometric bead array. RESULTS: The cytokine profile of UA→RA differs from the healthy individual cytokine profile. It is possible to observe specific cytokine pattern characterizing each patient, which alters during course of disease. Specifically, we can distinguish three UA→RA cohorts: the group of patients susceptible to the therapy, characterized by the drop of cytokine levels between 1st and 3rd visit with visible decrease of cytokines in 2nd visit and then secondary slighter increase in 3rd visit; the group of patients refractory or clinically worsening on the therapy, characterized by the highest cytokine levels at 2nd visit with secondary decrease in 3rd visit; and the group of patients with variable responses to the therapy without any specific common cytokine pattern. The cytokine patterns in supernatants of PBMC stimulated anti-CD3 for 72h and 120h are very similar. CONCLUSIONS: The personal profile including multiplexed cytokine patterns in serum and supernatant may be potentially used for optimization of therapy introduction and monitoring.