In this study, we evaluated gene expression patterns in ACPA-positive, MTX-naïve eRA and assessed the roles of cytokines as biomarkers of clinical treatment response. ACPA-positive, MTX-naïve eRA was associated with upregulation of IFN-γ and IL-17A gene signatures. Moreover, among patients with ACPA-positive, MTX-naïve eRA or established RA with moderate to high disease activity, plasma IFN-γ levels correlated well with other inflammatory cytokines, including IL-12 and IL-17A, and with clinical disease activity. Notably, in patients with RA with low disease activity, plasma IFN-γ and IL-17A levels may enable to discriminate the patients who can tolerate drug tapering or achieve drug-free remission.
Over 100 genes have shown to be associated with RA , and many RA-associated risk factor genes are dominantly expressed in CD4+ T cells . Additionally, the gene enhancer region (H3K4me1) is uniformly overlapped with most effector and memory CD4+ T cells , highlighting the roles of CD4+ T cell in RA pathogenesis. In this study, we showed that IFN-γ- and IL-17A-associated genes were significantly increased in these patients, suggesting the potential roles of Th1 and Th17 cells in early stage RA pathogenesis. Interestingly, five upregulated genes in patients with ACPA-positive, MTX-naïve eRA were related to both IFN-γ and IL-17A signalling, and these genes may provide important insights into molecular targeting in RA.
IFN-γ plays crucial roles in primary immune defence against microbial infection and is considered a pro-inflammatory cytokine. However, IFN-γ has been shown to have both pro-inflammatory and anti-inflammatory activities under different biological contexts . Importantly, RA synovium exhibits increased IFN-γ expression , and IFN-γ+ CD4+ T cells are dominant in RA synovial fluid . In patients with RA, collagen type II reactive T cells produce more IFN-γ than IL-4, suggesting an autoreactive T-cell skew toward the Th1 phenotype . However, animal models of RA have shown conflicting results; some have suggested that IFN-γ induces arthritis , whereas others have demonstrated that IFN-γ has protective effects against arthritis [13, 14]. Furthermore, IFN-γ reduces osteoclast formation, which is essential for joint destruction in RA . After the discovery of Th17 cells, IFN-γ was found to suppress arthritis by regulating Th17 cell development [33, 34]. A recent study found that five IFN-related genes (MxA, IFI6, OAS1, ISG15, and IFI44L) were highly expressed in MTX-naïve RA compared with that in established RA, and this upregulation was correlated with disease activity and predicted treatment resistance in MTX-naïve RA . In the current study, OAS1 and IFI44L were also significantly increased in ACPA-positive, MTX-naïve eRA. Furthermore, increased CXCL10 levels in MTX-naïve eRA have been found to be correlated with disease activity . Similarly, we showed that plasma CXCL10 levels were significantly elevated in MTX-naïve eRA compared with that in established RA. Our findings suggested that IFN-γ-related genes could play important roles in RA pathogenesis, particularly during the early phase.
Th17 cells are the primary producers of IL-17 among all CD4+ T cells . Additionally, Th17 cells are now considered the main pathological cells in RA pathogenesis [36, 37]. IL-17 induces osteoclastogenesis , and Th17 levels are correlated with RA disease activity . Moreover, IL-17 is involved in the pathological processes of early or preclinical RA; IL-17 blocking agents show inferior therapeutic responses compared with other biologic DMARDs, including TNF-α inhibitors, in established RA . Patients with RA with higher proportions of circulating Th1 and Th17 cells show poor clinical responses, indicating the potential predictive roles of Th1 and Th17 in treatment response . Discontinuation or tapering of DMARD is a major goal of RA treatment; however, few predictors of this strategy have been identified . In the current study, IFN-γ- and IL-17A-associated genes and CXCL10 were significantly elevated in ACPA-positive, MTX-naïve eRA. Plasma IFN-γ and IL-17A levels were significantly elevated in ACPA positive MTX naïve eRA even in patients with a low inflammatory status. Additionally, plasma IFN-γ and IL-17A levels in established RA with low disease activity were associated with drug-free or dose reduction at 6 months. Thus, these cytokines may play crucial roles in early RA pathogenesis and may be useful for prediction of tapering or discontinuing RA medication.
Dual overexpression of IFN-γ- and IL-17A-related gene signatures is an interesting phenomenon. Th1 and Th17 signals typically suppress each other [43, 44]; however, in this study, both IFN-γ- and IL-17A-related genes were upregulated in ACPA-positive, MTX-naïve eRA. This result could be explained by several hypotheses. First, Th1 and Th17 signals could both be upregulated and both suppress the other, and this could be described as “sleeping with enemy”. In vitro, autoreactive PTM peptide promotes Th17 induction in RA [45, 46]. Moreover, autoreactive T-cell responses to type II collagen show skewing toward Th1 . In animal models, collagen injection induces Th1 polarisation , and STAT3 inhibition ameliorates arthritis severity by reducing the Th17 population in a collagen-induced arthritis (CIA) model . In addition, type II collagen-stimulated CD4+ T cells from patients with RA exhibit increased IFN-γ and IL-17 production . Theoretically, PTM autoreactive peptide can induce both Th1 and Th17 dominant responses. IFN-γ suppresses Th17 differentiation in CIA and experimental autoimmune encephalitis by inducing IDO in APCs or via suppressor of cytokine signalling (SOCS) [33, 50]. SOCS has several subtypes and acts as an intracellular regulator for the Janus kinase (JAK)/STAT pathway. SOCS1 is critical a negative regulator of IFN-γ/STAT1 signalling and suppresses Th1 differentiation . Furthermore, SOCS1 simultaneously enhances Th17 differentiation via STAT3 signalling, and SOCS3 modulates Th1/Th17 responses with SOCS1 . Interestingly, IFN-γ also accelerates Foxp3+ regulatory T cell (Treg) induction and enhances the regulatory functions of Tregs . The protective roles of Tregs on systemic autoimmunity have been extensively studied, and Tregs have been shown to reduce Th17-mediated autoimmune responses . In contrast, IL-17 downregulates Th1 induction via IL-12Rβ2 suppresion , and the Th17-inducing transcription factor STAT3 blocks IL-12/p35 gene expression, thereby suppressing the Th1 response . Citrullinated peptide reacts with the HLA-DRβ shared epitope and stimulates CD4+ T-cell responses; therefore, these PTM peptide-mediated CD4+ T-cell responses may be only observed in ACPA-positive RA. We hypothesise that initially enhanced Th1 and Th17 responses induced by the PTM peptide could counteract each other, particularly during the early stages of ACPA-positive RA pathogenesis. Second, recent studies have shown that some Th17 cells can produce IFN-γ . These IFN-γ-positive Th17 cells are increased in the synovial fluid of patients with juvenile inflammatory arthritis , suggesting the pathological roles of this Th17 subset in inflammatory arthritis. Our study demonstrated the importance of IFN-γ and IL-17A in ACPA-positive eRA and highlighted the roles of these cytokines in drug-free remission. However, further studies are needed to determine the specific mechanisms of ACPA-positive eRA pathogenesis.
MCAs have several advantages compared with conventional ELISA. Because the minimal detectable range of MCA is lower than that of ELISA, MCA can be used to identify relatively low levels of cytokines in blood, and detect multiple cytokines at the same time using the same sample. MCA using antibody capture with magnetic beads and razor detection can be used to measure multiple cytokines simultaneously and can detect these cytokines at levels close to the physiological concentration . In our study, combinations of 5 cytokines (IFN-γ, IL-6, IL-17, TNF-α, IL-12) can detect the stable reference ranges of the same analytes and enable to determine the precise concentrations of these cytokines in patients with ACPA-positive, MTX-naïve eRA.
There were several limitations to the current study. First, the microarray data for ACPA-positive, MTX-naïve eRA were obtained from a relatively small sample size. However, our results were consistent with a previous study of MTX-naïve eRA . Second, the follow-up duration for patients with established RA with low disease activity was only 6 months. Third, all patients were seropositive RA; therefore, the results from the current study cannot be generalised to seronegative RA.