Clinical Impact of Interferon-γ in Anti-Citrullinated Protein Antibody-Positive Rheumatoid Arthritis

Background: CD4 + T cells are crucial for the pathogenesis of rheumatoid arthritis (RA). The roles of gene overexpression in CD4 + T cells and the predictive roles of Th1- and Th17-related cytokines have not been clearly dened in patients with RA. Here, we investigated gene expression levels in CD4 + T cells in methotrexate (MTX)-naïve early RA (eRA) and evaluated changes in CD4 + T-cell-related cytokines during eRA. Methods: Patients with anti-citrullinated protein antibody (ACPA)-positive MTX-naïve eRA were recruited. Patients with osteoarthritis were evaluated as controls. Microarray analysis was used to identify overexpressed genes in CD4 + T cells, and reverse transcription quantitative polymerase chain reaction was used to validate the identied genes. Plasma cytokine levels were measured, and correlations with disease activity were assessed. Results: Thirty-four genes showed overexpression in CD4 + T cells from patients with ACPA-positive MTX-naïve eRA compared with controls. Nineteen were related to interferon (IFN)-γ, and nine were related to interleukin (IL)-17A; ve were related to both IFN-γ and IL-17A. Plasma levels of ve cytokines were elevated in the ACPA-positive MTX-naïve eRA group compared with those in the control group, and C-X-C motif chemokine ligand 10 was signicantly elevated in the ACPA-positive MTX-naïve eRA group compared with that in the established active RA and OA groups. In established RA with low disease activity, the drug-free remission group/drug reduction group showed signicantly lower IFN-γ and IL-17A levels than the drug maintenance group and relapse after drug reduction group. Conclusion: Our T2 research of IFN-γ emphasized that, not only the Th17 immune response, but the Th1 immune response is also very important for the RA pathogenesis. Concurrent increase in IFN-γ and IL-17 were observed in active stage of ACPA-positive MTX-naïve eRA, and the plasma levels of them could be

pathogenesis of RA [5]. These modi ed antigens activate B cells to produce autoantibodies and stimulate antigen-presenting cells (APCs), FLSs, and T cells.

Helper T cells (cluster of differentiation [CD] 4 + T cells) are important elements in adaptive immune
systems and can be divided into several subtypes [6]. In in amed RA synovium, CD4 + T cells are predominant [7], suggesting that CD4 + T cells may be cornerstone immune cells in RA pathogenesis. Type 17 helper T cells (Th17 cells) have been identi ed in RA [8,9], and before identifying Th17 cells, interferon (IFN)-γ-secreting Th1 cells have been thought as major pathologic cells in RA development [10]. IFN-γ is the main cytokine produced by Th1 cells [6], and autoreactive Th1 cells are increased in the peripheral blood and synovial uid of patients with RA [11,12]. However, the effects of IFN-γ in animal models are unclear [13][14][15]. Furthermore, trials of IFN-γ and IFN-γ-blocking therapies have shown con icting therapeutic potentials in patients with RA [16,17]. Thus, IFN-γ may have various effects in RA, and further studies are required.
Recent genetic and epigenetic studies have revealed genes associated with the pathogenesis of RA [18].
For example, human leukocyte antigen (HLA)-DRB1 was the rst genetic risk factor shown to be associated with RA development [19], and such genetic predisposition facilitates autoimmunity of PTM autoantigens [20]. Based on a genome-wide association study, approximately 100 genetic susceptibility single nucleotide polymorphisms (SNPs) for RA were found [21]. However, most of these SNPs were not resided in protein-coding regions, and further studies of the functions and effects of the identi ed genes are required. Relevantly, signal transducer and activator of transcription 3 (STAT3)-inducible genes are overexpressed in CD4 + T cells from patients with undifferentiated arthritis who eventually developed seronegative RA [22]. Expression quantitative trait loci (eQTLs) are useful tools for identifying SNPs affecting disease-speci c gene expression. Notably, RA-related eQTLs have been shown to be enriched in CD4 + T cells [23,24]. Moreover, the IFN gene signature has shown to be increased in the whole blood of patients with methotrexate (MTX)-naïve RA compared with that in patients with established RA, and these changes were correlated with RA disease activity in MTX-naïve RA [25]. However, gene expression levels in CD4 + T cells in patients with MTX-naïve seropositive RA have not been clari ed.
Accordingly, in this study, we investigated gene expression levels in CD4 + T cells in ACPA-positive MTXnaïve eRA and evaluated changes in CD4 + T-cell-related cytokines during eRA. The goal of this study is present to translate research from animal models (T1 research) to real patients and to identify novel biomarkers for future T2 research.

Patients
Patients were recruited from Konkuk University Hospital and Ho-Youn Kim's Clinic for Arthritis and Rheumatism. Inclusion criteria for MTX-naïve, eRA were as follows: (1) ful lled the 1987 American College of Rheumatology (ACR) revised classi cation criteria for RA or 2010 ACR/European League Against Rheumatism classi cation criteria for RA [26,27]; (2) ACPA positive; (3) over 18 years old; (4) never taken disease-modifying antirheumatic drugs (DMARDs) prior to enrolment; (5) symptom duration of arthralgia less than 1 year; and (6) presence of synovitis con rmed by ultrasonography or magnetic resonance imaging. Exclusion criteria were as follows: (1) presence of another autoimmune disease; (2) presence of infection; and (3) presence of malignancy. Patients with established RA were also recruited and were all positive for either ACPA or rheumatoid factor. Patients with established RA were divided into low disease activity or moderate to high disease activity; patients who achieved a disease activity score (DAS) 28-erythrocyte sedimentation rate (ESR) less than 3.2 were de ned as having low disease activity, whereas those with DAS28-ESR of greater than 3.2 were de ned as having moderate to high disease activity. Patients with osteoarthritis (OA) were recruited as a control group. Synovial uid was obtained from patients with RA or OA when performing arthrocentesis for therapeutic purposes. In patients with ACPA-positive, MTX-naïve eRA, cytokine levels were analysed before DMARDs were started. In patients with established RA with low disease activity, cytokines were measured at the time when DAS28-ESR less than 3.2 was achieved. The disease course in patients with established RA with low disease activity was followed for 6 months, and patients were divided into the drug-free remission group, drug reduction group, drug maintenance group, and relapse after drug reduction group. Detailed information for each group is summarized Enzyme-linked immunosorbent assay (ELISA) We measured C-X-C motif chemokine ligand 10 (CXCL10) protein concentrations in plasma samples isolated from the whole blood of patients by ELISA (Abcam, Cambridge, MA, USA), according to the manufacturer's instructions. Absorbance at 450 nm was read on a VersaMax microplate reader with SoftMax Pro software (Molecular Devices, Sunnyvale, CA, USA).

RNA preparation
Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) and puri ed using RNeasy columns (Qiagen, Valencia, CA, USA). RNA samples were then quanti ed, aliquoted, and stored at -80 °C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis and analysis of the optical density at 260/280 using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA (500 ng), extracted as described above, was reverse transcribed into cDNA with a RevertAid First Strand cDNA synthesis kit (Thermo Scienti c, Waltham, MA, USA) according to the manufacturer's instructions. RT-qPCR was conducted in a 20-µL total volume containing 7.2 µL PCR-grade distilled water, 0.4 µL forward primers and reverse primers, and 10 µL SYBR Green I Master mix (Roche Diagnostics, Mannheim, Germany). PCR conditions were as follows: 95 °C for 10 min; followed by 35 cycles of 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s. All primers were synthesised by Bioneer Corp. (Daejeon, Republic of South Korea). The relative mRNA expression levels were normalised to the levels of β-actin mRNA.
Isolation of peripheral blood mononuclear cells (PBMCs) and CD4 + T cells Whole blood was collected using standard blood draw procedures. PBMCs were isolated from whole blood using Ficoll-Paque density medium (GE Healthcare, Chicago, IL, USA). The heparinised blood was diluted 1:1 in phosphate-buffered saline (PBS) and layered over the density gradient. The tube was centrifuged for 30 min at 1,800 rpm at room temperature. The whitish cell layer was collected and washed in PBS. To purify CD4 + T cells from PBMCs, the cells were incubated with anti-human CD4 microbeads (Miltenyi Biotec, Auburn, CA, USA). CD4 + T cells were then separated magnetically using a MACS column.
Gene expression pro ling and data analysis Gene expression analyses were performed in CD4 + T cells isolated from PBMCs using Affymetrix GeneChip Human Gene 2.0 ST oligonucleotide arrays. RNA (300 ng) was applied to the Affymetrix GeneChip as recommended by the manufacturer (http://www.affymetrix.com). The Affymetrix GeneChip Human Gene 2.0 ST oligonucleotide array was then scanned using an Affymetrix Model 3000 G7 scanner, and image data were extracted and analysed using Affymetrix Command Console software 1.1. For normalisation, we used the robust multi-average algorithm implemented in Affymetrix Expression Console software.

Statistical analysis
All data were expressed as means ± standard deviations or medians with interquartile ranges. Continuous variables were compared using Student's t tests or one-way analysis of variance and Bonferroni's multiple comparisons test. Correlation analysis was performed using Pearson's correlation coe cients. In all analyses, differences with P values of less than 0.05 were considered signi cant, and analyses were performed using SPSS version.20.0 (SPSS, Chicago, IL, USA). Upregulated genes in ACPA-positive, MTX-naïve eRA We performed microarray analysis of peripheral blood CD4 + T cells from ACPA-positive, MTX-naïve eRA (14 patients) and OA (9 patients); expression data for 103 genes were obtained. A heatmap of total gene expression is shown in Fig. 1A. In total, 101 genes were upregulated by more than 3-fold in ACPA-positive, MTX-naïve eRA compared with those in OA; two genes were downregulated (Fig. 1B). We further selected genes that were overexpressed by at least 6-fold (34 genes; Table 1). Among the 34 genes, we clustered gene sets according to associations with IFN-γ or IL-17A. Nineteen genes were related to IFN-γ signalling, and eight were related to IL-17A signalling (Tables 2 and 3). Interestingly, ve genes (IFIT3, RSAD2, GBP1, CCL20, and OASL) were related to both IFN-γ and IL-17A.

Plasma cytokine measurement via MCA in patients with established RA and OA controls
We measured the plasma levels of 5 cytokines in patients with established RA (N = 58) and OA (N = 16) group. Among established RA, 34 of 58 patients achieved low disease activity (DAS28-ESR ≤ 3.2) after DMARDs treatment. Both IFN-γ and IL-17A levels were elevated in patients with established RA with low disease activity compared with those in OA group ( Supplementary Fig. 1A). In contrast, 25 of 58 patients remained with moderate to high disease activity (DAS28-ESR > 3.2), all ve cytokines were signi cantly increased, compared with the OA group (Supplementary Fig. 2A). In patients with established RA with low disease activity, plasma IFN-γ levels were correlated with plasma IL-17A, IL-6, IL-12, and TNF-α levels ( Supplementary Fig. 1B). Disease activity was not signi cantly correlation with plasma IFN-γ, IL-6, IL12, or TNF-α ( Supplementary Fig. 1C). In patients with established RA with moderate to high disease activity, plasma IFN-γ levels were correlated with IL-17A, IL-12, and TNF-α ( Supplementary Fig. 2B). DAS28-ESR levels were signi cantly correlated with all ve cytokine plasma levels ( Supplementary Fig. 2C).
Notably, in comparison of OA, the mean levels of IFN-γ was signi cantly higher in early stage of RA as well as in established RA with low disease activity. Therefore, in addition to IL-17, plasma IFN-γ levels could be useful for diagnostic biomarker in early stage of RA, as well as responsible for chronic in ammatory status of patients with DMRDs treated established RA.
Evaluation of clinical outcome according to plasma IFN-γ in patients with low disease activity After 6 months follow up, RA patients with LDA (N = 34) were able to divide into 3 group of clinical outcome. To evaluate the impact of IFN-γ on clinical outcome, the plasma levels of IFN-γ and IL-17A were measured at time of achieving low disease activity and compared with 3 different clinical outcomes after approximately 6 months. The plasma levels of IFN-γ in drug free / drug reduction group were lower than drug maintenance and relapse group (

Discussion
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 ACPApositive, MTX-naïve eRA or established RA with moderate to high disease activity, plasma IFN-γ levels correlated well with other in ammatory 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 [28], and many RA-associated risk factor genes are dominantly expressed in CD4 + T cells [23]. Additionally, the gene enhancer region (H3K4me1) is uniformly overlapped with most effector and memory CD4 + T cells [24], highlighting the roles of CD4 + T cell in RA pathogenesis. In this study, we showed that IFN-γ-and IL-17A-associated genes were signi cantly increased in these patients, suggesting the potential roles of Th1 and Th17 cells in early stage RA pathogenesis. Interestingly, ve 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 proin ammatory cytokine. However, IFN-γ has been shown to have both pro-in ammatory and antiin ammatory activities under different biological contexts [29]. Importantly, RA synovium exhibits increased IFN-γ expression [30], and IFN-γ + CD4 + T cells are dominant in RA synovial uid [31]. 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 [12]. However, animal models of RA have shown con icting results; some have suggested that IFN-γ induces arthritis [15], 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 [32]. 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 ve 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 [25]. In the current study, OAS1 and IFI44L were also signi cantly increased in ACPA-positive, MTX-naïve eRA. Furthermore, increased CXCL10 levels in MTX-naïve eRA have been found to be correlated with disease activity [35]. Similarly, we showed that plasma CXCL10 levels were signi cantly elevated in MTX-naïve eRA compared with that in established RA. Our ndings 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 [8]. Additionally, Th17 cells are now In the current study, IFN-γ-and IL-17A-associated genes and CXCL10 were signi cantly elevated in ACPA-positive, MTX-naïve eRA. Plasma IFN-γ and IL-17A levels were signi cantly elevated in ACPA positive MTX naïve eRA even in patients with a low in ammatory 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 [12]. In animal models, collagen injection induces Th1 polarisation [47], and STAT3 inhibition ameliorates arthritis severity by reducing the Th17 population in a collagen-induced arthritis (CIA) model [48]. In addition, type II collagen-stimulated CD4 + T cells from patients with RA exhibit increased IFN-γ and IL-17 production [49]. 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 [51]. Furthermore, SOCS1 simultaneously enhances Th17 differentiation via STAT3 signalling, and SOCS3 modulates Th1/Th17 responses with SOCS1 [51]. Interestingly, IFN-γ also accelerates Foxp3 + regulatory T cell (Treg) induction and enhances the regulatory functions of Tregs [52]. The protective roles of Tregs on systemic autoimmunity have been extensively studied, and Tregs have been shown to reduce Th17-mediated autoimmune responses [53]. In contrast, IL-17 downregulates Th1 induction via IL-12Rβ2 suppresion [54], and the Th17-inducing transcription factor STAT3 blocks IL-12/p35 gene expression, thereby suppressing the Th1 response [55]. 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 [56]. These IFN-γ-positive Th17 cells are increased in the synovial uid of patients with juvenile in ammatory arthritis [57], suggesting the pathological roles of this Th17 subset in in ammatory 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 speci c 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 [58]. 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 [25]. 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.

Conclusions
In conclusion, our ndings showed that increased IFN-related gene signalling in CD4 + T cells was critical Informed consent was obtained from all patients, and the study was approved by the Human Research Ethics Committee of the Konkuk University Medical Center (approval no. KUH1010511) and the Clinics for Arthritis and Rheumatism (approval no. 0301202005BR00101).

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

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