Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

Abstract Background FURIN belongs to the proprotein convertase family that processes proproteins and is involved in many diseases. However, the role of FURIN in rheumatoid arthritis (RA) remains unknown. In this study, we investigated the association between circulating FURIN and disease activity in patients with RA and the effect of FURIN in THP‐1‐derived macrophages. Methods A total of 108 RA patients and 39 healthy controls participants were included in this study. RA patients were divided into four disease activity groups determined by the Disease Activity Score of 28 joints (DAS28). FURIN expression in peripheral blood mononuclear cells (PBMCs) and serum was detected by using quantitative real‐time polymerase chain reaction (qRT‐PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. Western blotting and qRT‐PCR were used to detect cytokines level after interfering FURIN expressed in THP‐1‐derived macrophages. Results Both FURIN mRNA and protein levels were significantly higher in RA patients than in healthy controls participants (P < .001). No significant difference in FURIN expression was observed among the four RA groups (P > .05). Spearman correlation revealed that FURIN positively correlated with transforming growth factor‐β1(TGF‐β1), rheumatoid factor (RF), and anti‐cyclic citrullinated peptide (anti‐CCP). Moreover, the inhibition of FURIN in THP‐1‐derived macrophages promoted the caspase‐1 and IL‐1β expression (P < .05). Conclusion FURIN levels were significantly increased in the peripheral blood of RA patients and were not associated with disease activity. The inhibition of FURIN in THP‐1‐derived macrophages with elevated IL‐1β levels shows that FURIN may have an anti‐inflammatory effect.


| INTRODUC TI ON
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation. Its clinical manifestations include progressive and destructive polyarthritis associated with serological evidence of autoreactivity. One of the pathological causes of RA is hyperactive immune cell responses that result in elevated levels of inflammatory cytokines. Evidence suggests that inhibitors of special cytokines, for example, TNF-α, IL-1, IL-17, and IL-6, provide improvement in RA patients. 1,2 FURIN is one of the nine members of the proprotein convertases subtilisin/kexin (PCSK) family and cleaves various precursor proteins in several physiological processes. 3,4 FURIN is involved in regulating the secretion of cytokines, is usually co-expressed with transforming growth factor-β1 (TGF-β1), and is a vital regulator of the TGF-β1 cytokine. 5,6 Furthermore, T cell-expressed FURIN is essential for the maintenance of peripheral immune tolerance and is critical for overall health and regulatory function of the body. 7 Thus, the dysregulation of FURIN levels may lead to different diseases and disorders, 8 such as primary Sjogren's syndrome disease (pSS), systemic lupus erythematosus (SLE), and RA. [9][10][11] A study on RA demonstrated, the overexpression of FURIN in fibroblast-like synoviocytes (FLS). 9 Further, systemic administration of exogenous FURIN in mice with collagen-induced arthritis (CIA) mitigates the symptoms of RA. 12 However, the relationship between circulating FURIN and the disease activity of RA is unclear. Hence, we studied peripheral blood mononuclear cells (PBMCs) and serum FURIN levels in RA patients and healthy controls participants, and analyzed the correlations between FURIN expression and RA disease activity in a Chinese population. Moreover, the effect of FURIN on cytokines (IL-1β, TNF-α, and TGF-β1) production was also studied in THP-1-derived macrophages.

| Enzyme-linked immunosorbent assay (ELISA)
Serum samples were stored at −80°C until the levels of the cytokines were detected. FURIN was detected in 108 RA patients and 39 healthy control participants by using a commercial Human FURIN Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Sigma Aldrich,  .480

TA B L E 1 The primer sequence
Note: Data were described as n (%) or median (interquartile range, 25th-75th). DAS28 28 joint count Disease Activity Score Using ESR.
Abbreviations: anti-CCP, antibodies to cyclic citrullinated peptides; CRP, C-reactive protein; DMARDs, disease-modifying antirheumatic drugs; ESR, erythrocyte sedimentation rate; NSAIDs, non-steroidal anti-inflammatory drugs; RF, rheumatoid factor. a CRP data were found for 93 of RA patients due to a lack of CRP data in fifteen patients.
b RF data were found for 100 RA patients due to a lack of RF data in eight patients.
c 70 anti-CCP positive was found in 96 RA patients due to a lack of anti-CCP data in twelve patients.

#
Comparison between RA patients and healthy. *Comparison among the four RA groups.
USA), and the results are presented as pg/mL. The concentrations of IL-1β, IL-4, IL-6, TNF-α, and IL-10 were determined by using ELISA kits (Mlbio) and TGF-β1 ELISA kits (Elabscience) according to the manufacturer's instructions.

| Transient transfection of siRNA in THP-1derived macrophages
THP-1 monocytes cell line was obtained from China Center for Type Culture Collection (Wuhan, China). Cells were seeded in 12-well plates at a density of 5 × 10 5 cells/mL and treated with 100 ng/mL PMA (Sigma) for 48 hours and then were transfected with siRNA (Santa Cruz Biotechnology) that are specific for FURIN and control siRNA(Santa Cruz Biotechnology) for 24 hours, and this was followed by treatment with 1 μg/mL LPS (Escherichia coli O55:B5, Sigma) and 5 mmol/L ATP (Solarbio) for 30 minutes. The cells were then harvested and used to extract total RNA and protein. with the ImageQuant LAS 500 system (Thermo).   (Table 2).

| FURIN expression was significantly higher in RA patients than in healthy control participants
FURIN mRNA expression levels in PBMCs were significantly upregulated in RA patients compared to those of healthy control participants (P < .001, Figure 1A). Serum FURIN levels were significantly higher in RA patients than in the healthy controls (P < .001, median 190.6 pg/mL vs. 85.9 pg/mL, Figure 1B). However, both FURIN mRNA and protein levels were not significantly different among the four RA disease activity groups (P > .05, Figure 1C,D). The statistical powers of serum and mRNA levels of FURIN between 108 RA patients and 39 healthy control participants were 0.997 and 1.000, respectively.

Studies have reported that FURIN is involved in cytokine secretion
and that FURIN is an important enzyme in TGF-β1 maturation. We analyzed the relationship of FURIN with TGF-β1 and other cytokines.
As shown in Table 3 and Table S1, FURIN mRNA expression was found to be closely correlated with TGF-β1 in PBMCs (r = .769, P = .000).
But no correlation was observed in serum protein level (r = −.091, P = .356, Figure 2). FURIN was not correlated with the concentra-

| D ISCUSS I ON
In this study, we demonstrated that the levels of both FURIN protein and mRNA in PBMCs were significantly higher in RA patients than in healthy control participants. There was no correlation The differences among the four RA groups were assessed by using the Kruskal-Wallis H nonparametric test. Significant differences are marked with asterisks: *P < .05, **P < .01, and ***P < .001 been reported to supplement FURIN activity. 5 Additionally, a positive feedback loop between FURIN and TGF-β1 has been reported in synoviocytes, which upregulates "A disintegrin and metalloprotease with thrombospondin motif-4" (ADAMTS-4); this eventually leads to the destruction of arthritis. 21,22 However, a recent report showed that TGF-β1 suppresses human RANKL-induced human osteoclast development and bone resorption and identified a potential therapeutic target for RA. 23 We believe that the opposing effects of TGF-β1 may be due to difference in the experimental cells used in previous studies. 23 Macrophages are the major source of IL-1β and contribute to tissue destruction and pain in RA patients. In this study, LPS and ATP treatment contributed to the inflammatory environment in THP-1-derived macrophages, and IL-1β and caspase-1 levels were higher following the inhibition of FURIN expression. Previous studies have shown that FURIN is multifunctional. 24 FURIN knockout mice were more sensitive to challenge, and expression levels of IL-1β

TA B L E 3 Correlation between FURIN and disease activity and clinical data
were higher than in wild-type mice. 16 Likewise, myeloid cells have been reported to express proprotein convertase FURIN which could inhibit the secretion of cytokine of IL-1β and attenuated inflammation. 16 Furthermore, exogenous FURIN reduced local IL-1β production and enhanced the function of regulatory T cells(T reg ), revealing the protection of FURIN in RA patients. 12 Although FURIN inhibited the production of certain cytokines, in this study, both FURIN and IL-1β were upregulated. Elevated FURIN levels in RA patients may be associated with conditions that arise from an abnormal immune and indicate that FURIN may be have the potential to augment the immune response in RA patients.
The limitations of this study are as follows. First, the study included a relatively small group size of the samples; a large number of samples are essential to further confirm our findings. Second, our samples were limited to Chinese patients who were treated, and further research must be conducted to verify these findings in untreated patients with RA. Third, there was no overexpression of FURIN in THP-1-derived macrophages that have more strongly supported the anti-inflammatory effect of FURIN.
In conclusion, we demonstrated that the levels of FURIN in peripheral blood were higher in RA patients but unrelated with disease activity. FURIN may have an anti-inflammatory effect by inhibiting IL-1β, it may be a potential target for inflammatory diseases.