In rheumatoid arthritis (RA), joint destruction is the consequence of an imbalance between the catabolic and anabolic pathways. Associated with chronic inflammation of the synovium, these processes involve cytokines, growth factors, and matrix-degrading enzymes that are produced by effector cells. Similarly, the disequilibrium of the Th1/Th2 balance affects the production of cytokines that orchestrate the immune response (1, 2). Indeed, the shift toward Th1 promotes the production of proinflammatory cytokines (interleukin-1 [IL-1], tumor necrosis factor α [TNFα]) and reduces the production of antiinflammatory cytokines (IL-10, IL-4). Although recent therapies designed to inhibit inflammatory cytokines do provide significant protection against structural damage in RA, the persistence of synovitis and the high rate of nonresponse to biologic therapies still result in joint destruction (3), which means that there is still a need to develop an alternative approach to controlling the cytokines secreted by effector cells.
Furin is a ubiquitous proprotein convertase involved in the proteolytic processing of a wide range of precursor proteins, including growth factors and their receptors, adhesion molecules, and various metalloproteinases (4). The cleavage of furin substrates, such as insulin-like growth factor 1 and its receptor and several proteinases, including stromelysin 3 (5), MT1-MMP (6), and ADAMTS (7, 8), is crucial for the mediation of their functions. Unprocessed forms of some of these molecules are biologically active, and in certain cases, such as endothelial lipase (9), matrix metalloproteinase 2 (MMP-2) proforms (10), and fibroblast growth factor 23 (11), their processed forms are inactive or even mediate the opposite biologic action via specific receptors (12). In addition to its implication in the activation of molecules involved in several diseases such as cancer and infections, furin has been shown to mediate the proteolytic activation of several proteins involved in cartilage remodeling (13). Indeed, it was previously proposed that furin could play a role in joint remodeling by its ability to generate active forms of MMPs (14) and to reduce cartilage degradation ex vivo (13). On the other hand, furin was also found to inhibit the activity of active MMP-2 (10), a well-established extracellular matrix (ECM)–degrading proteinase.
Recently, the loss of furin was found to reduce peripheral tolerance in mice in vivo (15). Therefore, in vivo, furin exerts different local effects in tissue, which may influence the outcome of the disease. Indeed, furin might affect the development of arthritis as a result of positive regulation of peripheral tolerance and, in contrast, by enhancing the intracellular maturation of proteolytic enzymes that may lead to degradation of the cartilage matrix. We undertook this study to evaluate the final in vivo effect of furin during inflammation and joint degradation in arthritis.
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We found that the systemic administration of furin reduced arthritis in conjunction with a reduction of the synovial pannus. The prevention of arthritic lesions by furin is mediated through the restoration of the Th1/Th2 balance in the joint, and is also associated with an enhanced proportion of Treg cells in the spleen, indicating systemic regulation of proliferation of the local pannus.
Initially, we observed that furin was highly expressed in samples from RA patients and from mice with arthritis. Furin expression in these tissues suggests that furin might promote joint inflammation or act as a compensatory mechanism in alleviating the inflammatory process. Therefore, we used exogenous furin to investigate the role of furin in the development of arthritis. We found that furin did not affect the general health or the behavior of the mice throughout these short-term experiments. No macroscopic alterations in other organs were noted at the time of death. The weight loss observed in mice treated with vehicle or furin inhibitor might be related to the inflammation conditions. However, ascertainment of information regarding toxicity due to prolonged or repeated administration of furin such as cell transformation requires additional investigations.
Systemic administration of furin was found to reduce local joint inflammation and damage and to promote the restoration of Treg cell numbers. In contrast, α1-PDX was found to enhance the severity of arthritis and reduce the proportion of Treg cells. This systemic effect is further shown by the fact that bone loss was prevented by furin and worsened by α1-PDX administration. Treg cells are involved in the suppression of both Th1 and Th2 pathogenic immune responses and control T cell homeostasis (2, 19–22) as well as the maturation of T effector cells (23). Furin has recently been found to play an important role in immunity through Treg cell activation, as revealed in studies using conditional deletion of furin in T cells, which resulted in impairment of the function of Treg cells as well as that of effector cells (15). Deletion of furin in CD4+ cells in mice resulted in the development of inflammatory bowel disease and lymph node hypertrophy, leading to Th1 activation as confirmed by serum cytokine patterns.
Here we show that in a context of systemic inflammation, exogenous furin enhanced the generation of Treg cells in a peripheral lymphoid organ such as the spleen, indicating that this enzyme plays an active role in immune tolerance, which is illustrated by an improvement of joint lesions. Our results provide further evidence that Treg cells have a role in regulating the immune tolerance that prevents the expansion of other T cells and the activation and functioning of autoreactive T cells (24). Th1 cells constitute a group of CD4+ T helper cells that are actively involved in the inflammatory immune responses that occur during intracellular infections, organ transplantation, and autoimmune responses (25), and they drive the immune responses that lead to joint damage. In these responses, furin reversed the local Th1/Th2 balance, as demonstrated by the reduced local levels of inflammatory cytokines, while the levels of antiinflammatory cytokines were enhanced. The shift toward Th1 with α1-PDX observed here further showed that blockade of enzymatic activity is one of the mechanisms driven by furin and is consistent with the invalidation of the furin gene in lymphocytes. IL-17, which is produced by Th17 cells, is a dominant cytokine involved in the development of arthritis (22, 26). However, we failed to demonstrate any regulation of IL-17 production by furin or its inhibition, suggesting that IL-17 is not involved in immune mediation in this particular condition.
Our finding of immune-mediated response to furin in Treg cells and in the Th1/Th2 balance is related to its enzymatic activity, which could be reversed by α1-PDX. Indeed, in studies using various in vitro and cellular models, this inhibitor has previously been reported to inhibit the activity of furin, leading to the inability of the enzyme to induce the proteolytic cleavage of furin substrates or to induce its downstream effectors (27). The inhibition of furin was found to reduce the expression and activation of various molecules, including cytokines, adhesion molecules, and growth factors (28–30). The in vivo integrative approach used here showed that exogenous furin affects immune regulation more than local tissue regulation.
The joint destruction in RA is a consequence of an imbalance between the catabolic and anabolic pathways, involving local factors and matrix-degrading enzymes induced by effector cells (2, 19). Produced in the form of precursors, most of these molecules are proteolytically activated by furin (31, 32), whereas the processing of others inhibits their activity and functions (4). Previously, in chondrocytes, furin was found to interact with the proADAMTS-4 precursor form and to mediate its intracellular activation (33), whereas its inhibition resulted in reduced release of transforming growth factor β (TGFβ) (34). Therefore, endogenous furin promotes the maturation of some catalytic enzymes that might impair the balance of cartilage homeostasis. Similarly, metalloproteinases are central enzymes in matrix degradation and remodeling, which are key events in joint destruction and inflammation. Immunohistochemistry analysis performed on sections from arthritic mice revealed that the expression of MMP-2, MMP-9, and MMP-14 was increased, but was reduced in arthritic mice receiving systemic furin. Further analysis of tissue derived from the joints of mice revealed an association between increased immunoreactivity toward MMP-2 and MMP-9 and the level of MMP-2 and MMP-9 activity as assessed by zymography. In arthritic mice the injection of furin significantly reduced the enzymatic activity of both MMP-2 and MMP-9.
Although the mechanism of action of the inhibitory effect of systemic furin on MMP-2 expression and activity is unknown, the presence of furin in the circulation might inhibit its well-established local function on MMP-2 and MMP-9 processing and activation (13). These differences might be related to the ability of furin to activate the Treg cells that subsequently release mediators involved in the inhibition of MMP expression/activation, such as IL-4 and IL-10, or that reduce the expression of MMP inducers, such as TNFα. Therefore, although furin is involved in the activation of molecules that mediate matrix degradation, its protective effect might be related to its ability to inhibit the expression of these matrix degradation molecules. In addition, a previous study indicated that deletion of furin in CD4+ cells was found to impair T cell function by altering the production of TGFβ (15). Indeed, TGFβ promotes naive T cells to a Treg cell phenotype (35), and TGFβ-secreting cells suppress the development and severity of CIA (36). Therefore, our observations raise the hypothesis that exogenous furin might also affect the processing of TGFβ, resulting in protection against arthritis. We await further investigations into this.
In conclusion, these data indicate that systemic administration of furin promotes Treg cells and restores the Th1/Th2 balance through the inhibition of the conversion of MMP-9 and MMP-2 precursors into their active forms. Furin could therefore play a major role in reducing autoimmunity in RA, which raises the possibility of the potential use of furin or its derivatives in inflammation, thus providing a new therapeutic agent for autoimmune diseases such as RA.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cohen-Solal had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Lin, Khatib, Cohen-Solal.
Acquisition of data. Lin, Ah Kioon, Lalou, Larghero, Launay, Khatib, Cohen-Solal.
Analysis and interpretation of data. Lin, Khatib, Cohen-Solal.