CXC chemokine receptor 3 antagonist AMG487 shows potent anti-arthritic effects on collagen-induced arthritis by modifying B cell inflammatory profile

Highlights

• We investigated the effect of AMG487 on the key mediators of RA.

• AMG487 treatment downregulated NF-κB signaling in collagen-induced arthritis mice.

• AMG487 treatment significantly decreases proinflammatory cytokines in CIA mice.

• AMG487-treated mice show upregulation of anti-inflammatory cytokines.

• AMG487 exhibits anti-arthritic activity and is a viable therapeutic option for RA.

Abstract

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19⁺ B cells in the spleen through flow cytometry. We also evaluated the mRNA and protein expression levels of these molecules using RT-PCR and western blotting in the knee tissues. Our results revealed that AMG487-treated mice showed decreased NF-κB p65-, NOS2-, MCP-1-, and TNF-α-, and increased IL-4-, and IL-27-producing CD19⁺ B cells compared with the control mice. Additionally, AMG487 treatment significantly down regulated NF-κB p65, NOS2, TNF-α, and IFN-γ, and upregulated IL-4 and IL-27 mRNA and protein expression levels compared with the control. Thus, our study shows that AMG487 exerts its anti-arthritic effect by potently downregulating inflammatory B cell signaling. Based on our observations, we propose that AMG487 could serve as a potential novel therapeutic agent for inflammatory and autoimmune diseases, including RA.

Introduction

Rheumatoid arthritis (RA), an inflammatory disease characterized by synovial inflammation, cartilage damage, and bone destruction, affects 1% of the global population [1]. Proinflammatory mediators play an important role in both initiation and development of RA [2]. The development and pathogenesis of RA is attributed to the association of several types of cells, including T cells, B cells, and autoantibodies [3,4]. Activated B cells have been reported to play important roles in pathogenesis and progression of RA [5,6]. In fact, the importance of B cells in RA has been emphasized by the success of therapeutic approaches using anti-CD20 monoclonal antibodies [7]. However, none of the currently available treatments provide a drug-free and long-lasting remission of RA.

Nuclear factor (NF)-κB transcription factor is a key factor inducing chronic inflammatory responses in the pathogenesis of RA [8]. NF-κB is highly activated in synovial tissues of RA patients [9] and induces production of several inflammatory mediators including cytokines and chemokines [10]. Thus, the inhibition of NF-κB signaling could be an effective therapeutic approach for RA treatment. A previous study also showed that inflammatory mediators such as inducible nitric oxide synthase (iNOS) increased during RA development [11]. Moreover, iNOS as a therapeutic target has demonstrated amelioration of RA disease progression [12]. Based on these observations, it has been hypothesized that inhibiting iNOS production could serve as the basis for the potential development of anti-arthritic drugs. Monocyte chemotactic protein-1 (MCP-1) is a potent mediator of chronic inflammation [13] and a crucial factor triggering inflammation in RA [14]. Thus, the modulation of inflammatory signaling could be an effective approach for treating RA and other chronic inflammatory diseases.

A previous study has shown that TNF-α plays a critical role in bone destruction in RA [15]. Indeed, an inflammatory response in RA is characterized by increased production of TNF-α [16]. Moreover, TNF-α was previously investigated as a target in the efforts to treat RA [17]. A previous study also evidenced that blocking of TNF-α could have therapeutic response on the RA progression [18]. The blockage of increased IL-1β production is associated with the maintenance of joint destruction of RA patients [19]. IL-4 is a potent anti-inflammatory cytokine that acts by inhibiting the production of pro-inflammatory cytokines [20]. IL-27 was recently discovered as an anti-inflammatory cytokine that impacts RA disease progression [21]. Previous studies also showed that administration of IL-27 ameliorates collagen-induced arthritis (CIA) [22] and inhibits NF-κB ligand (RANKL)-mediated osteoclastogenesis [23]. IL-4 is a signature cytokine of Th2 cell-mediated immune responses and is a potent anti-arthritic cytokine that inhibits cartilage damage and osteoclastogenesis [24,25]. These reports indicate the protective role of IL-4/IL-27 in the pathogenesis of RA.

Chemokine receptors play an important role in the recruitment of leucocyte subsets in inflammatory joint diseases. They are significantly expressed on B cells and have been implicated in the pathogenesis of RA [26]. A selective CXCR3 antagonist, AMG487 has been used to test different animal models to inhibit CXCR3 expression [27,28]. We have also previously shown that AMG487 attenuates joint inflammation in mouse model of CIA [29]. However, the mechanism by which AMG487 regulates the immune response remains unclear. Here, we investigated whether the anti-arthritic effects of AMG487 are mediated by the regulation of B cell inflammatory signaling.