Chemokines and chemokine receptors as promising targets in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease that commonly causes inflammation and bone destruction in multiple joints. Inflammatory cytokines, such as IL-6 and TNF-α, play important roles in RA development and pathogenesis. Biological therapies targeting these cytokines have revolutionized RA therapy. However, approximately 50% of the patients are non-responders to these therapies. Therefore, there is an ongoing need to identify new therapeutic targets and therapies for patients with RA. In this review, we focus on the pathogenic roles of chemokines and their G-protein-coupled receptors (GPCRs) in RA. Inflamed tissues in RA, such as the synovium, highly express various chemokines to promote leukocyte migration, tightly controlled by chemokine ligand-receptor interactions. Because the inhibition of these signaling pathways results in inflammatory response regulation, chemokines and their receptors could be promising targets for RA therapy. The blockade of various chemokines and/or their receptors has yielded prospective results in preclinical trials using animal models of inflammatory arthritis. However, some of these strategies have failed in clinical trials. Nonetheless, some blockades showed promising results in early-phase clinical trials, suggesting that chemokine ligand-receptor interactions remain a promising therapeutic target for RA and other autoimmune diseases.


Introduction
Chemokines are a family of small chemotactic cytokines (approximately 8-15 kDa). Chemokine ligand-receptor interactions control leukocyte migration during inflammation, promoting migration from the circulation into the extravascular space in inflamed tissues (1,2). Nearly 50 chemokines have been identified in mammals (3), commonly formed by four conserved cysteine residues-the first and third and the second and fourth forming disulfide bridges. Chemokines are divided into four subclasses according to the position of the first two conserved N-terminal cysteine residues: CC-chemokines (b-chemokines), having adjacent cysteine residues; CXC-chemokines (a-chemokines), having two cysteine residues separated by another amino acid; CX 3 Cchemokines (d-chemokines), having two cysteine residues separated by three other amino acids; and C-chemokines (g-chemokines), with only the second and fourth cysteine residues (4,5). The glutamateleucine-arginine (ELR)-positive (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) but not ELR-negative CXC chemokines (CXCL4, CXCL4L1, CXCL9, CXCL10, and CXCL11) have three amino acid residues (Glu-Leu-Arg) before the first conserved cysteine residue. The ELR motif is important for angiogenesis (6,7). Some chemokines are activated by matrix metalloproteinasemediated (MMPs)-mediated proteolysis (8).
Chemokine receptors are expressed on the surface of immune cells. "Classical" chemokine receptors are G-protein-coupled transmembrane receptors (GPCRs) and induce cell migration, whereas "atypical" chemokine receptors (ACKRs) are not coupled to G proteins and regulate cell migration (9,10). ACKRs scavenge chemokines to regulate chemokine gradients and dampen inflammation in a G protein-independent manner (3,11,12). Chemokine ligand-receptor interactions are presented in Table 1 (13).
The chemokine system may play a central role in rheumatoid arthritis (RA) pathogenesis. Several chemokines are highly expressed in the blood and inflammatory tissues, such as arthritic joints, of patients with RA. Furthermore, some genes encoding chemokine ligands and receptors have been reported as risk factors for RA development , and their expression is associated with clinical disease activity and severity (43-69). The regulation of immune cell recruitment into joints represents a major hallmark for therapeutic intervention, as the inhibition of the chemokine system can suppress the characteristic inflammation of RA, thereby halting its pathogenesis.
In this review, we summarize the pathogenic roles of chemokines and their receptors in RA. In addition, we provide evidence from recent human clinical trials using inhibitors of the chemokine system in RA and discuss the potential clinical benefits of chemokine blockade in patients with RA.

Rheumatoid arthritis
RA is an autoimmune disease characterized by autoantibody production, leading to the settlement of inflammatory processes with cytokine and chemokine production. This results in synovial inflammation, hyperplasia and swelling, cartilage and bone destruction and deformity, and systemic features, such as cardiovascular, pulmonary, and skeletal disorders (70).
Inflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a, play important roles in RA development. Biological agents, such as TNF-a and IL-6 inhibitors, have revolutionized RA therapies (71). However, approximately 50% of patients with RA are non-responders to these therapeutic approaches (72). Therefore, there is an ongoing need to identify novel targets and treatment strategies for RA.
Animal models of inflammatory arthritis have provided determinant information for the understanding of RA pathogenesis and development of RA therapeutics. Models such as type II collagen-induced arthritis (CIA) (73), collagen antibody-induced arthritis (CAIA) (74), K/BxN arthritogenic serum transfer model of arthritis (K/BxN) (75), and adjuvant-induced arthritis (AIA) (76) show RAlike arthritic phenotypes, including synovial hyperplasia with leukocyte infiltration and bone destruction. Furthermore, models of inflammatory arthritis and RA also show upregulated expression of chemokine ligands and their receptors in the serum, immune cells, and synovium (77-84). Thus, these animal models are useful for elucidating the pathogenic role of chemokines in RA.

Chemokine receptors in RA
Multiple chemokine receptors as well as chemokines contribute to RA pathogenesis (Table 3). Polymorphisms in CCR2, CCR5, CCR6, and CCR7-encoding genes are considered risk factors for RA development (29-42). CD4 + cells expressing CCR5 are increased in the blood of patients with active RA compared with that of patients with inactive RA patients and HD. Furthermore, CD4 + cells expressing CX 3 CR1 are decreased in patients with RA, and the CD4 + CD95 + T cell subset expressing CCR7 is associated with disease activity (63). In addition, CXCR4 and CXCL12 show higher expression in the serum and joints of patients with active RA than in those of HD and patients with RA remission. Moreover, the expression of these chemokines in the synovium has been correlated with disease score in patients with RA treated with TNFa inhibitors (54, 55). Chemokine

The pathological function of chemokine receptors in RA
Chemokines and their receptors control lymphocyte recruitment to inflamed joints in RA patients and animal models ( Figure 1). In RA patients, the recruitment of T cells into the synovium is controlled by CCR4, CCR5, CXCR3, CXCR4, and CXCR6 (95,97,102,126,127,129,(151)(152)(153)(154). Inhibition of CCL2, CCL5, or CXCL12 suppresses Th1 cell migration in vitro, suggesting that these chemokines might promote Th1 cell recruitment to the RA synovium (129). CD4 + T cells of patients with RA treated in vitro with anti-CCL22 antibodies differentiate into regulatory T cells (Tregs) via STAT5 activation (85). In SCID mice implanted with human RA synovium, recruitment of CD4 + CD28 -T cells, resembling effector memory T cells, is controlled by CCL5 and CXCL12 (127). CCR6 promotes Th17 cell recruitment into the inflamed joint in SKG arthritic mice (155), myostatindeficient (KO) mice, TNF-a transgenic (Tg) arthritic mice (156), and chemotactic ability of Th17 cells derived from patients with RA in vitro model (155,157). In addition, the CCR4 blockade suppresses Th17 cell migration to the arthritic joints in CIA mice (84). The CIA model also shows joint infiltration of CCR6 + type 3 innate lymphoid cells (iLC3s), which highly express IL-17A and IL-22. Furthermore, the number of CCR6 + iLC3s in the SF of patients with RA is correlated with disease activity (158).
Follicular helper T (Tfh) cells contribute to the formation and maintenance of germinal centers (GC). CXCR5 + Tfh cells are increased in the blood of patients with RA and CIA mice. Furthermore, the number of these cells correlates with the levels of clinical RA markers, such as C-reactive protein, rheumatoid factor, and erythrocyte sedimentation rate (159). In transwell experiments, the CXCL13/CXCR5 axis showed chemotactic activity in B cells of patients with RA (130). CXCR5 KO mice are resistant to CIA development; B cell-specific CXCR5 deficiency leads to mild arthritis with impaired germinal center (GC) response and antibody production, whereas T cell-specific CXCR5 deficiency promotes resistance to arthritis development by impaired GC response, antibody production, and inflammatory cytokine response (160).
CCL2 and CXCL8 enhance neutrophil chemotactic ability in cells from patients with RA, and CCR2 KO mice are resistant to AIA model through the suppression of CCL2/CCR2-induced neutrophil recruitment (163). CCL3 expression is associated with the neutrophil number in the SFs from patients with RA (106). Furthermore, the chemotactic activities of CCL4 and CCL5 are also correlated with the number of neutrophils in the SFs from patients with RA (94). An amino-terminal-modified methionylated form of CCL5 (Met-RANTES) antagonized the binding of CCL3 and CCL5 to their receptors CCR1 and CCR5, respectively, and the blockade inhibited arthritis in AIA rats via the suppression of neutrophil and macrophage migration into the joints (164).
ELR-positive CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) regulate neutrophil migration and angiogenesis via the receptor CXCR2 (6). CXCL5 expressed in RA SFs promotes neutrophil recruitment to EC in vitro (165). CXCL1 and CXCL5 induce neutrophil migration into the articular cavity of AIA mice, and chemotaxis is inhibited by the blockade of CXCR1/CXCR2 with repertaxin (79). CXCL1 and CXCL8 induce neutrophil chemotaxis in vitro, which is also inhibited by the blockade of CXCR1/CXCR2 and DF 2162, the later inhibiting neutrophil recruitment in zymosan-induced arthritis in mice and AIA in rats (166,167). Furthermore, in vitro, the ligand for CXCR1, CXCR2, CXCL2 enhances murine neutrophil migration, and the CXCL2-neutralizing antibody inhibits migration (139). Both CXCR2 and CCR1 are expressed in mice neutrophils, and their abrogation attenuates inflammatory arthritis in K/BxN mice (168). Recent in vivo imaging of joints showed that CCR1 promotes neutrophil crawling on the joint endothelium, whereas CXCR2 amplifies late neutrophil recruitment and survival in the joint (169). CXCL9 blocking peptide decreases neutrophil recruitment into the joints of AIA mice (170).
Increased OC differentiation and activity lead to bone loss and joint destruction in patients with RA. CCL3 enhanced osteoclastogenesis via OC migration and activation in the AIA rat model (174). CCL11/CCR3 induced OCP migration and bone resorption in vitro (175). CCL19 and CCL21, increased in RA SFs and serum, and their receptor CCR7, expressed in murine OCPs.
These chemokines did not affect OC differentiation but promoted OC migration and increased OC resorption activity in vitro and in vivo (176). The CCL25/CCR9 axis initiates the transformation of OCPs into mature OCs in vitro (100). CXCL2 promotes monocyte recruitment and osteoclastogenesis in RA samples in vitro, as well as in mouse bone marrow-derived macrophages (90,177). CXCL10 KO and CXCR3 KO in mice ameliorated arthritis in CAIA model by suppressing macrophage and T cell accumulation in arthritic joints. In addition, CXCL10 and CXCR3 inhibition decreased osteoclastogenic cytokine levels in the serum and spleen of CAIA (154). Furthermore, in vitro, CX 3 CL1/CX 3 CR1 regulates monocyte, DC, and OCP differentiation into osteoclasts (125, 178).
Chemokine receptors are also expressed in stromal cells, although their functions remain unknown. Angiogenesis is determinant for RA pathogenesis, namely for synovial proliferation and pannus formation (182). CCL21, in vitro, induces human microvascular ECs angiogenesis and migration via CCR7, suggesting that the CCL21/ CCR7 axis may contribute to angiogenesis in RA (140). The CXCL12/ CXCR4 axis also showed angiogenic activity in RA SFs in Matrigel in vivo (145) and the CXCL13/CXCR5 axis facilitated EC migration and angiogenesis in CIA mice (142). CCL28 and CCR10, highly expressed in RA synovium, regulate angiogenesis by EC recruitment, and CCL28 blockade inhibits EC migration and capillary formation (141). The CXCL16/CXCR6 axis promoted chemotactic and angiogenic activity in human umbilical vein ECs (HUVEC), which is a cell line (147). The CXCL12/CXCR4 and CXCL12/CXCR7 axes promote angiogenic activity in HUVEC, contributing to RA angiogenesis. CXCR7 is also expressed on ECs in the RA synovium. Furthermore, CXCR7 blockade ameliorated arthritis in CIA mice by suppressing angiogenesis (183).

Targeting the chemokine system in RA
In general, the signaling of "classical" G protein-coupled chemokine receptors is mediated by activating pertussis toxinsensitive Gi-type G proteins. Activated G proteins regulate multiple downstream signaling cascades, such as the JAK/STAT pathway and PI3K phosphorylation (188). In contrast, signaling of "atypical" chemokine receptors is independent of G proteins and remains somewhat unclear. In this section, we provide an update on arthritis animal models and clinical trials using drugs targeting chemokines and their receptors while discussing their potential as therapeutic targets.

Targeting the chemokine system in animal models of inflammatory arthritis
Animal experiments are useful in the testing and development of new therapeutic agents and treatment approaches. Some chemokine ligands and receptors in KO, Tg, and naturally mutant mice are used as arthritic models (Table 4). For instance, CCL3 KO mice showed milder clinical and histopathological scores in the CAIA model (189), whereas plt/plt mice, a naturally occurring CCL19 and CCL20 mutant strain, also showed mild arthritis in CIA model (190). CXCL10 KO mice showed mild arthritis in CAIA model through the inhibition of macrophage and T-cell migration into the synovium (154). CXCL14 Tg mice showed exacerbated autoimmune arthritis in a CIA model, caused by an excessive immune response against type II collagen (191).
CCR2 KO in the DBA/1J background exacerbated the CIA model because of the enhanced Th17 cell response and increased autoantibody production (192,193). CCR2 deficiency in IL-1Ra KO mice enhanced neutrophil migration (194). Furthermore, CCR2 deficiency in DBA/1J caused severe arthritis in CIA with cutaneous M. avium infection (195). In contrast, CCR2 KO in C57BL/6 mice showed decreased neutrophil infiltration into arthritic joints in AIA model (163). CCR4, CCR6, CCR7, CCR9, CXCR5, and CXCR6 deficiency ameliorated arthritis in CIA mice by suppressing the migration of Th17 cells (CCR4), DC (CCR7), and CD11b + splenocytes (CCR9) (82, 84,160,190,196,197). CCR5 KO mice showed conflicting results, with a reduced clinical score in CIA model in one study (198) and no changes in others (193). Although CCR6 KO mice were resistant to CIA model, the deficient CCR6 did not improve in an animal model of K/BxN and TNF-a Tg mice (196). In addition, CCR7 inhibition decreased autoantibody production and T cell proliferation in AIA mice (199). CXCR3 KO mice showed mild arthritis in CAIA model via the inhibition of both macrophage and T cell migration into the synovium (154). CXCR4-conditional KO in T cells reduced arthritic symptoms in CIA mice by inhibiting T cell migration (200). T cell-or B cell-specific CXCR5 KO mice, as well as fully CXCR5 KO mice, were resistant to both CIA and AIA models (160). CXCR6 KO mice showed resistance to K/BxN serum-induced arthritis and CIA model (147).
Regarding chemokine receptors, CCR1 antagonist J-113863 decreased the arthritic score but did not affect auto-antibody production in CIA mice (208). Small-molecule inhibitors of CCR2 combined with Methotrexate (MTX) treatment reduced both the arthritic score and bone destruction via the suppression of OC activity in CIA mice (209). Compound 22, a CCR4 inhibitor, ameliorated arthritis by reducing Th17 cell migration into the joints of CIA mice (84). A CCR5 antagonist (maraviroc) decreased the arthritic score and CD8 + T cell activation in CIA mice (210); however, other CCR5 antagonists (MCC22) did not change the arthritic score in K/B.g7 arthritic mice (211). In CIA monkeys, a CCR5 antagonist (SCH-X) reduced arthritic score but did not change biomarker expression (212). CCR9 antagonist (CCX8037) reduced the arthritic score by inhibiting CD11b + splenocyte recruitment into joints in CIA mice (82). The CXCR1/CXCR2 antagonist (SCH563705), but not the CCR2 antagonist (MK0812), reduced the arthritic score in CAIA mice (213). Furthermore, the blockade of CXCR1 and CXCR2 (DF 2162) ameliorated arthritis by inhibiting neutrophil migration in AIA rats (167). Anti-CXCR3 antibody reduced the arthritic scores and T cell influx into joints in adaptive transfer-induced arthritic rats (214). The CXCR3 antagonist (AMG487) contributed to the modulation of the Th17/Treg cell balance in CIA mice (215). Other CXCR3 antagonists, such as SCH 546738 and JN-2, also treated arthritis in CIA mice (216,217). A CXCR4 antagonist (T140) reduced the arthritic score and auto-antibody production in CIA mice (218).

Clinical trials of chemokine-targeted therapy in human RA
Based on valuable animal research, various therapeutic agents against chemokine ligands or their receptors have been developed and tested in patients with RA (219). However, several chemokines or   (Table 6). For instance, the CCL2-blocking antibody (ABN912) did not promote clinical improvements in patients with RA (220). In addition, the CCR2 antibody (MLN1202) failed at phase IIa of the clinical trial due to the reduction of monocyte levels and no changes in synovial biomarkers (221). Animal experiments have suggested CCR5 as a good RA therapeutic candidate (198,(210)(211)(212)222). However, reports showed that CCR5 is not determinant for RA development (223)(224)(225), and all clinical trials using CCR5 antagonists failed (226-228).
A phase II clinical trial with a CCR1 antagonist (CCX354-C) showed good efficacy in the ACR20 response in patients with abundant CCX354-C in plasma but not in those with poor CCX354-C plasma concentration. However, ACR responses did not significantly vary between placebo-and CCX354-C-treated patients (229). CCR1 antagonist (MLN3897, 10 mg, once, daily) combined with MTX had no discernible effects on the disease, despite high MLN3897 plasma concentrations and receptor occupancy of the therapeutic target (230). Another trial using a CCR1 antagonist (CP-481,715) and MTX also failed in phase II (231). CCR1 ligands, CCL3 and CCL5, can bind to other chemokine receptors, CCR3, CCR4, and CCR5 (Table 1). Therefore, even though CCR1 on leukocytes might be inhibited, other chemokine receptors can still promote leukocyte recruitment into inflamed joints in RA. This could explain the failures in the use of CCR1 as a therapeutic target.
In contrast, the combination of CXCL10 blocking antibody (MDX-1100) and MTX showed a mild therapeutic effect on the ACR20 response; however, ACR50, ACR70, and EULAR were not

Target
Therapeutic effect CCL2 Recombinant monomeric mutant CCL2 (p8A-MCP-1) protein reduced arthritic score and cytokine production in AIA rat CCL3 Anti-CCL3 antibody did not affect arthritic score in AIA rat CCL5 Met-RANTES reduced arthritic score in CIA mice Met-RANTES reduced arthritic score and macrophage infiltration into STs in AIA rat Recombinant CCL5 mutant ( 44 AANA 47 ) protein did not affect arthritic score in AIA rat Anti-CCL5 antibody reduced arthritic score in AIA rat CCL24 Anti-CCL24 antibody reduced arthritic score in AIA rat CXCL5 Anti-CXCL5 antibody reduced arthritic score and inflammatory cytokine production in AIA mice CXCL8 CXCL8-based decoy protein reduced arthritic score and neutrophil recruitment in AIA mice Anti-CXCR3 antibody reduced arthritic score and T cell influx into joints in adaptive transfer-induced arthritic rat CXCR3 antagonist (AMG487) reduced arthritic score and modulated Th17/Treg cell balance in CIA mice CXCR3 antagonist (SCH 546738) reduced arthritic score in CIA mice CXCR3 antagonist (JN-2) reduced arthritic score and cytokine production in CIA mice CXCR4 CXCR4 antagonist, 14-mer peptide T140 reduced arthritic score and autoantibody production in CIA mice significantly different between the treatment and placebo groups. The frequency of adverse events (AEs) in MDX-1100-treated patients was higher than that in placebo-treated patients; but any MDX-1100treated patients experienced serious AEs (232). Phase III of the clinical trial using MDX-1100 has not yet been launched.
The clinical trial using CX3CL1 blocking antibody (E6011, 200-400 mg) was effective for ACR20, ACR50 and ACR70 responses in RA patients with an inadequate response to MTX. The incidence of AEs and treatment-related AEs (TEAEs) were similar across the four treatment groups (placebo, E6011 100 mg, 200 mg, and 400/200 mg groups). Nonetheless, the incidence of serious AEs was similar between E6011-and placebo-treated patients. AEs such as nasopharyngitis, upper respiratory tract infections, bronchitis, pharyngitis, stomatitis, and back pain occurred in over 5% of the overall patients (233). However E6011 was no clear benefit in the ACR20 response rate was observed in RA patients with an inadequate response to biological DMARDs (234).
In addition to the above-mentioned blockade agents, other inhibitors of chemokine ligands or their receptors have demonstrated therapeutic effects on arthritis in RA models. Thus, these chemokine ligands and respective receptors may be promising targets for new RA therapies.

Conclusion
In this review, we summarize the functional roles of chemokine ligand-receptor interactions in arthritic joints of animal models and RA patients. Although several inhibitors of chemokines and/or their receptors have shown therapeutic effects in animal models of arthritis and clinical trials of patients with RA, limited therapeutic effects have been reported, suggesting that chemokine-targeted therapy still requires improvement. In targeting chemokine receptors, the choice of the most relevant receptor and ensuring high receptor occupancy at all times might be the key to therapeutic effects. In addition, inhibition of a single chemokine alone may not be sufficient to completely suppress leukocyte migration due to the functional overlap between chemokine systems. Therefore, the combined targeting of multiple chemokines and/or their receptors may be a more effective approach for human RA. Our previous study in animal models demonstrated that broadly cross-reactive chemokine-blocking antibodies for CXCR2 ligands dramatically ameliorated inflammatory arthritis compared with inhibition with antibodies against a single chemokine (241).
Further understanding of the importance of different chemokines at different stages of RA is required for the development of drugs that effectively target the system. We have previously developed an in vivo imaging technique to fully dissect the functional roles of chemokines and their receptors in inflamed joints in animal models (242). Interestingly, CXCR2 and ACKR1 are required for neutrophil apoptosis in the joint space, whereas the classical C5aR1 and atypical C5a and C5aR2 receptors are required for neutrophil apoptosis in the joint (146,169). Altogether, the development of effective inhibitors of chemokines and their receptors has untapped therapeutic potential in RA.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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