MCP-1/CCL2 is a chemokine that plays an important role in recruiting monocytes/macrophages and recruits monocytes to the site of inflammation through CCR2[5]. Inflammatory responses through the CCL2–CCR2 pathway and associated damage cause various diseases such as auto-immune disorders including RA, cancer, atherosclerosis, myocardial infarction, viral infections, and OA.
There are two forms of CCR2, CCR2A and CCR2B, which have structural differences in the C-terminal tails; each form has a different mode of action and mechanism[14]. The expression of CCR2A was increased by CCL2 in an experiment on synoviocytes extracted from RA patients[15]. The binding affinity of CCR2 depends on the extracellular region, with the N-terminal region and E3 domain playing important roles[16–18].
CCR2 has dual functions. Blocking CCR2 leads to improvement in clinical signs and histological scores in the early phase of OA (days 0 to 15) but aggravates clinical and histological signs in the delayed phase (days 21 to 36), the latter which is caused by a humoral immune response involving regulatory T cells[19]. In addition, collagen-induced arthritis in CCR2-null mice exhibited a more severe RA pattern, and it was confirmed that CCR2 also plays a protective role in RA[20]. As such, CCR2 has both pro- and anti-inflammatory effects, and special care is required when developing therapeutic drugs that target this protein[8].
Recently, the roles of cytokines and chemokines in OA pathogenesis have garnered increasing attention. Unlike RA, CCL2–CCR2 plays an important role in the progression of OA compared to other chemokine receptors[4, 21]. Thus, studies on OA treatments that target the CCL2–CCR2 pathway have been increasing. Xu et al. (2015) sampled chondrocytes from OA patients and performed MCP-1 stimulation; they found increased expression of MCP-1, CCR2, and MMP-13, as well as the induction of apoptosis of OA chondrocytes. Based on these studies, various drugs that block CCL2–CCR2 as therapeutic agents for OA as well as for RA are being developed and tested. As mentioned above, blockade using various molecules such as monoclonal antibodies or soluble receptors of CCL2 and CCR2 in RA has been effective in many animal experiments but has not obtained therapeutic effects in clinical trials[13]. There may be various explanations depending on each experiment, but the redundancy of action between chemokines and chemokine receptors composed of multiple ligands and receptors is likely the major cause[22]. Because of this redundancy, when a single ligand or receptor is targeted, the specificity decreases, leading to ineffective results[23, 24]. Izhak et al.(2009) demonstrated through experiments that a fusion protein comprising as few as 20 amino acids of the E3 domain of the CCL2 receptor increases binding affinity compared to conventional CCR2 antagonists. In addition, in animal experiments, circulating blood levels of CCL2 increased only 1.8-fold after E3-Ig administration, indicating that E3-Ig can act as a more effective therapeutic agent. By contrast, a 2000-fold increase was observed in other studies using anti-CCL2 monoclonal antibodies [16, 25]. Based on these results, researchers have been able to obtain meaningful results by conducting experiments in which sCCR2 E3 gene therapy was applied to an OA model.
Transfection of the sCCR2 E3 gene into MSCs sampled from an OA patient was followed by treatment with LPS and MCP-1. It was confirmed that SOX9 increased whereas MMP-1 and MMP-3 decreased. This means that the sCCR2 E3 gene induced a chondrogenic effect and inhibited catabolic factors by blocking the action of MCP-1 in MSCs. Thus, MCP-1 is increased in OA and induces a pathologic condition, whereas CCR2 inhibits OA pathogenesis by blocking this action of MCP-1.
In OA patients, the suppression of inflammation and the control of pain play very important roles in the quality of life. Fundamentally, treatment with nonsteroidal anti-inflammatory drugs targets inflammation but has an insufficient effect on controlling joint cartilage destruction, so a treatment that simultaneously treats pain and cartilage destruction is urgently needed. In a previous OA surgical model, MCP-1 and CCR2 increased significantly in L3–L5 dorsal root ganglia, which govern pain signals from peripheral joints, and the calcium response in neurons increased significantly upon MCP-1 stimulation. Comparison of the CCR2-null and CCR2-antagonist groups with the wild-type (WT) destabilization of the medial meniscus (DMM) group confirmed that there was a significant decrease in pain and an increase in travel distance. This further confirms that the regulation of MCP-1/CCR2 can help with controlling pain in the peripheral joints[26]. In this study, based on the above results, we attempted to confirm the efficacy of controlling MCP-1 in pain reduction and cartilage protection. To this end, we conducted experiments comparing Mock, full-length sCCR2, and sCCR2 E3 gene therapy in an OA rat model, where MIA was administered to the knee joint to maximize pain and joint destruction. PWT, PWL, and weight-bearing measurements showed that the pain severity decreased, and there was no difference between the groups receiving full-length sCCR2 and sCCR2 E3 gene therapy. Through micro-CT analyses of the three groups, it was confirmed that bone resorption was decreased in the sCCR2 E3 group, with a greater decrease observed in the full-length sCCR2 group. When the knee joint tissues of the three groups were sampled and stained with safranin O, proteoglycan depletion was less severe in the sCCR2 E3 group compared to the full-length sCCR2 group. The OARSI and Mankin scores were also lower, confirming that cartilage destruction was prevented more effectively. In analyses of the inflammatory cytokines and catabolic factors IL-1β, IL-6, and MMP-13 based on IHC staining of samples from the three groups, the sCCR2 E3 group exhibited a greater decrease in levels compared to the full-length sCCR2 group, indicating that sCCR2 E3 had a more effective anti-inflammatory effect. Other previous studies have demonstrated that CCL2–CCR2 is associated with OA and plays a key role in the inflammatory response; however, no clear evidence on its association with chondropathy has been provided. MiotlaZarebska et al. (2017)performed histological analyses and scoring of CCL2−/− and CCR2−/− mice after DMM, but no significant differences with the WT mice were observed [12].Xu et al. (2015) injected a CCR2 antagonist (Sigma) in an MIA-induced OA rat model, but there was no significant improvement based on micro-CT analyses and the pathology score. In this study, we demonstrated through improvement in micro-CT outcomes and OARSI and Mankin scores that sCCR2 E3 gene therapy can prevent cartilage destruction.
In each of the four groups (WT, Mock, sCCR2, full-length sCCR2), the small intestine was sampled at 14 days after treatment administration, and the degree of damage according to the levels of epithelial damage and inflammatory cell infiltration was measured. The least damage was observed in the sCCR2 E3 group. Also, the expression of MCP-1, CCR2, and IL-17 in the intestine was confirmed through IHC staining, with the sCCR2 E3 group exhibiting the greatest inhibition. Through this, we demonstrated that intestinal damage and inflammation were most suppressed via sCCR2 E3 gene therapy targeting the knee joint. Regarding the connection between the knee joint and intestine, based on existing studies, the experiment was conducted based on expectations of a connection via the joint-brain-intestine pathway. When joint inflammatory and nociceptive signals are produced, they are transmitted to the brain through an afferent arc, and the brain stimulates the vagus nerve through an efferent arc. Then, acetylcholine is secreted, resulting in a cholinergic anti-inflammatory response [27]. In this process, the afferent arc is stimulated by cytokines secreted from the joint, with CCL2 expected to play a key role. Although the role of CCL2 has not yet been demonstrated, the above speculation can be made based on reports of disruption in the integrity of the blood-brain barrier via the CCL2–CCR2 pathway[28, 29]. When the brain is stimulated by a joint pathology, it is predicted that changes in the intestinal immune environment will occur through changes in hormone homeostasis resulting from alterations in the acetylcholine/epinephrine balance [28, 30]. In this study, we were able to partially prove the association.
OA has a different immunopathology compared to RA as a low-grade inflammatory disorder, and the CCL2–CCR2 pathway plays a more important role in the former[21]. Therefore, CCL2–CCR2 blockage through sCCR2 E3 is expected to have better therapeutic effects in OA than in RA. In this study, we proved that sCCR2 E3 can block the CCL2–CCR2 pathway more specifically than can other CCR2 antagonists in OA because of its high binding affinity, which solves some of the redundancy problems between multiple ligands and receptors. However, because the inflammatory response within the knee joint is a function of many different multiple pathways, its effect may be limited in actual clinical trials[31]. The redundancy problem will need to be resolved to obtain a meaningful therapeutic effect, and various approaches and studies, such as multiple targeting, as well as a method for increasing the specificity between chemokines and ligands are required.