Functions of interleukin‐34 and its emerging association with rheumatoid arthritis

Summary

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic, synovial inflammation affecting multiple joints, finally leading to extra‐articular lesions for which limited effective treatment options are currently available. Interleukin‐34 (IL‐34), recently discovered as the second colony‐stimulating factor‐1 receptor (CSF‐1R) ligand, is a newly discovered cytokine. Accumulating evidence has disclosed crucial roles of IL‐34 in the proliferation and differentiation of mononuclear phagocyte lineage cells, osteoclastogenesis and inflammation. Recently, IL‐34 was detected at high levels in patients with active RA and in experimental models of inflammatory arthritis. Blockade of functional IL‐34 with a specific monoclonal antibody can reduce the severity of inflammatory arthritis, suggesting that targeting IL‐34 or its receptors may constitute a novel therapeutic strategy for autoimmune diseases such as RA. Here, we have comprehensively discussed the structure and biological functions of IL‐34, and reviewed recent advances in our understanding of the emerging role of IL‐34 in the development of RA as well as its potential utility as a therapeutic target.

Abbreviations

AA

adjuvant arthritis

CCL

CC chemokine ligand

CCR

C‐C chemokine receptor type

CIA

collagen‐induced arthritis

CSF‐1R

colony‐stimulating factor‐1 receptor

CXCL

chemokine (C‐X‐C Motif) ligand

DAS28

28‐joint Disease Activity Score

DMARDs

disease‐modifying anti‐rheumatic drugs

FLS

fibroblast‐like synovial cells

IL‐34

interleukin‐34

IP‐10

interferon‐inducible protein 10

MCP‐1

monocyte chemotactic protein‐1

PTP‐ζ

receptor‐type protein‐tyrosine phosphatase ζ

RA

rheumatoid arthritis

RANKL

receptor activator of nuclear factor‐κB ligand

TNF‐α

tumor necrosis factor α

Introduction

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by progressive inflammatory disorders of the joints and surrounding tissues, finally leading to irreversible joint destruction.1 It has an estimated prevalence of ~ 1% and affects women two to three times more often than men. Previously, patients with newly diagnosed RA were typically treated with non‐steroidal anti‐inflammatory drugs to reduce pain and joint swelling. Currently, treatment with disease‐modifying anti‐rheumatic drugs (DMARDs), or DMARDs in conjunction with biological agents has been shown to be effective in the normalization of cytokine levels, and alleviation of several clinical outcomes. However, a large proportion of patients with RA cannot tolerate these treatments, and some individuals also continue to have active disease even following therapy. Furthermore, major complications are caused by cumulative adverse effects, especially with non‐steroidal anti‐inflammatory drugs and corticosteroid treatments.2 Hence, effective alternative therapies are essential for the treatment of moderate to severe RA.

Although the exact aetiology and pathogenesis of RA remain unclear, it is generally accepted that inflammatory mediators play a pivotal role in driving synovial cell activation that leads to inflammation and joint destruction during arthritis. Accumulating evidence has disclosed high levels of several pro‐inflammatory mediators including tumour necrosis factor α (TNF‐α), interleukin‐6 (IL‐6) and IL‐1β, which are present at very high levels in the serum and synovial fluid of patients with RA and which enhance the recruitment of inflammatory cells into damaged tissues and aggravate the joint destruction in the disease process.2, 3 Therapeutic strategies involving inhibition of inflammatory cytokines using biological agents are indeed effective for patients with RA.4, 5 Inhibitors of TNF‐α (Infliximab, Etanercept, Adalimumab, Certolizumab, Golimumab),6 IL‐6 inhibitors (Tocilizumab, Sarilumab, Sirukumab)7 and IL‐1 inhibitors (Anakinra, Canakinumab)8 have been employed to treat RA in patients with an inadequate response to conventional DMARDs (Table 1), but are not effective in all cases. In addition, cytokine antagonists for RA are known to increase the risk of serious infections, especially in cases where patients have co‐morbidity factors,9 raising significant safety concerns about the use of currently available biological compounds, in particular, anti‐TNF‐α agents. Therefore, characterization of novel cytokines that are aberrantly expressed in animal models of RA and human patients is paramount for the development of effective therapeutic targets and treatments for moderate to severe RA.

Table 1.

Examples of antibodies tested for the treatment of rheumatoid arthritis

Targets	Agent	Types	Mechanism	References
TNF‐α	Infliximab Etanercept Adalimumab Certolizumab Golimumab	Recombinant IgG1 mAb Extracellular domain of TNF receptor II and the Fc portion of IgG1 fusion protein Recombinant IgG1 mAb TNF‐α specific Fab antibody fragments Human mAb	Binds to TNF‐α and prevents it from binding to its receptor Functions as a decoy receptor to TNF‐α Binds to TNF‐α and prevents it from activating TNF receptors Neutralizes the activity of TNF‐α Neutralizes TNF‐α bioactivity	92, 93 94, 95 96 97 98
IL‐1	Anakinra Canakinumab Gevokizumab LY2189102	Recombinant IL‐1Rα Human IgG1 mAb Humanized IgG2 mAb Humanized IgG4 mAb	Prevents binding of IL‐1β to IL‐1Rα Neutralizes the activity of IL‐1β Neutralizes the activity of IL‐1β Neutralizes the activity of IL‐1β	99 100 101 102
IL‐6	Tocilizumab Sarilumab Sirukumab	Humanized mAb Human anti‐IL‐6Rα mAb Human IgG1 mAb	Binds to IL‐6 receptor and blocks IL‐6 signalling Binds to IL‐6 receptor and blocks IL‐6 signalling Neutralizes the activity of IL‐6	103 104 105
IL‐17	Secukinumab Ixekizumab Brodalumab	Human Ig G1k mAb Humanized Ig G4 mAb Human anti‐IL‐17RA mAb	Selectively binds and neutralizes IL‐17A Neutralizes IL‐17A Inhibits the activity of IL‐17	106 107 108
CD20+ B cells CTLA‐4	Rituximab Abatacept	Chimeric mAb Extracellular domain of CTLA‐4 and the Fc region of IgG1 fusion protein	Depletes CD20+ B cells Binds to CD80 and CD86 receptors on the APC and blocking the binding to CD28 on T lymphocytes and preventing the immunological response	109 110

APC, antigen‐presenting cell; CTLA‐4, cytotoxic T‐lymphocyte‐associated protein 4; IL‐1, interleukin1; IL‐1R, interleukin‐1 receptor; mAb, monoclonal antibody; TNF‐α, tumour necrosis factor‐α.

Recently, IL‐34 was functionally identified as a cytokine by comprehensive proteomic analyses, and was shown to act as an alternative ligand of colony‐stimulating factor‐1 receptor (CSF‐1R) although it shares no sequence homology with CSF‐1.10, 11 Interleukin‐34 binds to CSF‐1R and promotes the proliferation, survival and differentiation of phagocytes, such as macrophages, osteoclasts and Langerhans cells.12, 13, 14, 15 Emerging findings indicate that IL‐34 levels are abnormally increased in serum and synovial fluid and strongly associated with antibody levels in patients with active RA, including rheumatoid factor and anti‐cyclic citrullinated peptide antibody, suggesting utility as an important mediator in RA as well as a successful therapeutic target from a clinical perspective.16, 17, 18 Additionally, IL‐34 produced by human fibroblast‐like synovial cells in RA promotes osteoclastogenesis.19 In this review, we have discussed the structure and functions of IL‐34, and summarized recent advances in understanding the role of IL‐34 in the pathogenesis of RA, and evaluating whether blocking IL‐34 can serve as a novel promising therapeutic strategy.

Structure and functions of IL‐34

Structure of IL‐34

Mice that are deficient for CSF‐1R (CSF‐1R^−/−) display a more severe phenotype compared with CSF‐1‐deficient (CSF‐1^op/^op) mice, suggesting that the activation of the receptor is mediated independently of CSF‐1, possibly through a second ligand.20 Interleukin‐34, a cytokine identified by a functional screening approach, was recently shown to serve as a second ligand for CSF‐1R.11 The gene encoding IL‐34 is located on human chromosome 16q22.1 and is organized into 11 exons. The translated IL‐34 protein has been characterized as a homodimer composed of 241 amino acids with a molecular mass of 39 000. Human IL‐34 shows sequence identities of 71%, 72% and 99·6% with mouse, rat and chimpanzee IL‐34, respectively.11 Interleukin‐34 is evolutionarily distant from, but structurally related to, CSF‐1; they are both N‐glycosylated secreted proteins21 (Fig. 1). Although they are both glycosylated secreted proteins, CSF‐1 could be more stable than IL‐34 in the absence of glycosylation, underlying a higher importance of the glycosylation for IL‐34. At the amino acid level, IL‐34 is comparatively more conserved across species than CSF‐1.22 Interleukin‐34 has the smallest dimerization interface among the short‐chain helical cytokine family members. A ribbon representation reveals that the overall structure of IL‐34 as a distinctive antiparallel four‐helix bundle cytokine fold consisting of αA, αB, αC and αD in addition to four short helices and two β strands (α1, α2, α3, α4, β1 and β2).23 Moreover, the crystal structures of IL‐34 complexed with Fab fragments offer a structural rationale for their respective neutralizing and non‐blocking activities and provide novel insights into their utility as therapeutic targets.23

Figure 1.

Schematic structure of interleukin‐34 (IL‐34). A predicted N‐linked glycosylation site is indicated with an arrow symbol. Disulphide bridges are shown as solid purple line lines.

Receptors of IL‐34

CSF‐1R

CSF‐1R, a 165 000 glycoprotein, is encoded by the CSF‐1R proto‐oncogene.22 CSF‐1R is expressed predominantly in monocytes, tissue macrophages, osteoclasts, monocyte‐derived DCs and LCs, and is additionally distributed in neuronal cells and muscle precursors. Accumulating evidence has shown that IL‐34 expression is closely correlated with that of CSF‐1R.24, 25 Data from functional studies indicate that IL‐34 binds to the CSF‐1 receptor (also known as M‐CSF receptor, c‐fms or CD115) expressed on the cell surface of monocytes. CSF‐1R is a member of the class III receptor tyrosine kinase family (RTK‐III), which includes KIT receptor, the Fms‐like tyrosine kinase 3 receptor, and platelet‐derived growth factor receptor.26, 27 RTK‐IIIs are composed of a glycosylated extracellular region comprising five immunoglobulin‐like domains (D1–D5) in the extracellular ligand‐binding portion, a split intracellular kinase domain in the intracellular portion of the molecule, and a single transmembrane segment (Fig. 2). The interdomain flexibility between the D2 and D3 modules is a pivotal feature that allows CSF‐1R to bind to both CSF‐1 and IL‐34, despite significant differences in the specific interactions with the two molecules.23, 27 Functional analysis of the IL‐34–CSF‐1R interface disclosed that the biological activity of IL‐34 is dominated by hydrophobic interactions, rather than the salt bridge network. Interleukin‐34 binds to CSF‐1R at the cleft between D2 and D3, leading to autophosphorylation of specific tyrosine residues within the intracellular domain. CSF‐1R is phosphorylated at eight tyrosine residues (Y556, Y561, Y699, Y708, Y723, Y809, Y873 and Y923).28, 29 The majority of the phosphorylated residues lead to binding of other kinases or adaptator proteins that initiate a succession of signalling pathways necessary for survival, proliferation, differentiation migration and spreading of phagocytes, such as macrophages, osteoclasts and microglia.30 However, different intracellular domain tyrosines in the activated CSF‐1R receptor play distinct roles in regulation of macrophage function. For instance, mutation of Y559 in the juxtamembrane domain as well as Y807 in the activation loop markedly inhibited the proliferation and differentiation of macrophages in the presence of CSF‐1 whereas Y706, Y721 and Y974 mutations altered the responses of morphological phenotypes.28, 31

Figure 2.

Structure of interleukin‐34 (IL‐34)/ colony‐stimulating factor‐1 receptor (CSF‐1R) complexes and the role of IL‐34/CSF‐1R in regulation of downstream signalling pathways. IL‐34 binds to the interdomain flexibly between D2 and D3 module of CSF‐1R. Signalling pathways, including extracellular signal‐regulated kinase 1/2 (ERK1/2), AKT, focal adhesion kinase (FAK), and signal transducer and activator of transcription 3 (STAT3) are rapidly activated, which subsequently regulate target gene expression and function in cell differentiation, proliferation, inflammation and immune responses. TFBS, transcription factor‐binding sites; VEGF, vascular endothelial growth factor.

Protein‐tyrosine phosphatase‐ζ and other receptors

Interleukin‐34 is known to play a critical role in regulation of function in various cell types through interactions with CSF‐1R. However, the differential expression patterns of IL‐34 and CSF‐1R suggest that IL‐34 signalling may additionally involve an alternative receptor.32 Recently, Nandi et al.33 have found that IL‐34 binds specifically to the extracellular domain of receptor‐type protein‐tyrosine phosphatase ζ (PTP‐ζ), a chondroitin sulphate proteoglycan and cell surface receptor that is highly abundant in brain,34 and inhibits the proliferation and motility of CSF‐1R‐deficient U251 human glioblastoma cells in a PTP‐ζ‐dependent manner. In addition, Segaliny et al.35 demonstrated that IL‐34 binds to chondroitin sulphate. Among the proteoglycans with chondroitin sulphate chains, syndecan‐1 was able to modulate the IL‐34‐induced CSF‐1R signalling pathways. The interactions between IL‐34 and the CSF‐1R were limited under a low/moderate expression of syndecan‐1, which can sequestrate IL‐34 at the cell surface using its chondroitin sulphate chains. Conversely, overexpression of syndecan‐1 significantly enhanced IL‐34‐induced activation of CSF‐1R. These findings indicate that alternative receptors interacting with IL‐34 may contribute to disease development.

Biological functions of IL‐34/CSF‐1R

Several studies have confirmed the ability of IL‐34 to bind CSF‐1R and affect various physiological and pathological processes, including cell differentiation, proliferation, inflammation and immune responses.24, 36

Cell differentiation

Cell differentiation is an important physiological process by which a cell changes from a less specialized to a more specialized type. Interleukin‐34 has been shown to induce the differentiation of monocytes into immunosuppressive macrophages with an IL‐10^high IL‐12^low phenotype, also designated alternatively activated/regenerative (M2) macrophages (CD163⁺).14, 37, 38 Moreover, IL‐34 induces M2 via CSF‐1R independently of CSF‐1. Several lines of evidence demonstrate that both IL‐34 and CSF‐1 have the ability to promote differentiation of human monocytes into macrophage phenotypes with subtle but clear differences, which are best exemplified by the differentiation marker C‐C chemokine receptor type 2 (CCR2).39, 40 Furthermore, IL‐34, but not CSF‐1, is intrinsically involved in CSF‐1R‐mediated development of follicular dendritic cell‐induced monocytic cells (induced CD11b⁺ cells) in vitro.41 A recent report indicated that IL‐34 stimulates osteoclast differentiation by phosphorylating extracellular signal‐regulated kinase 1/2 (ERK1/2) and Akt, and is able to substitute for CSF‐1 entirely in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis by promoting adhesion and proliferation of osteoclast progenitors.12, 42 Likewise, IL‐34‐induced macrophages switch memory T cells into T helper type 17 cells in a membrane IL‐1α‐dependent manner.43 The data collectively indicate a vital role of IL‐34/CSF‐1R in regulation of differentiation.

Cell proliferation

Cell proliferation is a basic life activity of organisms. However, a series of growth disorders can occur at the cellular level in various diseases, such as cancer and RA, in which cells display uncontrolled growth and division beyond the normal limits.44, 45 Interleukin‐34 is known to promote the proliferation of human CD14⁺ monocytes, microglial cells and macrophages through interactions with CSF‐1R.12, 46, 47 A recent report showed that IL‐34 has more cross‐species specificity and is considerably less potent than CSF‐1 in inducing CSF‐1R‐mediated macrophage proliferation.48 In addition to immunological cells, IL‐34 is associated with the proliferation of human osteosarcoma in vivo. Moreover, IL‐34 directly stimulates endothelial cell proliferation in vitro.49

Inflammation

Inflammation is a major biological process that enables tissues to develop a host defence mechanism for protecting against injury, infectious agents, cancers or immune dysregulation diseases, including RA. Perpetuation of inflammation is a harmful process that damages tissue, and necrosis can induce inflammation.50, 51 Interleukin‐34 has been identified as a novel and moderate inflammatory cytokine that significantly induces macrophage activation and migration.13, 37 Expression of IL‐34 is increased with inflammation in patients with inflammatory bowel disease and in experimental colitis, and is associated with elevated TNF‐α and IL‐6 expression in lamina propria mononuclear cells as well as with CCL20 production in colon epithelial cells through the ERK1/2 pathway.24, 52, 53 The cytokine is overexpressed in the inflamed salivary glands of patients with Sjögren syndrome, a chronic immune disorder typically affecting exocrine glands, and regulates monocytes and/or macrophages involved in the pathogenesis of salivary gland inflammation.54 Moreover, IL‐34 dose‐dependently induces transforming growth factor‐β in microglia,46 while suppressing Candida albicans induced TNF‐α production in M1 macrophages by down‐regulating Toll‐like receptor 2 and Dectin‐1 expression.55 Another study has demonstrated that IL‐34 dramatically induces pro‐inflammation cytokine (IL‐6) and chemokine [IL‐8/CXCL8, interferon‐inducible protein 10 (IP‐10)/CXCL10, and monocyte chemotactic protein‐1 (MCP)‐1/CCL2] production from human whole blood, indicating a significant role in inflammatory disease processes.56

Angiogenesis

Angiogenesis, a hallmark of wound healing and inflammatory diseases, plays a central role in various physiological processes, ranging from reproduction and fetal growth to wound healing and tissue repair.57, 58 Interleukin‐34 is highly expressed in the pro‐inflammatory environment, e.g. that of the cancer osteosarcoma, indicating potential involvement in the pathogenesis of osteosarcoma. The cytokine modulates focal adhesion kinase, Src, Akt and ERK1/2 signalling pathways in endothelial cells, and as expected, these signal molecules appear critical for IL‐34‐mediated angiogenesis. In addition, IL‐34 is proposed to modulate angiogenesis by stimulating the secretion of vascular endothelial growth factor, IP‐10, MCP‐1 or IL‐8 and promote osteosarcoma progression by increasing the tissue vasculature and enhancing recruitment of macrophages and their differentiation into the M2 phenotype. Consequently, IL‐34 appears to act as a pro‐metastatic regulator in osteosarcoma. Moreover, IL‐34 may play a major role in inflammatory disease progression through regulating mononuclear phagocyte adhesion to the endothelium, angiogenesis and macrophage recruitment.

Cell adhesion and migration

Cell adhesion and disassembly drive the migration cycle by activating small guanosine triphosphates of the Rho family.59 Migration is initiated by cell polarization and formation of membrane protrusions and contributes to tissue infiltration during the inflammatory diseases including RA.60 Exogenous IL‐34 is an established mediator of monocyte/CD34⁺ cell adhesion to activated human umbilical cord monolayers.49 Interleukin‐34 has been shown to induce a significant increase in migration of myeloid cells (THP‐1 and M2a macrophages), through a syndecan‐1‐dependent mechanism35 and promotes adhesion of osteoclast progenitors as well as migration of murine macrophage J774A.1 cells.12, 61

Regulation of IL‐34 expression

As aberrant expression of IL‐34 in various immune cells may lead to cancer progression or autoimmune disease, targeting of the cytokine may provide a successful therapeutic strategy for RA. However, effective delivery of IL‐34 inhibitors to specific cell types remains a considerable challenge. Elucidation of the regulatory mechanisms of IL‐34 expression may therefore be beneficial for treatment. Expression of IL‐34 was markedly up‐regulated by TNF‐α, IL‐1β and IL‐17 in RA fibroblast‐like synoviocytes (FLS), compared with patients with osteoarthritis.19 Chemel et al.17 further investigated the mechanisms underlying regulation of IL‐34 by TNF‐α and IL‐1β signalling in primary human RA FLS. The group found that TNF‐α and IL‐1β enhance IL‐34 expression by activating nuclear factor‐κB and c‐Jun N‐terminal kinase (JNK) signalling pathways in a dose‐ and time‐dependent manner. However, p38 or ERK1/2 inhibitors did not affect the TNF‐α‐induced IL‐34 mRNA level, indicating that these pathways are not involved in the regulation of IL‐34.19 Moreover, the increase in IL‐34 level was more marked in TNF‐α‐stimulated RA FLS than in IL‐17‐stimulated or IL‐1β‐stimulated RA FLS, and IL‐34 mRNA expression was significantly higher in TNF‐α‐stimulated RA FLS, compared with controls.19 Blockade of TNF‐α in patients with RA using infliximab also led to reduced levels of serum IL‐34.16 However, the issue of whether IL‐1 inhibitors (the IL‐1 receptor antagonist, anakinra, and anti‐IL‐1 monoclonal antibody, canakinumab) can suppress IL‐34 expression in human RA FLS remains to be established.

Similar to induction patterns of IL‐34 in FLS, Eda et al.62 showed that pro‐inflammatory cytokines, including IL‐1β and TNF‐α, markedly stimulate IL‐34 mRNA expression through JNK and ERK1/2 but not p38 signalling in human osteoblasts. Additionally, IL‐34 levels were maximally increased after stimulation with TNF‐α (~74‐fold). MC3T3‐E1 mouse osteoblastic cells produced IL‐34 in response to TNF‐α through the nuclear factor‐κB signalling pathway in a dose‐ and time‐dependent manner.63 Other than pro‐inflammatory cytokines involved in IL‐34 induction, recent data suggested that 2‐methylene‐19‐nor‐(20S)‐1α,25(OH)₂D₃ (2MD), a potent analogue of 1α,25‐dihidroxyvitamin D₃, can significantly enhance IL‐34 expression, in not only mouse spleen but also bone, via vitamin D receptor‐mediated signals. Notably, both splenectomy and IL‐34 knockdown inhibited 2MD‐induced osteoclastogenesis.64 Equine infectious anaemia virus S2 protein also enhanced the IL‐34 response in macrophages.65 Further studies focusing on the relationship between microRNAs (miRNAs) and IL‐34 disclosed that miR‐28‐5p overexpression leads to marked suppression of IL‐34 levels in hepatocellular carcinoma cells.66

Recently, Hwang et al. showed that elevated levels of IL‐34 in FLS of RA patients are the result of autocrine stimulation by TNF‐α.19 Since TNF‐α was the most potent stimulator of IL‐34 expression in RA synovial fibroblasts, infliximab or eternacept (TNF‐α blockade therapy) was sufficient to reduce the serum concentrations of IL‐34 and disease activity of RA patients.16, 67 The data suggest that IL‐34 produced by FLS is a downstream cytokine of TNF‐α. Furthermore, IL‐34 elevation in plasma from RA patients was markedly decreased after DMARD treatment,consistent with a decrease in DAS28 activity.19, 68 These results highlight the value of exploring the key pathways involved in regulation of IL‐34 expression. Based on the information obtained, therapeutic agents, such as antibodies for pro‐inflammatory molecules, may be effectively used to regulate the expression of IL‐34 in RA.

Role of IL‐34 in the pathogenesis of RA

Aberrant expression of IL‐34 in RA

Rheumatoid arthritis is a complex autoimmune disorder mainly characterized by symmetric inflammation of synovial joints. Abnormal inflammatory and immune responses account for the majority of clinical manifestations in patients with active RA, although the aetiology and pathogenesis of the disease remain to be clarified.69 Cytokines clearly play a key role in driving synovial cell activation that leads to joint destruction. The pleiotropic effects of IL‐34, a recently discovered cytokine, on the immune system are at the preliminary stages of investigation. Interleukin‐34 produced by epithelial lineage cells is indispensable for the development of tissue macrophage‐like cells.70 Interestingly, recent studies disclosed that IL‐34 is also expressed in synovial fibroblasts and the synovial sublining and intimal lining layer from patients with arthritis, and significantly associated with synovitis severity.17, 71 Levels of IL‐34 were markedly higher in FLS, serum and synovial fluid of RA patients compared with healthy individuals and osteoarthritis patients,16, 19, 72 and IL‐34 levels were correlated with the total leucocyte count in synovial fluid.17 Furthermore, a positive correlation was observed between serum levels of IL‐34 and rheumatoid factor as well as anti‐cyclic citrullinated peptide antibody titres in RA patients.18 More recently, IL‐34 levels in synovial fluid were shown to be markedly higher in RA patients with a high 28‐joint Disease Activity Score (DAS28 ≥ 3·2) than in those with lower DAS28 (< 3·2).68 Hence, unusual IL‐34 levels may represent an effective marker of active RA. A quantitative real‐time PCR study further disclosed that IL‐34 is highly expressed by osteoblasts.15 Taken together, the findings to date clearly suggest that IL‐34 functions in the pathogenesis of RA.

Role of IL‐34 in bone erosion and cartilage destruction

Pro‐inflammatory cytokines promoting inflammation and osteoclastogenesis in the arthritic joint play a major role in the pathogenesis of RA and other autoimmune diseases.73 Identification of individuals with severe joint damage progression is necessary for physicians to tailor treatment strategies, as RA has a heterogeneous course. Several studies have been performed to identify effective predictors of aggressive and erosive RA. Osteoclasts are specialized cells that differentiate from the monocyte/macrophage haematopoietic lineage in response to RANKL as well as CSF‐1, involved in a variety of inflammatory bone degenerative diseases, including RA.74 Bone and cartilage destruction of RA are mediated by bone‐resorbing osteoclasts at the bone–pannus interface of the synovium owing to chronic inflammation of multiple synovial joints in RA.75 These chronic inflammatory conditions may promote osteoclast formation and a subsequent increase in resorbing activity.76 RANKL is a key molecule in the pathogenesis of RA, which participates in osteoclastogenesis and activation of bone resorption.77 Interestingly, accumulating evidence suggests that IL‐34 contributes to the pathogenesis of RA due to its pro‐inflammatory and osteoclastogenesis‐inducing properties.12, 17, 42 A recent study by Chen et al.15 showed that recombinant mouse IL‐34 combined with RANKL not only promotes the differentiation of mouse osteoclast‐like cells from splenocytes and bone marrow in a dose‐dependent manner but also forms osteoclasts with bone‐resorbing activity. Moreover, IL‐34 induces osteoclast differentiation from human peripheral blood mononuclear cells. Another report showed that IL‐34 and conditioned medium (containing soluble IL‐34 secreted from RA FLS cultured with TNF‐α) promotes chemotactic migration of peripheral blood mononuclear cells and subsequent osteoclast formation; inhibition of functional IL‐34 with a specific antibody also suppresses osteoclast formation.19

A multivariate logistic regression analysis study reported that IL‐34 is associated with the rate of progression of erosion and joint space narrowing scores reflecting cartilage loss of RA joints, supporting its potential as a biomarker for predicting subsequent radiographic progression in patients with active RA.72 Another investigation demonstrated that the serum level of IL‐34 is significantly higher in the third stage of X‐ray progression than in the second, and positively associated with anti‐cyclic citrullinated peptide antibody production and MMP‐3 levels, which are related to the severity of RA bone destruction.78 Notably, both AFS98 (an anti‐CSF‐1R monoclonal antibody) and GW2580 (a CSF‐1R kinase inhibitor) were recently shown to abrogate bone erosion, which was associated with depletion of osteoclasts in collagen‐induced arthritis (CIA), serum‐transfer arthritis or adjuvant arthritis.79, 80

Articular cartilage destruction, a central feature in joint diseases, is a formidable clinical problem.81 Chondrocyte apoptosis, metalloproteinase and aggrecanase‐mediated extracellular matrix degradation contribute to progressive cartilage degeneration in RA. Cartilage destruction is guided by inflammatory cytokines, which promote the expression of key factors that contribute to chondrocyte death as well as extracellular matrix metabolic imbalance. CSF‐1R is expressed in pre‐hypertrophic layers of developing articular chondrocytes, and expression of CSF‐1 is induced in articular chondrocytes under inflammation stimulation.82, 83 Importantly, blockade of CSF‐1R by AFS98 or GW2580 strongly protects against cartilage destruction in rats with adjuvant arthritis and in mice with CIA.79, 80 Further studies are required to determine whether IL‐34 is expressed in articular chondrocytes and its cell‐specific roles in RA.

The reports collectively suggest that IL‐34/CSF‐1R is also correlated with severity of RA bone erosion and cartilage damage and plays a vital role in the aetiology of RA by supporting osteoclastogenesis and adjusting the balance of bone metabolism. Clarification of the precise biological roles of IL‐34 in the bone loss of arthritis may aid in the identification of novel pharmacological targets.

Role of IL‐34 in the inflammatory process

Inflammation is a common pathophysiological event in several diseases, such as RA, inflammatory bowel diseases and cancer. Inhibition of the synthesis or activity of inflammatory mediators remains an effective clinical strategy for treating inflammation‐mediated diseases, in particular, RA. Pro‐inflammatory cytokines inducing inflammation in the joints contribute to RA pathophysiology.84 For instance, the pro‐inflammatory cytokines IL‐1β and TNF‐α promote the production of a range of cytokines, metalloproteases and chemokines in synovial fibroblasts. Accumulating evidence suggests that IL‐1β and TNF‐α enhance IL‐34 expression in synovial fibroblasts of RA patients.17, 19 Therefore, IL‐34 may be a downstream effector through which IL‐1β and TNF‐α exert their effects on inflammation.

Interestingly, IL‐34 is proposed to contribute to the pathogenesis of RA by regulation of autoimmune‐related components, such as pro‐inflammatory cytokines and chemokines. Expression of membrane IL‐1α may be up‐regulated by IL‐34 in a time‐dependent manner during differentiation of monocytes into macrophages. Membrane expression of IL‐18, another member of the IL‐1 family, has been concomitantly detected in human macrophages induced in the presence of IL‐34.43 Furthermore, IL‐34 triggers IL‐6 expression as well as production of IP‐10, IL‐8 and MCP‐1 to a marked extent in human whole blood. Eda et al.56 demonstrated that GW2580, a CSF‐1R kinase inhibitor, induces a significant decrease in IL‐34‐induced chemokine levels in a dose‐dependent manner.

A recent study reported that neither exogenous IL‐34 nor CSF‐1 promotes IL‐6 secretion by synovial explants from patients with RA, indicating that endogenous IL‐34 and/or CSF‐1 in synovial production of RA is saturating in regard to CSF‐1R availability.71 Additionally, single blockade of either IL‐34 or CSF‐1 did not affect IL‐6 secretion by synovial tissue. However, inhibition of CSF‐1R with huAb1 led to significant concentration‐dependent decrease in synovial explant IL‐6 secretion, suggesting that these two cytokines are largely redundant.71 Moreover, huAb1 treatment suppressed the production of chemokines (CCL‐2, GCP‐2, CCL‐7, CXCL‐8, IP‐10, MIG) in synovial explants as well as secretion of IL‐1β and TNF‐α, ENA‐78 and MMP‐2. These findings suggested that inhibition of CSF‐1R signalling or dual neutralization of IL‐34 and CSF‐1 may provide effective approaches to attenuate the generation of autoimmune‐related components in synovial tissue from patients with RA.

Interleukin‐34 was shown to increase the expression of microRNA‐21(miR‐21) through activating the signal transducer and activator of transcription 3 signal pathway in FLS, and blockage of miR‐21 expression induced resistance to apoptosis through a decrease in the Bcl‐2/Bax ratio.68 These changes could be reversed by inhibition of the CSF‐1R, suggesting that blockade of IL‐34/CSF‐1R can suppress proliferation of FLS, which reduces subsequent accumulation of inflammatory cytokines. However, further research is essential to delineate the precise roles and underlying mechanisms of IL‐34 in the inflammatory process associated with RA, both in vitro and in vivo (Table 2).

Table 2.

The role of interleukin‐34 in cell proliferation, differentiation, inflammation, angiogenesis, migration and adhesion

	Target cells	Roles	IL‐34/CSF‐1R inhibition	Mechanisms	References
Proliferation	Osteoclast precursors Microglia Macrophages Endothelial cells Osteosarcoma cells	→ → → → →	GW2580 GW258	p‐ERK1/2↑ TGF‐β↑, oAβ↓, IDE↑, HO‐1↑ p‐Src↑, p‐FAK↑, p‐ERK1/2↑, p‐P38↑	12 46, 47 11, 22, 48 49 49
Differentiation	Human CD14+ monocytes Murine CD11b+ cells Macrophages PBMC Memory T cells FDMCs	↗ ↗ ↗ ↗ ↗ ↗	GW2580 GW2580 anti‐IL‐34 antibody, IL‐34‐specific shRNA	TRAP↑, NFATc1↑, cathepsin K↑, CCR2↑, p‐Akt↑, p‐ERK1/2↑ TRAP↑, cathepsin K↑, p‐Akt↑, p‐ERK1/2↑ M2 macrophage polarization↑, Recruitment↑, Osteoclastogenesis↑ M2 macrophages↑, IL‐10↑, IL‐12↑, CD14↑, CD163↑ Th17 cells↑, CCR6↑, CCR4↑, CD161↑, membrane IL‐1α↑	12, 39 12 42, 49 14 43 41
Inflammation	IL‐34‐macrophages Colon epithelial cells LPMCs Microglia M1 macrophages HWB	┤ → → ┤ ┤ →	anti‐IL‐34 antibody anti‐IL‐34 antibody GW2580	TNF‐α↓, IL‐1β↓ CCL20↑, p‐ERK1/2↑, p‐P38↑ TNF‐α↑, IL‐6↑, p‐ERK1/2↑ TGF‐β↑ TNF‐α↓, TLR2↓, Dectin‐1↓ IL‐6↑, IP‐10/CXCL10↑, IL‐8/CXCL8↑, MCP‐1/CCL2↑	24 53 52 46 55 56
Angiogensis	Endothelial cell	→		Pseudotubes↑, VEGF↑, IP‐10↑, MCP‐1↑, IL‐8↑	49
Migration	THP‐1, M2 macrophages J774A.1 cells	→ →	GW2580	Syndecan‐1 dependent, M‐CSFR phosphorylation↑, p‐ERK1/2↑	35 61
Adhesion	Osteoclast progenitors Monocyte/CD34+ cell	→ →			12 49

oAβ, oligomeric amyloid β; IDE, insulin‐degrading enzyme; HO‐1, haeme oxygenase‐1; PBMC, peripheral blood mononuclear cells; FDMs, follicular DC‐induced monocytic cells; CCL, CC chemokine ligand; LPMCs, lamina propria mononuclear cells; TLR2, toll‐like receptor 2; HWB, human whole blood; TRAP, tartrate‐resistant acid phosphatase; IP‐10, interferon‐inducible protein 10; MCP‐1, monocyte chemotactic protein‐1; CXCL, chemokine (C‐X‐C Motif) Ligand; VEGF, vascular endothelial growth factor.

“↑”, up‐regulation; “↓”, down‐regulation; “→”, promote; “↗”, induce; “┤“, inhibit.

Therapeutic implications of IL‐34 in RA

Accumulating evidence obtained from both humans and mouse models has revealed a key role of IL‐34 in RA development and progression.16, 17, 78, 85 The differences in IL‐34 expression between patients and healthy controls support the potential utility of anti‐IL‐34 therapy for RA patients characterized by high levels of IL‐34. Significant discoveries regarding the effects of targeting IL‐34/CSF‐1R in autoimmune diseases have recently been reported. Before identification of IL‐34, administration of anti‐CSF‐1R antibody in experimental models of arthritis significantly suppressed bone and cartilage destruction and reduced pain‐related behaviours in an adjuvant arthritis model.79, 80, 86 Interestingly, Ohno et al.87 recently reported that anti‐CSF‐1R not only suppresses disease progression in a CIA mouse model, but also significantly attenuates pro‐inflammatory cytokine production (TNF‐α, interferon‐γ, IL‐1β and IL‐6). Consistent with this finding, oral CSF‐1R inhibitors (GW2580, Ki20227 and PLX3397) were shown to afford significant protection against bone erosion in mouse arthritis models.88, 89 An anti‐CSF‐1R antibody inhibited inflammation‐associated osteoclastogenesis in a KRN‐serum transfer arthritis mouse model90 and reduced the production of inflammatory mediators in RA synovial explants as well as pannus formation and bone destruction in the CIA.71 Another investigation showed that a specific antibody blocking functional IL‐34 induces significantly reduced osteoclast formation.19 More importantly, the efficacy of dual neutralization of IL‐34 and CSF‐1 was superior to single blockade of CSF‐1 or IL‐34 as well as anti‐TNF therapy in CIA.85 However, further studies are needed to examine the specific roles of IL‐34 in the development and treatment of RA using IL‐34 knockout (KO) mice in comparison to wild‐type mice, CSF‐1 KO mice and the double KO/or the CSF‐1R KO mice in RA model. Overall, the results indicate that specific inhibition of the IL‐34/CSF‐1R pathway may present an effective therapeutic strategy for autoimmune diseases, including RA (Fig. 3).

Figure 3.

Role of interleukin‐34 (IL‐34) in the pathological process of rheumatoid arthritis. IL‐34 acts through downstream mediators to exert biological effects on destruction of cartilage and bone and joint inflammation in rheumatoid arthritis.

Conclusions and future perspectives

Interleukin‐34 is a type of multifunctional cytokine that participates in a range of cellular processes including differentiation, inflammation, angiogenesis, adhesion and migration, and is consequently involved in the development of several diseases. Recent studies have demonstrated that IL‐34 plays a crucial role in bone destruction through promoting inflammation and osteoclastogenesis, as seen in RA. The IL‐34 level is markedly higher in both patients with RA and animal models. Conversely, IL‐34 and/or CSF‐1R suppression attenuate the severity of inflammatory arthritis in animal models.80, 85 These results support the targeting of IL‐34/CSF‐1R as a potentially effective therapy for RA.91 However, interventions for blocking IL‐34 should be considered in terms of not only beneficial effects but also the risk of potential detrimental consequences for the host. Current data on IL‐34‐mediated modulation in RA have mostly been obtained from murine models. Future investigations should focus on human systems to comprehensively elucidate the therapeutic potential of IL‐34 in RA.

Disclosures

The authors declare no financial or commercial conflicts of interest.