Emerging role of targeting macrophages in rheumatoid arthritis: Focus on polarization, metabolism and apoptosis

Abstract Macrophages maintain a dynamic balance in physiology. Various known or unknown microenvironmental signals influence the polarization, activation and death of macrophages, which creates an imbalance that leads to disease. Rheumatoid arthritis (RA) is characterized by the massive infiltration of a variety of chronic inflammatory cells in synovia. Abundant activated macrophages found in RA synovia are an early hallmark of RA, and the number of these macrophages can be decreased after effective treatment. In RA, the proportion of M1 (pro‐inflammatory macrophages) is higher than that of M2 (anti‐inflammatory macrophages). The increased pro‐inflammatory ability of macrophages is related to their excessive activation and proliferation as well as an enhanced anti‐apoptosis ability. At present, there are no clinical therapies specific to macrophages in RA. Understanding the mechanisms and functional consequences of the heterogeneity of macrophages will aid in confirming their potential role in inflammation development. This review will outline RA‐related macrophage properties (focus on polarization, metabolism and apoptosis) as well as the origin of macrophages. The molecular mechanisms that drive macrophage properties also be elucidated to identify novel therapeutic targets for RA and other autoimmune disease.

also influence macrophage activation, polarization and apoptosis.
The number of macrophages in RA synovium is significantly correlated with disease activity indicators (including c-reactive protein level, erythrocyte sedimentation rate, joint swelling count, synovial lining vascular density and thickness, and radiological severity). 3 Additionally, macrophage depletion can instigate chronic arthritis in both mouse and human systems. 4 In addition, inflammatory conditions and phosphoinositide 3-kinase (PI3K) signalling pathways may enhance the anti-apoptosis ability of macrophages mediated by Fas/ Fas ligand interactions or by cytokine withdrawal. 5 In RA synovium, the anti-apoptosis ability of pro-inflammatory macrophages is stronger than that of anti-inflammatory macrophages.
Multiple studies have demonstrated the potential role of macrophages as a novel therapeutic target in autoimmune disease. In RA, the changes in the number of synovial macrophages and the expression of inflammatory products reflect the therapeutic efficacy. 6 In addition to numbers, the polarization status of macrophages is closely related to RA. Noticeably, M1 and M2 macrophages appear to be two extreme situations for the dynamically changing macrophage phenotype. Therefore, it is necessary to use the ratio of M1/M2 to evaluate the disease activity and treatment efficacy, rather than the change of a single indicator of M1 or M2. However, an unbiased and systematic approach has not been undertaken to determine the molecular signatures and biological functions of macrophages with dynamically changed phenotypes that underlie the potential targeting of macrophages in RA treatment.
In this review, we discussed our expanding view of RA-related macrophage properties (focus on polarization, metabolism and apoptosis) as well as the origin of macrophages. The molecular mechanisms that drive macrophage properties also be elucidated to identify novel therapeutic targets for RA and other autoimmune disease.

| THE ORI G IN OF R A-REL ATED MACROPHAG E S
Peritoneal and bone marrow-derived macrophages are widely studied in arthritis animal models, while peripheral blood monocytes and synovial macrophages are widely studied in patients with RA ( Figure 1).
Initially, it was thought that there were two main macrophage subsets found in the peritoneal cavity of adult mice and named as large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). 7 Approximately 90% of peritoneal macrophages are LPMs which are derived from foetal liver macrophages and disappear rapidly from stimulation by lipopolysaccharide (LPS) or thioglycolate.
After the disappearance of LPM, SPM holds the dominant position, but SPMs are derived from bone marrow-derived monocytes. 7 More F I G U R E 1 The origin and marks of RA-related macrophages in mice. Peritoneal macrophages, bone marrow-derived macrophages, peripheral blood monocytes and synovial macrophages are widely studied in RA and animal arthritis model. These heterogeneous macrophages show different marks, roles and origins. Bone marrow-derived macrophages are derived from Ly6c hi monocytes, which can possess the phenotype of resident macrophages. Two main macrophage subsets found in peritoneal cavity of adult mice named as LPM and SPM. Approximately 90% peritoneal macrophage are LPMs which are derived from foetal liver macrophages, SPMs are derived from bone marrow-derived monocytes. Two types of SM were found: embryonic SMs (ESMs) which were F4/80 + CD11band appeared at a mid-embryonic stage; and bone marrow-derived SMs (BMSMs), which were F4/80 -CD11b + and appeared at a late-embryonic stage. ESMs expressed anti-inflammatory cytokines such as IL-4 and IL-10, and BMSMs expressed pro-inflammatory cytokines such as IL-1β and TNF recently, a study has detailed the subtypes of macrophages in the peritoneal cavity of mice. 8 There are multiple subsets of macrophages in the peritoneal cavity, which can be identified by F4/80, CD64, T-cell immunoglobulin domain and mucin domain 4 (TIM4), and lymphocyte antigen 6c (Ly6c) staining. 9 Ly6c -, F4/80 hi , CD64 + , and Tim4 -, and Ly6c -, F4/80 hi , CD64 + , and Tim4 + represent resident peritoneal macrophage populations. Tissue-resident macrophages are mainly derived from yolk sac macrophages and foetal liver monocytes. Recent studies have shown that tissue-resident macrophages can also be derived from embryos and sustain themselves for a long time through local proliferation independent of hematopoietic stem cells. 10-12 F4/80 lo/-, CD64 + and Ly6C + represent monocyte-derived macrophages. 9 Bone marrow-derived macrophages are derived from Ly6c hi monocytes, which can possess the phenotype of resident macrophages. 13 Under normal physiological conditions, tissue-resident macrophages with a certain proliferation capacity are in a state of low differentiation and low proliferation to maintain homeostasis balance. Under tissue-resident macrophage depletion, inflammatory conditions or physiological stress, abundant peripheral blood monocytes enter the tissues via blood and differentiate into M1 macrophages with TNF-α and inducible nitric oxide synthase (iNOS) as the main marker by upregulating their gene expression associated with the macrophage. 14 The number of peripheral macrophages raised through lymphatic vessels decreases after inflammation. 15 After the inflammatory response subsides, the bone marrow-derived macrophages in the liver will replace the resident Kupffer cells. 16 However, in most cases, the bone marrow-derived macrophages cannot replace the tissue-resident macrophages, and most macrophages will undergo programmed death after the inflammatory response subsides. 17 A small proportion of bone marrow-derived macrophages will continue to survive, displaying strong plasticity, and can directly differentiate into M2 macrophages. However, this mechanism remains unclear. 18 In in vitro experiments, macrophage colony-stimulating factor (M-CSF) can usually be used to induce monocytes in the bone marrow to differentiate into bone marrow-derived macrophages. 19 The normal synovial membrane is comprised of two distinct parts: the intimal lining layer and synovial sublining layer. The intimal lining layer consists of fibroblast synovial cells (FLS) and macrophage-like synovial cells (MLS). The synovial sublining layer consists of synovial macrophages (SMs), blood vessels and other cells. 20 Transcriptome analysis of FLS and MLS isolated from patients with F I G U R E 2 Macrophage polarization in RA. CD163 and CD32 are highly expressed in the synovial lining macrophages of RA patients, CD163, CD32 and CD64 are highly expressed in the macrophages in the lining layer. The synovial macrophages of RA patients highly express MMP12, TNF-α and the transcription proteins PHD3 and CCR2 of pro-inflammatory gene EGLN3 and show lower expression of M2 macrophage indicator CD209. RA synovial fluid macrophages express pro-inflammatory genes (INHBA, FCER1A, SLC2A1, MMP12, EGLN3, CCR2), while express low expression of anti-inflammatory genes (IGF1, HTR2B, FOLR2, CD36). CD163 and FRβ are expressed in synovial fluid macrophages, while CD209 is not expressed. Some studies confirm that RA synovial fluid highly express M1 macrophage indicators, including HLA-DR, CD40, CD80, CD86 and CD276. M1 macrophage indicators HLA-DR, CD86, CD64 and CCR5 are highly expressed in mononuclear macrophages in peripheral blood of RA patients, while M2 macrophage indicators CD163 shows low expression, and CD200R and CD16 show no difference. Peritoneal macrophages from AA rats produce high levels of TNF-α, IL-1β, IL-6 and IL-23 and low levels of TGF-β1. Peritoneal macrophages from CIA mice express high levels of CD86 and iNOS and low levels of CD206 and Arg1 RA confirms that MLS is a macrophage with a strong inflammatory tendency. 21 At first, despite advances in identifying the development of tissue-resident macrophages, the origins of SMs are elusive.
Beige mice carry a gene (bg) that encodes the presence of large intracellular particles in many cell types, 22 transplanted bone marrow from beige mice into irradiated normal mice. They observed giant granules in the synovial lining cells of bone marrow recipients that were similar to those of the beige mouse, which indicated that bone marrow-derived cells enter through the synovial lining. Giant granulosa-bearing cells are also seen beneath the intimal lining layer. Bone marrow recipients were observed by electron microscopy in MLS, and giant granulosa were observed in MLS and FLS in the donor material (beige). 23 Their research suggests that only MLS originates from bone marrow, possibly because they resemble mononuclear phagocytes elsewhere. However, it is still possible that MLS also originates from the bone marrow, but at a slower rate of replacement. Another study has shown that macrophage populations in the synovial lining may be embryonically derived and bone marrow-derived through immunotyping, 24 though further investigations are required to verify this. Recently, the origin of synovial macrophages has been revealed by our group. 25 Two types of SMs were found: embryonic SMs (ESMs) which were F4/80 + CD11band appeared at a mid-embryonic stage; and bone marrow-derived SMs (BMSMs), which were F4/80 -CD11b + and appeared at a late-embryonic stage.

| MACROPHAG E P OL ARIZ ATI ON IN R A
The concept of M1/M2 macrophage polarization is originally proposed to explain the difference in the function of macrophages induced by different external stimuli in vitro. At present, it is believed that macrophages can also be polarized in vivo. 14 Depending on phenotypes and secreted cytokines, macrophages have been classified into many types ( Figure 2). However, they are first classified into M1 (inflammatory macrophages) and M2 (anti-inflammatory macrophages). M2 are further divided into M2a, M2b and M2c for the different stimuli. A study by Mosser et al 27 have divided macrophages into classically activated macrophages (equal to M1), alternatively activated macrophages (equal to M2a) and regulatory macrophages (equal to M2b/c). In normal physiology, the phenotype of macrophages maintains a dynamic balance. However, the inflammatory immune response is complex and rapid, and the composition of M1/M2 varies at different times, stages of differentiation and tissues 28 (Table 1) 35 The lack of activated STAT-1 results in the negative expression of many M1 markers. 31,35 NF-κB is the key in M1 polarization, which can regulate the transcription of IL-1, TNF-α, IL-6 and cyclooxygenase-2 (COX-2). 29 IRF is the participating media in macrophage polarization. High level of IRF5 found in M1, involved in the toll-like receptor (TLR) pathway, inhibits the expression of IL-10 and promotes Th1-Th17 responses. 30,36 IRF1, which has an antagonistic role on IRF4, cooperates with NF-κB and responds to multiple pro-inflammatory factors, while IRF4 is key in M2 polarization 32,36 PPARγ, a type of nucleus receptor, plays an important role in M2 polarization. 33 The liver X receptor (LXR), which is similar to PPARγ, can also increase the expression of arginase-1 (Arg1). 37

| Macrophage polarization in RA synovium
Mass immune cells, such as macrophages and lymphocytes, are observed in both the lining and sublining layer of patients with RA. A  39 These abnormal indicators suggest that M1 macrophages are dominant in the RA synovium ( Figure 2).

| Macrophage polarization in RA synovial fluid
Soler et al 39 38 indicating that the mononuclear macrophages in the peripheral blood of patients with RA tend to be M1 macrophages ( Figure 2).

| Macrophage polarization in peripheral blood of RA patients and peritoneal cavity of arthritis model
Our previous study found that peritoneal macrophages from AA rats produce high levels of TNF-α, IL-1β, IL-6 and IL-23 and low levels of transforming growth factor-β1 (TGF-β1). 42 Peritoneal macrophages from CIA mice express high levels of CD86 and iNOS and low levels of CD206 and Arg1 43 ( Figure 2).

| THE S PECIFI C ROLE OF MACROPHAG E S IN R A
Macrophages interact with a microenvironment to participate in RA inflammation development (Figure 3). When activated, macrophages produce cytokines, chemokines, metabolites and other factors to participate in the RA process.

| Macrophage mediator in RA
Mediators secreted by macrophages have been extensively summarized. Here, we focus on mediator newcomers secreted by macrophages in RA. tion. 54 In addition to the media described above, macrophages produce some other mediums which are not discussed in detail.  57 Succinic acid can combine with the GPR91 of itself and other macrophages and trigger them to release IL-1β. 57 High levels of succinic acid are found in the synovial fluid of patients with RA, and mice macrophages cultured in this synovial fluid secret a high level of IL-1β. 57 A previous study has identified increased markers of hypoxia and glycolytic metabolites in RA synovial fluid. 55 Elevated hypoxia levels were found to be negatively correlated with increased synovial inflammation. 55 Oxidative stress is conducive to glycolysis, which helps to accelerate the occurrence of inflammation and subsequent angiogenesis dysfunction in RA. 60

| Macrophage death in RA
The death of activated macrophages has been implicated in the pathogenesis of RA. Reportedly, the anti-apoptosis ability of proinflammatory macrophages is higher than that of anti-inflammatory macrophages in the synovial fluid of patients with RA. 60 Enhancing the anti-apoptosis ability or increasing the anti-inflammatory macrophage population is a potential treatment for RA.
Apoptosis is a widely accepted procedure of cell death which depends on the activation of cysteine aspartate-specific protease lack of A20 in macrophages significantly enhances NLRP3-mediated caspase-1 activation, pyroptosis and IL-1β secretion. 78 The deletion of NLRP3 and caspase-1 improves RA-associated inflammation and cartilage destruction in A20myel-KO mice.

| THE DE VELOPMENT OF ANTIRHEUMATIC DRUG S
Current treatments for RA involve DMARDs, NSAIDs, biologicals, glucocorticoids and botanical agents ( Table 2). No drugs are specific and safe for macrophages under clinical conditions, but they do inhibit some effects of macrophage activation, such as the production of inflammatory macrophage cytokines including TNFα, IL-1 and IL-6. Inhibition with antibodies or soluble receptors has been used for patients with RA for many years. Novel agents that target these factors are more efficacious in RA. Tocilizumab is the first fully human monoclonal antibody to target IL-6 receptors directly and was approved for treatment in patients with active moderate to severe RA in 2010. 79 Adalimumab and tocilizumab also impact on the hepcidin-mediated alteration of iron homeostasis. 80    polarization. Altogether, targeting physiological macrophages in RA will lead to the development of RA precision treatment.

ACK N OWLED G EM ENTS
The study was supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T S
The authors declare that there is no conflict of interests.

AUTH O R CO NTR I B UTI O N S
Study concept and design: WW, YC, and. XY; Writing of the paper: XY, YC and WW; All authors reviewed the manuscript prior to submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.