Exploring the Role of Macrophages and Their Associated Structures in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease characterized primarily by persistent synovial inflammation, bone destruction, joint deformity, and even functional impairment. According to current epidemiological surveys on RA, the overall incidence continues to show a rising trend, with a higher prevalence in females compared to males [1, 2]. Fundamentally, the pathological hallmark of RA is synovial inflammation and pannus formation; however, the exact pathogenesis remains incompletely understood. Numerous studies have indicated that genetic factors, environmental factors, and immune responses are closely associated with the development of RA. Macrophages, as key participants in innate and cellular immunity, possess functions such as phagocytosing pathogens, presenting antigens, and secreting various cytokines, playing a significant role in various immune and inflammatory responses [3, 4]. Due to their high plasticity, macrophages can polarize into classically activated (M1) or alternatively activated (M2) macrophages under different microenvironmental influences. These two phenotypes respectively exert proinflammatory and anti-inflammatory effects, balancing each other [5]. Disruption of this balance is one of the critical reasons leading to sustained inflammation. Additionally, when activated macrophages encounter pathogen invasion, they release a web-like structure called macrophage extracellular traps (METs) during their terminal stages to trap pathogens. However, excessive release of METs can act as autoantigens, activating immune responses and promoting inflammatory effects [6]. Furthermore, upon sensing pathogen infection, macrophages undergo a GSDMD-mediated proinflammatory and programmed cell death event known as pyroptosis. This process leads to cell swelling, plasma membrane rupture, and massive release of inflammatory substances, thereby promoting inflammatory reactions [7]. Macrophages also secrete exosomes that participate in intercellular communication and signaling, thus promoting or inhibiting disease progression [8]. Given the persistent presence of immune and inflammatory responses throughout the course of RA, the aforementioned macrophages and macrophage-derived structures may significantly impact the progression of RA. Hence, this review summarizes the research progress regarding the roles, mechanisms, and potential therapeutic strategies of macrophages and their associated structures in RA.

Macrophages, as one of the primary immune cells in both innate and adaptive immunity, exert their functions through processes such as phagocytosis of pathogenic microorganisms, antigen presentation, and production of various cytokines, thereby playing roles in host defense, alleviation of inflammation, removal of apoptotic cells, and tissue repair in the normal immune microenvironment [4]. However, in RA, when the monocyte chemoattractant protein-1 (CCL2) and CX3C motif chemokine ligand 1 (CX3CL1) expressed by fibroblast-like synoviocytes (FLS) interact with the CC chemokine receptor 2 (CCR2) and CX3C chemokine receptor 1 (CX3CR1) expressed by monocytes, it promotes the substantial recruitment of macrophages into the synovium, thus advancing the progression of synovial inflammation [9, 10]. Within the synovium, the accumulating macrophages produce increasing amounts of proinflammatory cytokines and chemokines. The interaction between chemokine receptors and their ligands further promotes macrophage migration in a self-perpetuating cycle, which in turn sustains the chronic inflammatory environment. In addition, macrophages also play a role in clearing the burden of neutrophils. During the early stages of inflammation, neutrophils accumulate in large numbers; the proteases they contain can degrade the extracellular matrix that serves as a support scaffold for infiltrating cells. Moreover, neutrophils produce large quantities of reactive oxygen species (ROS), leading to excessive oxidative reactions that can result in additional tissue damage [11]. Macrophages can induce apoptosis in neutrophils and actively phagocytose these apoptotic cells, thereby contributing to tissue repair. Furthermore, synovial tissue macrophages, which constitute a significant part of the synovial tissue, can be divided into several different subpopulations based on their origins and functions [12]. Research has identified two subpopulations of synovial tissue macrophages that possess unique transcriptomic profiles capable of producing lipid mediators that promote the resolution of inflammation and enhance the reparative activities of synovial fibroblasts, thus contributing to the amelioration of inflammation in RA [13].

In RA, besides the aforementioned functions, macrophages also engage in interactions with synovial fibroblasts. The joint synovium comprises two layers: the first is a continuous dense layer, known as the lining layer, formed by tissue-resident fibroblasts and macrophages; the second is a sparse sublining layer composed of tissue-resident fibroblasts, macrophages, adipocytes, blood vessels, and lymphatic vessels [14]. In the lining layer, a high concentration of tissue-resident macrophages connects tightly, forming an immunological barrier. Beneath this barrier, tightly packed fibroblasts secrete hyaluronic acid and lubricin into the joint space to lubricate the joint cavity. When RA develops, inflammatory responses are initiated, beginning with the disruption of the immunological barrier formed by tissue-resident macrophages. Subsequently, fibroblasts proliferate and invade the joint space. Simultaneously, pathogenic fibroblasts and Mer tyrosine kinase (MERTK) macrophages that produce alarmins expand in the sublining layer, leading to the infiltration of inflammatory cells such as neutrophils, creating a proinflammatory microenvironment. This results in the proliferation and hypertrophy of the synovium, ultimately forming pannus tissue [15], as shown in Figure 1. The pathophysiological mechanisms of RA are shown in Figure 2.

Macrophages possess high heterogeneity and plasticity; therefore, in different microenvironments and under various stimuli, they can differentiate into two distinct categories: M1 (classically activated) macrophages and M2 (alternatively activated) macrophages. These two types of macrophages have opposing yet complementary roles, as shown in Figure 3. The signaling pathways associated with macrophage polarization are shown in Figure 4.

First, M1 macrophages are induced by stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Upon formation, they initiate Th1 immune responses by secreting ROS, nitric oxide (NO), and high levels of proinflammatory cytokines, such as IL-1β, to trigger and sustain inflammatory responses and recruit other immune cells to the inflamed tissue [16]. M1 macrophages have bactericidal and phagocytic properties, as well as antitumor activity. After phagocytosis, these dead cells fuse with lysosomes to form vesicles, which are then degraded by lysosomal hydrolases [17]. Additionally, M1 macrophages are activated by pathogen-associated molecular patterns via pattern recognition receptors (PRRs) and by T cell-derived cytokines, such as IFN-γ, to induce the expression of inducible nitric oxide synthase, thereby playing a role in controlling parasitic infections [18]. However, macrophages exhibit a certain degree of tolerance, which serves to limit inflammation-induced tissue damage [19]. When M1 macrophages lose self-tolerance and secrete excessive proinflammatory factors, it can lead to an overactive Th1 immune response, resulting in chronic inflammation.

Second, M2 macrophages primarily exist within a Th2 immune environment and are induced by Th2 cytokines, such as IL-13 and IL-4. M2 macrophages release angiogenic mediators such as transforming growth factor-beta (TGF-β) and vascular endothelial growth factor, along with large amounts of anti-inflammatory cytokines, such as IL-10. Therefore, M2 macrophages play a role in promoting angiogenesis, tissue repair, and anti-inflammation. However, studies have shown that tumor-associated M2 macrophages can promote tumor growth, proliferation, and metastasis [20]. Consequently, in addition to their distinctly different roles in inflammation, M1 and M2 macrophages also have completely opposite effects on tumors. For example, researchers have targeted the IL4 receptor to allow albumin-bound paclitaxel, a commonly used agent for treating malignant tumors, to reprogram M2 macrophages into an M1 phenotype via ROS-HMGB1-TLR4 axis, thereby enhancing antitumor immunity and inhibiting tumor growth and metastasis [21].

However, unlike M1 macrophages, M2 macrophages can differentiate into four distinct subtypes – M2a, M2b, M2c, and M2d – upon exposure to different stimuli. Each of these subtypes, once induced by different substances, secretes distinct cytokines and exerts different effects. Specifically, M2a macrophages are induced by IL-4 and IL-13 and secrete IL-10 and TGF-β, contributing to type 2 immunity, allergy, and fibrosis promotion. M2b macrophages are induced by Toll-like receptors (TLRs) and IL-1Ra, secreting IL-10 and TNF-α, among others, to activate Th2 immune responses and promote infection and tumor progression [22]. M2c macrophages are induced by glucocorticoids and IL-10 and exert potent anti-inflammatory effects by releasing TGF-β and IL-10, also contributing to tissue repair and matrix remodeling [23]. Lastly, M2d macrophages, also known as tumor-associated macrophages, are induced synergistically by TLRs and adenosine A2A receptors, secreting high levels of IL-10, TGF-β, and vascular endothelial growth factor, which facilitate tumor angiogenesis and metastasis [3].

The primary pathological changes in RA are persistent synovitis and bone destruction. In the synovial tissue of RA patients, macrophages account for 30%–40% of the total cellular content. Studies have shown that in RA patients, peripheral blood exhibits a mixed M1 and M2 phenotype, and during periods of disease activity, the proportion of M1 macrophages increases, disrupting the dynamic balance between M1 and M2 macrophages, thereby contributing to the progression of RA [24].

During the active phase of RA, the imbalance in the M1/M2 ratio is primarily due to the heightened activity of M1 macrophages. M1 macrophages can secrete high levels of proinflammatory cytokines and chemokines, recruiting a large number of inflammatory cells and stimulating the abnormal activation of FLS, leading to synovial hyperplasia. This hyperplasia results in increased infiltration of inflammatory cells, thereby accelerating the inflammatory response. Additionally, M1 macrophages can promote T-cell activation through their antigen-presenting function and stimulate B cells to produce more anti-citrullinated protein antibodies (ACPAs) [25]. Numerous studies have elucidated that the increase in ACPAs leads to sustained autoimmune and inflammatory responses, making the formation of ACPAs a critical step in the development and progression of RA.

There is a reciprocal relationship between the skeletal system and the immune system. Within the bone microenvironment, osteocytes and immune cells share the influence of cytokines and signaling factors, working together to maintain the dynamic balance of osteoimmunology and bone metabolism. Besides the dynamic balance of M1/M2 macrophages in osteoimmunology, there is also a dynamic balance between osteoblasts (OBs) and osteoclasts (OCs) in bone metabolism. Insufficient activation of OBs and excessive activation of OCs disrupt the OBs/OCs ratio, leading to bone formation being less than bone resorption, which results in decreased bone density and increased bone fragility, thereby creating conditions for skeletal diseases [26]. The balance of macrophage polarization and the balance among bone cells have a causal relationship, and the imbalance in both systems contributes to the formation of a pathological joint microenvironment, mediating the occurrence and development of RA. During the polarization process of M1 and M2 macrophages, TNF-α and IL-6 secreted by M1 can inhibit the activity and formation of OBs; in contrast, M2 macrophages can secrete bone morphogenetic protein-2 and TGF-β to induce OB differentiation and stimulate precursor OBs to mature into OBs [27].

However, during the entire polarization process, a greater impact is exerted by OCs. Fukui et al. [28] indicated that in RA patients, the M1/M2 ratio in vitro is positively correlated with the number of differentiated OCs, suggesting that an imbalance in the M1/M2 ratio promotes osteoclastogenesis. Numerous studies have confirmed that both macrophages and OCs originate from myeloid progenitor cells within the monocyte/macrophage system, where they compete and interact with each other [29]. OCs differentiate under the synergistic action of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), whereas macrophages differentiate under the sole action of M-CSF. Thus, macrophages can serve as a source for OC formation [30]. During periods of inflammatory activity, under various stimulatory influences, macrophages polarize toward the M1 phenotype and secrete numerous biological factors that affect OC differentiation. Among these, TNF-α, IL-1β, and IL-23 promote the differentiation and survival of OCs and enhance RANKL secretion; IL-12 inhibits the differentiation and survival of OCs; the chemokine CXCL2 promotes the proliferation of OC precursors; CXCL10 enhances the expression of RANKL and TNF-α; low levels of NO promote OC formation, while high levels inhibit it; and ROS promote OC formation [23]. During the resolution phase of inflammation, macrophages predominantly polarize to the M2 phenotype, and the IL-10 they secrete can inhibit the formation of OCs, reduce their activity, and downregulate the levels of RANKL and M-CSF, blocking the production of OC activation factors; TGF-β can inhibit the formation and activity of OCs [31].

Extracellular traps (ETs) are web-like fiber structures formed by immune cells upon stimulation, composed of DNA, proteases, histones, antimicrobial peptides, and other components. Subsequent research discovered a new form of programmed cell death, termed “ETosis.” In 2010, it was first reported that mature and differentiated macrophages can also produce ETs, referred to as METs [32]. At present, most studies focus on neutrophil extracellular traps (NETs), and research elucidating the compositional structure of METs is limited. Scholars [33] have summarized the known types of monocytes and macrophages that produce METs as well as the cellular proteins identified within the MET structures. For example, macrophages derived from human monocytes can produce METs containing histone H4; METs derived from human alveolar macrophages contain matrix metalloproteinase 12; and METs formed by human peripheral blood mononuclear cells include a variety of proteins such as histones H2 and H3, elastase, myeloperoxidase, and lactoferrin. Therefore, compared with NETs, the compositional structure of METs is largely similar [34, 35].

Currently, reports on METs are limited, and their formation mechanism remains unclear. However, considering the structural similarity between METs and NETs, it is speculated that the mechanisms of MET formation are broadly similar to those of NETs, mainly categorized into lytic and nonlytic processes. The lytic process involves the rearrangement of the cytoskeleton, degradation of the nuclear envelope, and rupture of the plasma membrane under the influence of ROS [36]. Depending on the pathway of ROS generation, this can be further subdivided into NOX (NADPH oxidase)-dependent and mitochondrial-dependent mechanisms. The nonlytic process, on the other hand, does not rely on the action of oxidants, nor does it involve mitochondrial release; it does not require cell lysis or plasma membrane rupture. Instead, it involves the formation of vesicles from the inside out by the nuclear envelope, followed by the expulsion of chromatin through these vesicles [37].

Researchers [38] have summarized that under different microenvironmental conditions and upon exposure to different stimuli, the mechanisms and effects of MET formation vary. These differences are briefly described as follows: In a fungal environment, macrophages generate METs in an NOX-independent manner, utilizing mitochondrial DNA or nuclear DNA, as seen with Candida albicans [39]. In a bacterial environment, macrophages also employ an NOX-independent pathway to regulate elastase activity to form METs, as observed with Mycobacterium tuberculosis [40] and Escherichia coli [41]. In protozoan environments, macrophages generate ROS and myeloperoxidase through an NOX-dependent mechanism to induce MET formation, as exemplified by Eimeria japonica [42]. In diabetic or obese individuals, MET formation is closely associated with peptidyl arginine deiminase type 2 (PAD2) or peptidyl arginine deiminase type 4 (PAD4)-mediated hypercitrullination of histones, which can promote inflammatory responses and insulin resistance [43]. Additionally, the mechanisms by which the same organism forms METs can differ depending on the inducing agent. For instance, MET formation by Staphylococcus aureus induced by statins is related to inhibition of the sterol pathway [32], whereas MET formation induced by phosphomycin involves NADPH oxidase-dependent ROS [44]. M. tuberculosis, after interacting with human macrophages, can induce MET formation independently of the NADPH/ROS system but requires the virulence factor ESAT-6 (early secreted antigenic target 6 kDa) for induction [45]; however, when IFN-γ is added, both MET formation and M. tuberculosis aggregation depend on the ESAT-6 secretion system 1 [46]. Furthermore, METs formed by the same organism via the same pathway can exert different effects. For example, C. albicans can stimulate mouse macrophages to form METs via a non-NADPH/ROS-dependent mechanism, and these METs can both increase infection and exert trapping and killing effects [39], as well as capture microbes at the site of infection to prevent their invasion rather than directly killing them [41].

El Shikh et al. [47] examined synovial tissues from biopsy samples of collagen-induced arthritis (CIA) in mice and RA patients, detecting that macrophages in secondary lymphoid organs and ectopic lymphoid structures within the synovium release METs. They confirmed that METs contribute to citrullination of self-antigens and the production of ACPAs, thereby promoting the progression of autoimmune responses in RA. Bashar et al. [48] proposed that the formation of METs requires PAD2 and PAD4, and that METs serve as a source of citrullinated antigens bound by RA autoantibodies. Other researchers [49] used RNA sequencing and other molecular biology methods to demonstrate that METs can promote the proliferation and migration of FLS in RA patients through the cGAS-mediated PI3K/Akt signaling pathway, thereby accelerating inflammatory responses.

Pyroptosis is an inflammatory, programmed form of cell death characterized by plasma membrane pore formation, cell swelling, lysis, and the release of inflammatory contents. Three key components of the pyroptosis pathway play crucial roles in the formation of these pores. First, inflammasomes act as intermediaries in immune signaling and transmission, serving as multiprotein complexes that trigger the activation of inflammatory caspases and processing of pro-interleukin-1β (pro-IL-1β), such as NLRP1 (NLR family pyrin domain-containing 3), NLRP3 (NLR family pyrin domain-containing 3), and NLRC4 (NLR family CARD domain-containing protein 4) [50]. Their mechanisms of action differ; NLRP1 and NLRC4 interact directly with pro-caspase-1 via their CARD (caspase activation and recruitment domain) motifs, whereas NLRP3 interacts with pro-caspase-1 through the adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) [51]. Among these, NLRP3 is particularly relevant to RA due to its enhanced activity in the peripheral blood of patients with active RA [52]. Second, cysteine-aspartic acid protease-1 (caspase-1) is activated by the inflammasome and cleaves the precursors of IL-1β and IL-18, promoting their maturation, and subsequently cleaves gasdermin D (GSDMD), a key effector protein of pyroptosis [53]. Besides caspase-1, caspase-11/4/5/3 are closely associated with pyroptosis. Among them, caspase-11/4/5, similar to caspase-1, can cleave GSDMD, whereas caspase-3 is typically considered an apoptotic caspase that primarily executes apoptosis. However, when the levels of gasdermin E (GSDME) are high, caspase-3 can induce pyroptosis downstream of apoptosis through the cleavage of GSDME [54, 55]. Third, the Gasdermin (GSDM) consists of two domains connected by a flexible peptide: a pore-forming N-terminal domain and an inhibitory C-terminal domain. It acts as a pore-forming protein that creates functional pores in the cell membrane [56]. Within the Gasdermin family, only GSDMD and GSDME have been confirmed to be cleaved by caspases to form functional membrane pores [57]. The cleavage of GSDMD by caspase-1/11/4/5 induces pyroptosis following inflammasome activation, leading to its characterization as the “executioner protein” of pyroptosis, while the cleavage of GSDME by caspase-3 leads to pyroptosis initiated by apoptosis. Studies on caspase-3/GSDME-mediated pyroptosis have mainly focused on tumor immunity [58]. Although a few of studies have found that caspase-3/GSDME can influence the progression of RA by mediating macrophage pyroptosis in RA mouse models [59], current research remains limited, insufficient to fully substantiate the role of caspase-3/GSDME-mediated pyroptosis in RA. Therefore, the discussion below focuses solely on macrophage pyroptosis executed by GSDMD.

When pathogenic microorganisms invade or the body sustains damage, the organism can detect these “danger signals.” Intracellular PRRs can identify pathogen-associated molecular patterns and damage-associated molecular patterns. Subsequently, PRRs activate and recruit inflammasome assembly, which then activates caspase [60]. Pyroptosis is primarily mediated by caspase-1 and caspase-11 in mice (or caspase-4/5 in humans), and based on the stimulus recognized by PRRs and the activated caspase, it can proceed via two pathways: the canonical inflammasome pathway mediated by caspase-1 and the noncanonical inflammasome pathway mediated by caspase-11/4/5. Initially, the caspase-1-dependent canonical inflammasome pathway is initiated by various crystalline compounds, pathogens, potassium efflux, and calcium influx, triggering the assembly of the NLRP3 inflammasome [61]. Then, the NLRP3 inflammasome recruits and activates pro-caspase-1, leading to the maturation and release of IL-1β and IL-18 by activated caspase-1. It also cleaves the flexible peptide linking the N-terminal and C-terminal domains of GSDMD, relieving the inhibitory effect of the C-terminal domain. The remaining GSDMD-N terminus oligomerizes, binds to the membrane, and forms pores in the plasma membrane [62]. Following this, the cell swells and ruptures, releasing inflammatory contents and promoting inflammation. Second, the noncanonical inflammasome pathway-induced pyroptosis was first observed in 2011 in murine macrophages infected with Gram-negative bacteria and was found to be closely related to caspase-11 [63]. Further research revealed that human caspase-4/5 shares similar functions with murine caspase-11. In the noncanonical pathway, LPS, a component of Gram-negative bacterial cell walls, binds to the CARD domain of pro-caspase-11/4/5, activating caspase-11/4/5. Activated caspase-11/4/5 directly cleaves GSDMD, forming pores in the plasma membrane, similar to the canonical pathway [64]. However, during this process, caspase-11/4/5 cannot directly process pro-IL-1β to produce active inflammatory factors [65], as shown in Figure 5.

Gasdermin-mediated pyroptosis of macrophages can disrupt the intracellular environment necessary for pathogen replication, serving as an important defense mechanism against bacteria, yeast, and mammals. However, excessive pyroptosis can lead to the onset and progression of chronic inflammatory diseases, including RA. Given the presence of fibroblast-macrophage crosstalk in the RA synovium, and since synovial fibroblasts can express several GSDMs and produce IL-1β, Demarco et al. [66] hypothesized that synovial fibroblasts may also undergo pyroptosis, exacerbating inflammation, suggesting that pyroptosis might enhance fibroblast-macrophage cross talk. Zhang et al. [67] used multiplex immunohistochemistry to determine that, compared to osteoarthritis patients, RA patients exhibit increased levels of NLRP3, caspase-1, and cleaved GSDMD in synovial fluid macrophages, along with significantly elevated levels of IL-1β and IL-18, indicating that macrophage pyroptosis is a driving factor in RA inflammation. Other researchers [68] analyzed data showing that DNA polymerase beta (Pol β) is significantly downregulated in RA and that the lack of Pol β increases the likelihood of macrophage infiltration and bone destruction. Subsequently, in vitro experiments confirmed that Pol β deficiency exacerbates LPS-induced macrophage pyroptosis and upregulates the expression of NLRP3, IL-1β, and IL-18. Additionally, studies have confirmed that sodium chloride promotes macrophage pyroptosis through a specific signaling pathway, thereby exacerbating RA [69].

Exosomes are bilayered vesicles composed of complex RNA, proteins, lipids, and other substances and belong to the category of extracellular vesicles. Exosomes are formed when the outer membrane of multivesicular bodies, derived from endosomal invagination, fuses with the cell membrane and releases into the extracellular matrix [70], as shown in Figure 6. Exosomes can be produced by various cells under physiological or pathological conditions, such as epithelial cells, fibroblasts, and immune cells, and they can naturally exist in various bodily fluids [71]. Exosomes serve as important mediators of intercellular communication, regulating target cell function through interactions with target cells and participating in multiple physiological and pathological processes, such as inflammatory and immune responses. Macrophages can also form exosomes, which can influence the polarization of macrophages to M1 or M2 phenotypes, present cytokines, and participate in the progression of RA [72].

Macrophage-derived exosomes can activate TLRs), present inflammatory factors, or use the various inflammatory factors and chemokines they contain to activate the NF-κB signaling pathway, thereby regulating macrophage polarization [73]. Macrophage exosomes can not only promote the development of inflammatory responses but can also inhibit the expression of NF-κB and the activation of TLRs through three miRNAs they carry, thereby preventing the overactivation of the immune response. For example, researchers [74] loaded plasmid DNA encoding IL-10 and betamethasone phosphate sodium into exosomes derived from M2-type macrophages and injected these exosomes into an RA mouse model. Observations showed that compared to the control group, the levels of proinflammatory factors in the joints of the experimental group mice were reduced, while the levels of anti-inflammatory factors were increased, and the average inflammation index was lower in the experimental group, indicating that exosomes generated by macrophages can serve as excellent drug carriers for treating RA.

RA, a chronic autoimmune disease, is characterized by a prolonged and difficult-to-treat course. Currently, clinical management primarily involves nonsteroidal anti-inflammatory drugs, antirheumatic medications, and newer biological agents targeting specific pathways to control the disease. With recent advances in understanding the pathogenesis of RA, numerous potential targeted therapies aimed at different mechanisms have emerged. As the most abundant immune cells in the joint microenvironment of RA patients, macrophages are present throughout the course of the disease and significantly influence its progression. Therefore, targeting macrophage polarization, METs, macrophage pyroptosis, and macrophage exosomes provides potential therapeutic avenues for RA treatment.

The impact of macrophage polarization on RA is attributed to an increase in M1 macrophages, leading to an elevated M1/M2 ratio and an imbalance in polarization. Therefore, the general strategy for targeted therapy of RA through modulation of macrophage polarization aims to repolarize macrophages and restore the dynamic balance of the M1/M2 ratio. Numerous researchers have established and intervened in RA mouse models, demonstrating that compounds such as tripterygium glycosides, punicic acid, and gelsemium alkaloids can inhibit M1 polarization and promote M2 polarization, thus achieving macrophage repolarization and improving joint inflammation and bone destruction in RA [75‒77]. Increasingly, studies based on the principle of macrophage repolarization have developed novel biologics and applied them to RA rat models. By inhibiting M1 polarization or promoting M1 apoptosis, these interventions facilitate M1/M2 repolarization, effectively alleviating or treating RA [78‒80]. For example, Chen et al. [81] found that Au25-synthesized nanoclusters significantly promoted the polarization of M1 macrophages to M2, effectively reducing synovial hyperplasia and inflammatory cell infiltration, thereby alleviating RA.

Currently, most literature on METs focuses on how METs are formed, how different microorganisms induce METs, and the role of METs in various diseases, such as acute kidney injury [82], atherosclerosis [83], chronic obstructive pulmonary disease [84], secondary spinal cord injury [85], and cystic fibrosis lung disease [86]. However, there is a paucity of research specifically targeting METs for therapeutic purposes, and studies explicitly targeting METs in the treatment of RA are even rarer. The imbalance between the formation and clearance of ETs is a fundamental mechanism underlying the progression of RA. Therefore, the exploration of therapeutic strategies targeting ETs in RA can be summarized in two main directions: reducing the formation of ETs and increasing their clearance. At present, biological agents that have been proven to reduce the formation of NETs include ROS inhibitors, PAD inhibitors, anti-inflammatory/immunomodulatory agents, antithrombotic agents, and others [87, 88]. Additionally, multiple studies have shown that exogenous recombinant human deoxyribonuclease I (DNase I) can degrade the DNA-protein immune complexes produced during NET formation, aiding in their degradation and clearance [89‒91]. However, the efficacy and side effects of these biological agents still require extensive experimental validation. Since most current research focuses on the mechanisms and targeted therapies of NETs, the aforementioned treatments are based on NETs. Whether these NET-based treatments are effective against METs and whether summarizing all ETs under the umbrella of NETs is an oversimplification remain questions that need further investigation.

During pyroptosis of macrophages, caspase-1 activated by inflammasomes processes pro-IL-1β and IL-18 into mature inflammatory cytokines IL-1β and IL-18. When GSDMD is cleaved by caspase-1/11/4/5, it forms pores in the plasma membrane, causing cell rupture and the release of large amounts of IL-1β, IL-18, and other inflammatory contents, thereby exacerbating the inflammatory response in RA. This process is one of the significant sources of proinflammatory cytokines in the course of RA. Therefore, targeting the inhibition of macrophage pyroptosis may become a new therapeutic approach for RA. Ge et al. [92] treated CIA mice with punicalagin and discovered that punicalagin could mitigate pyroptosis in CIA mice by downregulating the expression of NLRP3 and caspase-1. This reduction in pyroptosis consequently improved inflammation in the CIA mice. Another study [93] treated adjuvant arthritis rats with paeoniflorin monomer derivatives and found that after treatment, the expression of TLR4, NLRP3, caspase-1, ASC, and GSDMD-N in macrophages was downregulated, and the ratio of macrophage pyroptosis decreased. Concurrently, a reduction in joint swelling and a significant relief of inflammation were observed, indicating that paeoniflorin monomer derivatives can effectively alleviate the severity of adjuvant arthritis by inhibiting macrophage pyroptosis. Two classes of pyroptosis inhibitors have been identified to date: the first class includes small molecule drugs that block NLRP3, such as MCC950, CY-09, BOT-4-ketone, and OLT1177, which are associated with arthritis; the second class includes new pyroptosis inhibitors that block GSDMD, such as the natural polyphenol punicalagin [61], necrosulfonamide [94], dimethyl fumarate [95], and disulfiram [96, 97].

Exosomes, acting as mediators of intercellular communication, can interact with target cells and exert targeted effects. In clinical treatments, exosomes are often used as carriers, loading drugs or biologics into or onto exosomes to form a new type of targeted formulation that is administered to the diseased site. According to the studies reported to date, some research has focused on macrophage-derived exosomes to develop a drug delivery platform, which is then tested in RA models. For example, Zhao et al. [98] prepared nanovesicles simulating M2 macrophage exosomes and loaded them with M1 macrophage membranes to create a hybrid nanovesicle, which was further loaded with black phosphorus nanosheets. After injecting these vesicles into an RA mouse model, they observed that the vesicles could inhibit proinflammatory mediators and release anti-inflammatory mediators, thereby suppressing the severity of RA. Additionally, some studies have simulated exosomes from other cells or developed biomimetic exosomes to serve as carriers for targeted regulation of macrophage polarization to achieve therapeutic goals. For instance, researchers [99‒101] have demonstrated that exosomes derived from bone marrow mesenchymal stem cells can promote the phenotypic transition of synovial macrophages from M1 to M2, regulate chondrogenesis, and inhibit inflammatory responses to alleviate osteoarthritis.

RA is highly disabling, and the chronic pain and progressive functional impairment that occur during the disease’s progression significantly impact the quality of life and mental health of patients. Current treatments for RA mainly involve the use of antirheumatic drugs (DMARDs) or surgical intervention. DMARDs include traditional synthetic DMARDs (e.g., methotrexate), biologic DMARDs (e.g., adalimumab), and targeted synthetic DMARDs (e.g., Janus kinase inhibitors) [102]. However, the side effects of these drugs and the significant risks associated with surgery are unavoidable, and balancing the effectiveness and safety of the drugs, as well as considering withdrawal reactions posttreatment [103], remains challenging.

Macrophages are the most abundant immune cells in the joint microenvironment of RA patients. Besides their roles in phagocytosis, antigen presentation, and cytokine secretion, they can polarize into M1 or M2 macrophages with proinflammatory or anti-inflammatory effects, respectively. An elevated M1/M2 ratio exacerbates the inflammatory response in RA. Additionally, activated macrophages can form METs upon stimulation, which trap and kill pathogens. However, when the formation and clearance of METs are imbalanced, excessive METs can exacerbate the inflammatory response and accelerate the progression of RA. Upon infection by pathogens, macrophages can also form pores in the plasma membrane, leading to cell swelling and rupture, and the release of large amounts of inflammatory substances, which further exacerbates the inflammatory response and accelerates the progression of RA – this process is known as macrophage pyroptosis. Moreover, macrophages can generate extracellular vesicles called exosomes, enhancing their ability to communicate with other tissues and cells and influencing processes such as cell proliferation and differentiation. Thus, avoiding an imbalance in the M1/M2 ratio, excessive accumulation of METs, excessive macrophage pyroptosis, and developing new biologics using exosomes as carriers represent promising new strategies for treating RA. However, the number of studies discussing the roles of macrophage polarization, ETs, pyroptosis, and exosomes in RA remains limited, especially those concerning METs, and most reported studies are still in the animal experimentation stage. Therefore, whether these potential treatments will be effective in humans and whether they will cause adverse reactions still requires more research and sufficient clinical trials to explore and verify. We believe that further elucidation of the mechanisms underlying RA and the influence of macrophages and their associated structures on the development of RA, as well as the conduct of additional clinical trials, will contribute to leveraging macrophages as biomarkers and targets for diagnosis and treatment in RA.

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

The authors declared that financial support was received for the research, authorship, and/or publication of this article. This study was supported by Anhui Province Traditional Chinese Medicine Heritage and Innovation Project (2024CCCX117), National Natural Science Foundation of China (NSFC) General Program (No. 82374117), Provincial Department of Education (Key Project) (No. 2023AH050858), Health Commission Outstanding Talent Project (2022) (No. 392), Anhui Province Higher Education Institutions Natural Science Foundation Project (No. KJ2019A0448), and Anhui Province Traditional Chinese Medicine Heritage and Innovation Research Project (No. 2020ccyb13). The funders had no role in the study design, execution and analysis, and manuscript conception, planning, writing, and decision to publish.

Conceptualization and writing – original draft preparation: Xin Tian; writing – review and editing: Jingjing Chen, Yujie Hong, Cao Yang, Jing Xiao, and Yan Zhu.