The signaling pathways implicated in the polarization of macrophages towards the M1 phenotype are extensively documented in the literature. Previous research has identified key pathways associated with inflammation mediated by M1 macrophages, such as the Notch, ERK, MAPK, JAK/STAT, and MAPK signaling pathways.

Notch signaling appears to be a causative factor in the imbalance between M1 and M2 macrophages in RA, thus playing a significant role in the pathogenesis of RA. Sun et al. (36) observed that M1 macrophages derived from bone marrow (BM) exhibit activated Notch signaling in the inflamed joints of TNF-α-transgenic mice. Furthermore, they found that RA synovial tissue promotes the activation of Notch signaling in BM-derived macrophages, leading to M1 polarization. Treatment with thapsigargin, a Notch inhibitor, reduces TNF-α-induced M1 macrophage polarization and mitigates inflammation and joint bone loss by promoting a switch from M1 to M2 macrophages. The Notch signaling pathway is crucial in regulating osteoclast differentiation and bone-resorbing activity by directly influencing osteoclast precursors, as well as indirectly affecting cells of the osteoblast lineage and immune system (37). In joints affected by RA, Notch1 is upregulated and activated in fibroblast-like synoviocytes, Th17 cells, and M1 macrophages, promoting the secretion of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-17. This cascade of events ultimately results in inflammation, bone degradation, and joint bone loss (38). The ERK signaling pathway, a member of the MAPK family, plays a significant role in regulating macrophage phenotype (39). Activation of the ERK1/2 pathway during LPS-induced inflammatory responses can lead to M1 polarization and suppression of inflammatory reactions (40). Nesfatin-1 induces c-c motif chemokine ligand 2 (CCL2) overexpression through the MEK/ERK pathway in RA synovial fibroblast, in which overexpressed CCL2 enhanced the polarization of M1 macrophages by treating THP-1-derived (M0) macrophages with synovial fibroblast conditioned medium (41). Nesfatin-1 identified as a potential risk factor for RA (42, 43). CCL2 levels are high in RA synovial tissue (44), which induces the recruitment and migration of monocytes to inflammatory sites in arthritis (45), thereby facilitating the progression of knee synovitis in affected individuals (46). The JAK-STAT pathway is stimulated by diverse inflammatory stimuli, influencing the differentiation of macrophages and the inflammatory response. Specifically, the activation of the JAK/STAT1 signaling cascade by IFN- γ facilitates the release of pro-inflammatory mediators by M1 macrophages. STAT1 activity is conducive to M1 polarization and its inhibition can lead to M2 polarization (47, 48).

Natural products, such as extracts derived from traditional Chinese medicine, have the potential to effectively manage RA by modulating the repolarization of M1 to M2 macrophages. Specifically, Tripterygium wilfordii glycosides have been shown to suppress the secretion of pro-inflammatory cytokines (IL-1, IL-6, CXCL8, TNF-α, and VEGF-A) by M1 macrophages while concurrently enhancing the expression of the anti-inflammatory cytokine IL-10 in M2 macrophages. The results of liquid phase chip quantitative analysis demonstrate that Triptolide decreases TNF in arthritis models, as well as levels of α, CXCL2, and VEGF, while increasing levels of IL-4 and IL-10. Additionally, Triptolide A inhibits NF- κB, PI3K/AKT, and p38 MAPK signaling pathways, thereby ameliorating RA joint inflammation (49). Wuweiganlu (WGL) is a renowned formulation primarily utilized for the management of RA and other chronic conditions as recommended by Tibetan medicine. In vitro experiments have shown that WGLWE induces the polarization of M1 macrophages towards the M2 phenotype, while also inhibiting the secretion of proinflammatory cytokines TNF-α and IL-6 (50).

In addition to pro-inflammatory cytokines, the pathogenesis of RA encompasses a multitude of pathological factors that synergistically contribute to the perpetuation of the inflammatory response and exacerbation of tissue damage. Metabolically, M1 macrophages predominantly rely on aerobic glycolysis, whereas M2 macrophages predominantly utilize oxidative phosphorylation (51). In the setting of joint inflammation, the formation of synovial pannus and the establishment of hypoxic inflammatory microenvironments significantly enhance the glycolytic metabolism of macrophages, driving their polarization towards the M1 phenotype. HIF-1α, NF-κB, Notch-1, and JAK-STAT have been identified as factors influencing alterations in macrophage metabolic phenotype. Of particular note, the activation of HIF-1α in macrophages has emerged as a crucial signaling mechanism governing aerobic glycolysis and M1 polarization in recent research (52). Inhibition of HIF-1 in macrophages suppresses glycolysis levels and M1 polarization, as well as impairs cell migration and bactericidal function (53, 54). The PI3K-AKT-mTOR-HIF-1α pathway serves as the fundamental mechanism for improving angiogenesis in RA (55). In comparison to synovial macrophages in healthy control groups, macrophages in RA synovium exhibit heightened expression of HIF-1 α (56), leading to enhanced transcription of glycolytic enzymes and upregulation of critical pro-inflammatory cytokines like IL-1 β (57). The accumulation of succinate, an intermediate metabolite of the tricarboxylic acid (TCA) cycle, is hypothesized to induce the activation of HIF-1 α, subsequently resulting in the generation of downstream IL-1 β (57). Recent studies have demonstrated that the reduction of citrate in M1 polarized macrophages (58) and the blockade of HIF-1α-related signaling pathways (59) can effectively suppress the overactivation of glycolytic metabolism in M1 cells. Additionally, the activation of the AMPK pathway results in the reprogramming of M1 glycolysis (60), inhibition of mTORC1 activity, suppression of protein synthesis, regulation of macrophage glucose metabolism and proliferation, and enhancement of mitochondrial enzyme activity to facilitate oxidative phosphorylation (61). Besides, lysine acetyltransferase 2A in synovial tissue promotes macrophage glycolytic reprogramming by inhibiting the activity of nuclear factor-erythroid 2-related factor 2 and downstream antioxidant molecules (62). These biological processes ultimately result in a phenotypic transition from M1 to M2.

In recent years, research has demonstrated that macrophages can modulate the phenotype of FLS via diverse biological mechanisms, including post-translational protein modifications. This highlights the regulatory role of macrophages in controlling the phenotype of FLS. The post-translational modifications of malondialdehyde acetaldehyde (MAA) and citrulline (CIT) are implicated in the pathogenesis of RA (RA). Upon modification by MAA and/or citrullinated fibrinogen, macrophages secrete soluble platelet-derived growth factor (PDGF) PDGF-BB subtypes, which can promote fibroblast-like synoviocyte (FLS) differentiation into invasive phenotypes. Compared to the control group, there is an upregulation of the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinase 1/2 (p-Erk1/2), phosphorylated protein kinase B (p-Akt) signaling pathways, as well as vimentin (VIM) and type II collagen (COL2A1), indicating an increase in mRNA expression of these genes (70). Extracellular traps (ET) are composed of histones, double-stranded DNA, myeloperoxidase, or elastase and are considered the primary source of citrullinated autoantigens in RA, leading to the production of anti-citrullinated protein antibodies (ACPA). Macrophages produce a specific type of ET known as macrophage extracellular trap (MET) (71). The stimulation of the DNA sensor GMP-AMP synthase (cGAS) in RA RA-FLS by macrophage-derived microvesicles (METs) from HP-1 cells triggers the activation of the PI3K/Akt signaling pathway, leading to enhanced proliferation, migration, invasion, and expression of inflammatory cytokines in RA-FLS. The findings indicate a significant upregulation of proinflammatory cytokines such as TNF and IL-1β, as well as matrix-degrading enzymes MMP-9 and MMP-13, in MET-stimulated RA-FLS compared to untreated controls (72). As single-cell sequencing and transcriptome techniques have advanced in the study of RA (RA), numerous investigations have explored the heterogeneity of macrophage subtypes, indicating that macrophage polarization extends beyond the traditional M1 and M2 classifications. Through extensive single-cell RNA sequencing (scRNA seq) analysis, thorough phenotypic, spatial, and functional assessments, Alivernini S et al. (73) identified two distinct populations within the macrophage-like synoviocytes (MLS), further stratified into nine clusters with distinct characteristics. The MerTK^negCD206^neg cluster elicits the production of pro-inflammatory cytokines and instigates inflammatory responses in FLS. The MerTK^posCD206^pos cluster in RA patients is associated with the production of lipid mediators during the continuous remission phase of the disease, which resolves inflammation and promotes the repair phenotype of FLS. This interaction between the MerTK^posCD206^pos cluster and FLS in the remission phase of RA plays a crucial role in maintaining joint immune homeostasis.

FLS has the capacity to induce macrophage polarization through distinct mechanisms, diverging from the traditional M1/M2 polarization paradigm. Prolonged exposure to pro-inflammatory conditions may result in heightened prostaglandin (PGE2) production in FLS. In conjunction with inflammatory mediators, macrophages have a tendency to polarize towards heparin-bound EGF-like growth factors (HBEGF) that deviate from the traditional M1 and M2 polarization phenotypes. The presence of HBEGF-enriched inflammatory macrophages in rheumatoid arthritis has been shown to give rise to distinct subpopulations of inflammatory mediators, including IL-1 and the growth factor HB-EGF, as well as surface embryonic protein. These macrophages also exhibit heightened expression of pro-inflammatory genes such as IL1B and CXCL2. Pathway analysis indicates that FLS may influence the metabolic profile of macrophages treated with TNF, resulting in a collective suppression of factors involved in oxidative phosphorylation. On the other hand, it has been observed that HBEGF inflammatory macrophages have the ability to stimulate the invasiveness of FLS, whereas EGFR inhibitors, originally designed for cancer treatment, have shown efficacy in inhibiting the fibroblast responses induced by macrophages in RA tissue (74). Extracellular vesicles (EVs) are small membrane-bound particles released by cells into the extracellular space, containing a variety of biomolecules such as RNA, lipids, proteins, and DNA. These EVs have the ability to transfer their contents to recipient cells, influencing their phenotype. Exosomes, a subtype of EVs with a diameter of ≤ 100-150 nm, are formed within multivesicular bodies. The PTX3 protein found in exosomes derived from RA-FLS does not impact the mRNA expression of TNF, IL6, and IL1B in M1 macrophages. However, experimental results from transwell migration assays indicate that PTX3 can enhance macrophage migration (75). PTX3 is an innate immune inflammatory modulator consisting of lipopolysaccharide, IL-1β, TNF-α, and other inflammatory factors. These components are associated with processes such as angiogenesis, atherosclerosis, cell proliferation, and tumor evasion (76). Furthermore, research conducted on animals has demonstrated that the interaction between MLS and FLS in inflammatory environments can trigger metabolic alterations, enhancing the longevity of MLS and contributing to the development of chronic inflammation in RA (77). Separate mouse arthritis tissue-derived synovial macrophages (ADSM) and arthritis tissue-derived synovial fibroblasts (ADSF), and culture ADSM in serum-free conditioned medium of ADSF. The expression levels of inflammatory macrophage markers Nos2, Tnf, Il-1b, and CD86 were significantly increased in ADSM. Metabolic flux analysis revealed upregulation of glycolysis and mitochondrial respiration in ADSMd, suggesting a potential long-lived phenotype in ADSM.

The crosstalk between MLS and FLS has been shown to impact the pathogenesis of RA by promoting bone destruction (78). Additionally, the interaction between macrophages and fibroblasts plays a role in mediating cartilage degradation and facilitating the migration of pathogenic osteoclast precursors to the inflamed synovium. Endothelial cells and synovial fibroblasts are significant sources of CX3CL1, a chemokine that has been implicated in these processes. Recent research has identified a specific subset of macrophages, characterized by high expression of CX3CR1 and low expression of Ly6C, F4/80, and I-A/I-E, known as arthritis-associated osteoclast macrophages (AtoMs), as a precursor population of pathogenic osteoclasts in arthritis. The bidirectional signal is produced between synovial macrophages and fibroblasts via the interaction of CX3CR1 CX3CL1. However, the precise mechanism remains unclear (79). Li and colleagues (80) conducted an examination of the influence of CCR2 expression on RA-FLS by co-culturing them with macrophages in an in vitro model. The results showed that treatment of RA-FLS with a CCR2 antagonist led to reduced expression of IL-1, IL-6, and TNF-α in macrophages, as well as induction of M2-type differentiation. The specific mechanism underlying these effects, potentially involving the downregulation of inflammatory cytokines and matrix metalloproteinases in RA-FLS, remains unclear.