Emerging role of leptin in joint inflammation and destruction

Abstract Rheumatoid arthritis (RA) is an autoimmune disease characterized by tumor-like hyperplasia and inflammation of the synovium, which causes synovial cell invasion into the bone and cartilage. In RA pathogenesis, various molecules in effector cells (i.e., immune cells and mesenchymal cells) are dysregulated by genetic and environmental factors. Consistent with the early stages of RA, these pathogenic cells cooperate and activate each other directly by cell-to-cell contact or indirectly via humoral factors. Recently, growing evidence has revealed essential role of adipokines, which are multifunctional signal transduction molecules, in the immune system. In this review, we summarize the current understanding of the cross-talk between leptin, one of the most well-known and best-characterized adipokines, and osteoimmunology. Furthermore, we discuss the contribution of leptin to the pathogenesis of RA and its potential mechanisms.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that can impair physical function by causing persistent synovial inflammation leading to joint destruction. In RA pathogenesis, various molecules in immune cells (e.g., T cells, B cells, and monocytes) and mesenchymal cells are dysregulated through the influence of genetic predisposition and environmental factors. From the onset of early RA to the progression of destructive synovitis, these pathogenic cells cooperate and activate each other directly via cell-to-cell contact or indirectly via humoral factors (e.g., cytokines and chemokines). For instance, tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1b play major roles in joint inflammation, and biological agents against these molecules have been developed in the field of RA therapy [1]. Adipokines are multifunctional signal transduction molecules, and their relationship with osteoimmunology has attracted attention in recent years [2][3][4][5][6][7][8][9][10][11]. In this review, we discuss the role and contribution of leptin, one of the best-characterized adipokines, in the pathogenesis of RA.

Source and signal transduction of leptin
Leptin is a 16-kDa non-glycosylated cytokine-like hormone encoded by LEP and is mainly secreted by adipocytes [12]; thus, its concentration often correlates with the white adipose tissue volume. However, leptin is produced not only in adipocytes, but also in other cell types (e.g., skeletal muscle cells, epithelial cells, macrophages, basophils, mast cells, and T cells) [13][14][15][16][17][18] and is modulated by factors affecting energy metabolism, including proinflammatory cytokines (e.g., TNF-a, IL-6, and IL-1b) [19]. For instance, human Foxp3 þ CD4 þ CD25 þ T regulatory (Treg) cells and CD4 þ effector T cells have been observed to produce leptin even in the steady-state and are enhanced under CD3/CD28 stimulation [20]. Furthermore, human lung tissue taken after infectious pneumonia has shown significantly greater leptin staining in epithelial cells compared with uninjured lungs [21]. Its primary function is to regulate food intake and energy consumption by inducing anorexigenic factors and suppressing orexigenic neuropeptides in the hypothalamus [22]. Moreover, leptin can function in the periphery by entering the bloodstream and modifying various biological activities such as basal metabolism, insulin production, atherosclerosis, bone and cartilage metabolism, immune responses, inflammation, and reproduction [23][24][25][26][27][28][29]. Synthesized leptin binds to the leptin receptor (Ob-R), a member of the class I cytokine receptor superfamily [30]. There are at least six receptor isoforms with common extracellular domains but have cytoplasmic domains of different lengths: a soluble isoform (Ob-Re), four short isoforms (Ob-Ra, Ob-Rc, Ob-Rd, and Ob-Rf), and a long isoform (Ob-Rb) ( Figure 1). Ob-Rb is the only receptor capable of transmitting canonical Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling [31], while the short and soluble receptors are thought to be pivotal for leptin transport and degradation [32]. Additionally, various alternative pathways are known to be involved in leptin signaling, such as the mitogen-activated protein kinase (MAPK), protein kinase C, Src homology region 2containing protein tyrosine phosphatase 2/growth factor receptor-bound protein 2, and phosphoinositide 3-kinases (PI3K)/Akt pathways [22]. These receptors are expressed in a wide range of cell types, such as immune cells, chondrocytes, synovial fibroblasts, and osteoblasts, suggesting that leptin is an important factor linking inflammation and joint destruction [33,34].

Leptin in immune systems
Leptin has been shown to regulate immune responses and inflammation in various manners [3,5,6]. Leptin has a positive effect on cell proliferation and survival, pro-inflammatory mediator secretion, and migration/chemotaxis of natural killer (NK) cells, monocytes/macrophages, dendritic cells, neutrophils, eosinophils, and basophils. Therefore, leptin appears to induce and amplify inflammation by modifying the innate immune system.

Natural killer (NK) cells
Leptin has also been reported to affect NK cell proliferation and cytotoxicity. Short-term leptin stimulation augmented the proliferation of human NK cells in an in vitro study [35], and in response to leptin administration, the number of NK cells in the peripheral circulation was markedly increased in both lean and obese rats [36]. In the mouse bone marrow, leptin signaling regulates NK cell development by enhancing immature NK cell survival via downregulating Bcl-2 expression and upregulating Bax transcripts [37]. Moreover, leptin has been reported to decrease IL-4 and IL-10 expression [38] and increase IL-2 and IFN-c production in human NK cells [39,40]. In addition, leptin increased the cytotoxic activity of human NK cells via STAT3 phosphorylation and expression of IL-2 and perforin [39], promoted actin rearrangement, which is essential for cell migration [41], and enhanced the expression of adhesion molecules represented by Lselectin [38].

Monocytes/macrophages
Leptin has been reported to enhance the inflammatory phenotype of monocytes/macrophages [42,43]. For instance, leptin promotes the phagocytic activity of macrophages by modifying the expression levels of cAMP [44] and reactive oxygen species [45]. In addition, chemotaxis of macrophages was enhanced by leptin via signal transduction pathways including JAK/STAT, MAPK, and PI3K/Akt signaling [46]).

Dendritic cells (DCs)
Leptin has been reported to promote the survival of DCs by inhibiting apoptosis [47,48]. In addition, leptin induces the expression of some pro-inflammatory molecules such as IL-1b, IL-6, IL-12, TNF-a, and MIP-1a in human DCs as well as stimulates the migration and chemotaxis of immature DCs [47,[49][50][51].

Neutrophils
Leptin induces the survival of neutrophils by delaying the cleavage of pro-apoptotic factors (i.e., Bid and Bax), mitochondrial release of cytochrome C, and activation of caspases through PI3K, NF-jB, and MAPK signaling [52]. Moreover, leptin mediates the migration and chemotaxis of neutrophils via p38 MAPK and Src kinases [53]. Accordingly, in individuals who are obese, neutrophils show enhanced release of superoxide and chemotactic activity [54].

Eosinophils
In eosinophils, leptin has been reported to act as a survival factor by delaying apoptosis [55]. Leptin also increased the secretion of the pro-inflammatory factors IL-1b, IL-6, IL-8, and MCP-1 and modulated the expression of adhesion molecules; that is, it upregulated ICAM-1 and CD18 and downregulated ICAM-3 and L-selectin [55]. In individuals who are obese, eosinophils demonstrated increased adhesive and migratory capacities towards eotaxin and CCL5 [56].

Basophils
In basophils, leptin promotes migratory activity, induces cytokine secretion (i.e., IL-4 and IL-13), increases the expression of CD63, one of the activation markers, and augments degranulation in response to aggregation of the high-affinity receptor for IgE [16].

Adaptive immunity
Studies have also focused on the effects of leptin on the adaptive immune system. Interestingly, mice deficient in leptin (ob/ob) and leptin receptors (db/ db) demonstrated thymus atrophy and T cell lymphopenia, which were rescued by leptin treatment in ob/ob mice [57,58].

T cells
Leptin promotes the differentiation of double-positive CD4 þ CD8 þ thymocytes into single-positive CD4 þ T cells [59] and stimulates the production of IL-2, IFN-c, and granulocyte-macrophage colonystimulating factor from thymocytes [60]. In addition, leptin enhances the proliferation of human naïve CD4 þ T cells [57], while inhibiting that of Treg cells [14,[61][62][63] and memory T cells [64]. Interestingly, in co-culture of CD8 þ T cells with leptin-treated Treg cells, leptin impaired Treg cells activity and restored CD8 þ T cell proliferation [65]. Moreover, leptin decreased the secretion of transforming growth factor-b and IL-10 from Treg cells. Accordingly, congenitally leptin-deficient children showed decreased numbers of circulating CD4 þ T cells and impaired T cell proliferation and cytokine expression, which were rescued by administration of recombinant human leptin [66]. In contrast, subjects who were obese demonstrated a reduced number of Treg cells [67].
Leptin can also polarize CD4 þ T cells into the Th1 phenotype through the synthesis of Th1-cytokines (i.e., IL-2 and IFN-c) [68][69][70][71]. In fact, CD4 þ T cells derived from ob/ob mice showed significantly decreased expression of Th1 cell-specific T-box transcription factor [72]. The expression of retinoic acid-related orphan receptor c t, a transcription factor crucial for the differentiation of Th17 cells, was enhanced by leptin in CD4 þ T cells [73]. In contrast, neutralizing the leptin antibody reduced the number of Th17 cells and expression of retinoic acid-related orphan receptor c t [74]. Another study demonstrated that CD4 þ T cells derived from ob/ob mice show diminished polarization of these cells into Th17 cells as a consequence of STAT3 signaling inhibition [75]. Similarly, Th17 cells from fasted mice showed reduced proliferative activity that was rescued by leptin administration [76].

B cells
In B cells, leptin suppresses apoptotic activity and induces cell proliferation by activating Bcl-2 and cyclin D [77]. In addition, leptin has been shown to augment the expression of pro-inflammatory molecules (e.g., TNF-a, IL-4, IL-6, and CXCL8) and antiinflammatory cytokines (e.g., IL-10) through the JAK/STAT, p38 MAPK, and ERK1/2 pathways [78][79][80][81]. Accordingly, db/db and ob/ob mice showed reduced numbers of B cells in the circulation and bone marrow. Moreover, in the bone marrow of fasted mice, the number of pro-B, pre-B, and immature B cells decreased while that of mature B cells increased, which were all modified by leptin treatment [82][83][84].

Leptin as a potential regulator of RA pathophysiology
Numerous studies have shown that the concentrations of leptin are elevated in blood samples from patients with RA compared to in those from control subjects [85][86][87][88][89][90][91][92][93], independent of BMI [90]. Furthermore, several studies reported a correlation between leptin levels and disease activity measures (i.e., DAS28 and CRP), whereas other did not show similar results [94][95][96][97]. In other studies, leptin was reported to be expressed in the synovium [98], and its concentration in the synovial fluid was higher in patients with RA than in patients with osteoarthritis (OA) [86,99]. The role of leptin in joint destruction in RA has also been examined. One study reported that patients with RA showing advanced erosion had higher serum leptin concentrations [100], whereas other studies showed inconsistent results [101][102][103][104][105]. The concentrations of leptin were lower in the synovial fluid than in paired serum from patients with non-erosive RA, but not in patients with advanced joint destruction [86]. This result proposed that local consumption of leptin in the joint could protect against erosive progression.
In addition to its effects on the immune system, leptin can modulate metabolism in the bone, cartilage, and synovium [106,107]. Leptin may promote the influx of immune cells into the synovium by regulating the expression of adhesion molecules on endothelial cells as well as on immune cells [108,109]. In addition, leptin stimulates chondrocytes to produce proteases such as matrix metalloproteinases, pro-inflammatory mediators including IL-1b, TNF-a, IL-6, IL-8, and MCP-1, and adhesion molecules such as VCAM-1 [110,111]. Chondrocytes of OA, a representative degenerative disease that is also known to be a common comorbidity of RA, produce higher levels of leptin compared to healthy donors [112] and are related to the severity of cartilage destruction [113,114]. Leptin also modulates osteoblast function and bone metabolism to lead to joint destruction in OA patients [107]. Moreover, leptin induces the expression of some cytokines (e.g., IL-6) and chemokines (e.g., IL-8) via JAK2/ STAT3, NF-jB, and AP-1 signaling in synovial fibroblasts [115,116].
The relationship between leptin and RA pathophysiology has been demonstrated in animal models. ob/ob mice showed less severe arthritis accompanied by reduced IL-1b and TNF-a levels [117]. In addition, articular injection of leptin enhanced Th17 cells in the joints of a collageninduced arthritis mouse model, which was associated with the severity of inflammation and development of arthritis [118]. A positive correlation between leptin and IL-17 in the plasma was reported in patients with RA [119].
Overall, these data indicate that leptin is a crucial modulator of joint inflammation and bone and cartilage remodeling associated with the RA pathophysiology. If the disease activity of RA remaining under the use of conventional synthetic diseasemodifying antirheumatic drugs (csDMARDs) or biologic DMARDs is associated with leptin signaling, at least part of the clinical effect of JAK inhibitors on multidrug-resistant cases could be mediated by blocking leptin signaling. Furthermore, differences in JAK selectivity have also been reported among JAK inhibitors. As described above, among the JAK/ STAT pathways downstream of leptin, JAK2/STAT3 is particularly important, and the difference in immunological properties between drugs with high JAK1 selectivity (e.g., upadacitinib, filgotinib) and JAK inhibitors including JAK2 (e.g., tofacitinib、 baricitinib、 peficitinib) is interesting. The blocking efficiency of leptin signaling by each JAK inhibitor and the resulting difference in clinical outcomes are for further study.

Conclusions
The risk of developing moderate-to-severe RA is reportedly higher in patients with obesity than in non-obese subjects [120][121][122]. However, the amount of subcutaneous and visceral fat does not necessarily correlate with the amount of intra-articular fat. Further research is required to clarify the effects of systemic hyperleptinemia on the immune system, as well as provide mechanistic insights into leptininduced inflammation and local destruction of RA joints.

Author contributions
H.T. wrote the manuscript. K.F. supervised and revised the manuscript. All authors read and approved the final manuscript.