The Role of Inflammation in the Pathogenesis of Osteoarthritis

A joint is the point of connection between two bones in our body. Inflammation of the joint leads to several diseases, including osteoarthritis, which is the concern of this review. Osteoarthritis is a common chronic debilitating joint disease mainly affecting the elderly. Several studies showed that inflammation triggered by factors like biomechanical stress is involved in the development of osteoarthritis. This stimulates the release of early-stage inflammatory cytokines like interleukin-1 beta (IL-1β), which in turn induces the activation of signaling pathways, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), and mitogen-activated protein kinase (MAPK). These events, in turn, generate more inflammatory molecules. Subsequently, collagenase like matrix metalloproteinases-13 (MMP-13) will degrade the extracellular matrix. As a result, anatomical and physiological functions of the joint are altered. This review is aimed at summarizing the previous studies highlighting the involvement of inflammation in the pathogenesis of osteoarthritis.


Introduction
Osteoarthritis or degenerative arthritis is a public health issue in an aging society. It is a chronic musculoskeletal disorder of the movable joints, such as knee and hip joints [1]. Osteoarthritis affects around 250 million people around the world, the majority of which are the elderly [2]. Changes in the joint tissues during aging can contribute to the development of osteoarthritis. For instance, an increase in cells manifesting senescent secretory phenotype leads to enhanced production of cytokines and matrix metalloproteinases (MMPs) in the joint environment [3]. Furthermore, reduced growth factors and the responsiveness of chondrocytes will cause less matrix synthesis and repair [3]. Other than aging, environmental, biomechanical, and biochemical factors can also contribute to the initiation of osteoarthritis. Osteoarthritis affects the entire structures of the joints, including articular cartilage, subchondral bone, meniscus, synovial membrane, and infrapatellar fat pad (IFP). The common structural characteristics of osteoarthritis are cartilage degradation, subchondral bone remodeling, osteophyte formation, and changes in the synovium and joint capsule [4].
Patients with osteoarthritis have typical clinical symptoms, such as severe joint pain, stiffness, and significantly reduced mobility, leading to decreased productivity and quality of life among the patients, as well as increased socioeconomic burden to the patients and the society [5]. As the prevalence of osteoarthritis increases with age, the aging population worldwide makes this disease a nonnegligible issue [6]. Current therapies for osteoarthritis are limited to symptom-relieving drugs and total knee arthroplasty for severe cases. Drugs addressing the underlying biological causes of osteoarthritis are not available in the market currently [7].
The involvement of immune cells in the development and progression of osteoarthritis has been highlighted in recent studies [8]. Inflammatory components, such as cytokines and chemokines, are produced by chondrocytes and synoviocytes in the joints of patients with osteoarthritis. Synovial fibroblasts are also a source of proinflammatory cytokines and matrix-degrading enzymes under osteoarthritis condition [9]. Moreover, IFP has been shown to contain a significant amount of immune cells like macrophage and T cells. As a result, IFP acts as a site of inflammatory mediators in osteoarthritic knee [10]. These inflammatory mediators alter cell signaling pathways, gene expression, and behavior of joint tissue [11]. The changes in cellular signal transduction lead to enhanced activation of the inflammatory pathway. Thus, more inflammatory compounds and enzymes are released. As a result, anatomical and physiological functions of the joint are altered [12].
This review is aimed at summarizing the recent clinical and preclinical studies performed previously to investigate the relationship between joint inflammation and the pathogenesis of osteoarthritis. The mechanism by which inflammation contributes to the pathogenesis of osteoarthritis will also be discussed.

Literature Search
A literature search on original articles written in English and published between 2014 and 2019 was performed using Scopus and PubMed database with the string: (osteoarthritis OR chondrocytes) AND inflammation. Both preclinical and clinical studies were included in this review. The search showed studies on human, animal, and cell lines investigating the involvement of inflammation in osteoarthritis by determining the differential expressions of genes, inflammatory components, and enzymes. Samples collected in human studies include synovial fluid and articular cartilage, from patients who underwent total knee arthroplasty, as well as blood (Table 1). For animal studies, agents, such as interleukin-1 beta (IL-1β) and monosodium iodoacetate (MIA), were used to induce osteoarthritis in the animal models (Table 2). Samples like articular cartilage and synovial tissue were taken after the treatment period. Agents such as IL-1β and tumor necrosis factor-alpha (TNF-α) were applied on cell lines to trigger inflammation (Table 3).

The Role of Immune Cells in Osteoarthritis
Immune cells like activated neutrophils and macrophages can secrete cytokines, such as IL-6 and IL-1β, which amplify the inflammatory process in osteoarthritis [13]. Increased infiltration of leukocytes (macrophages, T-lymphocytes, Blymphocytes, and neutrophils) in the synovium, particularly within the subintimal layer, is a characteristic of osteoarthritis [14]. Shan et al. reported elevated PD1+CXCR5+ CD4+ T cells, ICOS+CXCR5+CD4+ T cells, and IL 21+ CXCR5+CD4+ T cells in peripheral blood of patients with osteoarthritis [15]. CD4+ T cell is the T helper cell (Th cell) which may induce inflammation in the early stage of osteoarthritis. C-X-C chemokine receptor type 5 (CXCR5), inducible costimulator (ICOS), and programmed cell death 1 (PD-1) are known to be expressed by the Th cell [15].
Lymphocyte activation gene-3 (LAG-3+) regulatory T cells (Treg cells) have also been shown to increase in osteoarthritis [16]. Treg cells act as an immunoregulator in many inflammatory diseases [17]. It regulates the secretion of anti-inflammatory cytokines and expression of cytokine receptors [17]. Evidence showed that the response of Treg cells decreased in osteoarthritis in concurrent with an increase of LAG-3 expression in osteoarthritis. It has been postulated that LAG-3 molecules can reduce Treg function and boost inflammation [16]. CD3+ T cells are revealed as the predominant immune cells in IFP of dogs with canine cruciate ligament disease, which is associated with osteoarthritis, followed by CD14+ macrophages [18]. Both cell types can produce various cytokines such as IL-1β and IL-6 when activated. Immune cells and inflammatory mediators secreted in IFP will interact with other joint tissues, which can promote the pathological process of osteoarthritis [10]. At the same time, cartilage and the synovium are also shown to modulate the IFP. IL-1β stimulates increased proinflammatory cytokine secretion by IFP [19], suggesting that a cross-talk happens between IFP and joint tissues.
IL-1β is involved in a series of cellular activities, such as cell proliferation, differentiation, and apoptosis. It interferes with the production of essential structural proteins, including collagen type II and aggrecan, by influencing the activity of chondrocytes in the joint. Moreover, IL-1β affects MMPs' synthesis by chondrocytes, including MMP-1 and MMP-13, which, in turn, destroy the articular cartilage [35]. IL-1β was also shown to induce the production of reactive oxygen species, for example, nitric oxide (NO) [36]. Since IL-1β has been proven to play a significant role in the pathogenesis of osteoarthritis, it is commonly used to induce an in vitro osteoarthritis model in chondrocytes [37]. It stimulates expression of TNF-α and surface expression of TNF receptor (TNFR) in chondrocytes [38]. Binding of TNF-α to TNFR causes signal transduction and activates TNF receptorassociated factor 2 (TRAF2). TRAF2 will activate the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway involved in inflammatory diseases.
A study conducted among patients with osteoarthritis showed that IL-1β, IL-6, IL-8, IL-18, IL-17, IL-22, and transforming growth factor-beta 1 (TGFβ1) were increased in the inflamed synovium tissues compared to the noninflamed tissues [14]. IL-17 induces the release of IL-6, IL-8, and TNF-α by synovial fibroblasts and chondrocytes, leading to inflammation and cartilage breakdown [39]. It is secreted by T helper 17 cell (Th17), mast cell, and myeloid cell. Other than that, IL-17 promotes the recruitment and activation of neutrophils, which are the initial cell types recruited to the inflammation sites [40]. Activated neutrophils synthesize 2 Mediators of Inflammation      Mediators of Inflammation several inflammatory factors, which regulate the inflammation process in osteoarthritis. IL-17 has been shown to be present in the synovial fluid of a subset of patients with end-stage osteoarthritis [41]. Increased IL-17 and IL-22 levels are also detected in the synovial fluid of temporomandibular joint of patients with osteoarthritis [42]. The increase of these two cytokines is associated with the elevation of receptor activator of nuclear factor kappa-Β ligand (RANKL), which induces the differentiation of osteoclasts and resorption of subchondral bone, a layer of bone beneath the cartilage in joint [42]. IL-22 stimulates the proliferation of synovial cells and enhances the expression of MMPs in fibroblast-like synoviocytes (FLS) [43]. Interleukin 6 (IL-6) is known as a proinflammatory cytokine in chronic inflammatory diseases. In osteoarthritis, IL-6 released by joint tissue will bind to the soluble IL-6 receptor (IL-6R), leading to transsignaling [44]. As a consequence, the immune system is activated, whereby mononuclear cells like monocytes are recruited to the inflamed joint area [44]. IL-6 transsignaling will skew the differentiation of monocytes to macrophages through the upregulation of the macrophage colony-stimulating factor (M-CSF) receptor  [45]. A significant increase of IL-6 and IL-8 has been observed in patients with osteoarthritis [46][47][48][49][50][51]. For instance, Favero et al. determined the inflammatory molecules produced from coculture of meniscus tissue and synovial membrane from patients with early-stage (n = 5) and end-stage (n = 5) osteoarthritis [47]. They demonstrated the presence of IL-6 and IL-8 in patients at both stages, but their levels were higher among the end-stage patients [47]. In osteoarthritis, IL-8 in the synovial fluid plays a role in recruiting neutrophils and activating them. The activated cells will secrete enzyme elastase to degrade type II collagen crosslinks and proteoglycan in the articular cartilage [52].
IL-37 is a member of the IL-1 family, and it is an antiinflammatory cytokine [53]. Its level has been shown to elevate in osteoarthritis patients [53,54]. IL-37 can reduce the synthesis of proinflammatory cytokines and catabolic enzymes by osteoarthritic chondrocytes and synoviocytes. Mabey et al. collected blood and synovial fluid samples from patients with osteoarthritis (n = 32) and healthy controls (n = 14) to determine inflammatory cytokine levels in patients with knee osteoarthritis. They demonstrated that IL-2, IL-4, IL-6, and IL-10 levels were higher in patients with osteoarthritis compared to the controls [55]. Besides, a study by Xia et al. also showed an increased IL-10 level in lymphocyte activation gene-3 negative (LAG-3 -) regulatory T cells (Treg) from patients with knee osteoarthritis [16]. Conversely, He et al. demonstrated a decrease of IL-4 expression in articular cartilage from patients with osteoarthritis [56]. It may be due to the difference in the samples used between the signaling studies, whereby Mabey et al. used blood while He et al. used chondrocytes isolated from articular cartilage. The expression of IL-4 receptor had been shown to elevate in the serum of patients with osteoarthritis [57]. However, IL-4 was also shown to express at a decreased level in cartilage from patients with osteoarthritis compared to cartilage from healthy controls [58].
Other than the cytokines aforementioned, several other cytokines are involved in the pathogenesis of osteoarthritis. Some of the cytokines shown to increase in osteoarthritis are IL-18, TGFβ1 [14], IL-1 receptor (IL-1R) [59], and IL-1 alpha (IL-1α) [23]. Shan et al. showed enhanced serum IL-21, IL-17A, and IFN-γ levels in patients with osteoarthritis compared to controls [15]. IL-21 also upregulates RANKL expression, thereby stimulating bone marrow stem cells to differentiate into mature osteoclasts [60]. The interactions between pro-and anti-inflammatory cytokines were explored using macrophage conditioned medium (CM). Upregulation of proinflammatory cytokines (IL-1b, IL-6, MMP13, and ADAMTS5) and downregulation of cartilage matrix components (aggrecan, type II collagen) were observed in human osteoarthritic cartilage explants cultured with CM of proinflammatory macrophages expressing IFN-γ and TNF-α. However, CM of anti-inflammatory macrophages expressing IL-4 or IL-10 did not suppress the stimulation effects of conditioned media of proinflammatory cytokinestimulated explants [61].

The Role of Chemokines in Osteoarthritis
Chemokine is a subfamily of cytokine with low molecular weight. It is classified into four families, namely, CXC, CC, C, and CX3C families, depending on the position of cysteine (C) residues [62]. Chemokine functions as a chemoattractant, which directs the migration of immune cells to damaged or infected sites [62]. A study by Monasterio et al. revealed increased C-C motif ligand 5 (CCL5), CCL20, C-C motif receptor 5 (CCR5), and CCR7 in patients with temporomandibular joint osteoarthritis (TMJ-OA) [42]. These chemokines play a role in the recruitment of T helper cell type 1 (Th1), T helper cell type 17 (Th17), and T helper cell type 22 (Th22) to the affected joint in the study [42]. As a consequence, proinflammatory cytokines like IL-1β, IL-17, and IL-22 will be released in the joint and trigger the inflammation process.
In laboratory studies, chemokine receptor CXCR4 was shown by Sun et al. to reduce in chondrocyte culture induced with inflammation [29]. It is a specific receptor for stromalderived-factor-1 (SDF-1), also called CXCL12. Raghu et al. revealed that CCL2 and its receptor, CCR2, were increased in a mouse model of osteoarthritis [63]. The study suggested CCL7 is a monocyte chemoattractant, which has been shown to increase in the synovial fluid of patients with osteoarthritis [41]. Production of CCL7 from synoviocytes is enhanced by IL-17 [41]. Patients with facet joint osteoarthritis (n = 48) also demonstrated elevated CCL4 and C-C Motif Chemokine Ligand 4 Like 2 (CCL4L2) compared to healthy controls (n = 10) [64]. The study showed that CCL4 and CCL4L2 expressions were involved in the canonical NF-κB signaling pathway. Hou et al. discovered increased expression of CX3CL1 in synovial fibroblasts from patients with osteoarthritis [9]. CX3CL1 induced MMP-3 expression in a time-dependent and dose-dependent manner by reacting with its receptor, CX3CR1 [9]. Furthermore, Alaaeddine et al. showed enhanced expression of CCL20 and its receptor, CCR6, in cartilages compared to controls. This enhanced expression can further stimulate IL-6, MMP-1, and MMP-13 production [46].
Increased CCR3 and its ligand, CCL11, also known as eotaxin-1, have been detected in synovial cells from patients with osteoarthritis [21]. CCR3 is expressed by a few inflammatory cells such as T cells [65] and dendritic cells [66]. Besides, the study showed that CCL11 was able to stimulate the release of MMP-9 in synoviocytes from patients with osteoarthritis. Favero et al. observed the elevation of CCL21 and CCL5 in coculture of meniscus and synovial membrane from patients with osteoarthritis [47]. Conditioned media from osteoarthritis tissues like cartilage, IFP, meniscus, and synovium induced and enhanced production of CXCL8 and CCL21 in the synoviocyte cell line [67]. Several other studies showed an enhanced CCL2 level in patients with osteoarthritis [47,63,68]. CCL2 is also known as monocyte chemoattractant protein 1 (MCP-1). CCL2/CCR2 signaling will trigger monocyte trafficking into the inflamed joint area and, in turn, cause further inflammation. Arkestål et al. observed an increased CCR2 and a decreased CXCR3 expressions in the peripheral blood of patients with osteoarthritis compared to controls [69].
Expression of gelatinases such as MMP-2 (gelatinase A) and MMP-9 (gelatinase B) has been shown to associate with the pathogenesis of osteoarthritis. MMP-2 and MMP-9 are responsible to cleave ECM, cytokines, and chemokines, thus enhancing their activities [85]. MMP-2 level [14,24,86] and MMP-9 [21,82] increase in inflamed synovial tissues compared to noninflamed tissues. MMP-10, named as stromelysin-2, also increases in early-and end-stage osteoarthritis. Favero et al. showed a significantly increased level of MMP-10 in the meniscus and synovial coculture compared to meniscus alone [47]. The synovium was collected from the suprapatellar pouch while meniscus was isolated from the inner superficial zone of osteoarthritis patients who underwent total knee replacement. The two tissues were cocultured using a transwell to separate it and allow interaction between the two tissues.

The Role of Signaling Pathways and Other Inflammatory Components in Osteoarthritis
The nuclear factor-kappa B (NF-κB) transcription factor plays a central role in the pathogenesis of osteoarthritis [88]. It is triggered by proinflammatory cytokines and ECM degradation products [89]. The activated NF-κB will 11 Mediators of Inflammation modulate the expression of several cytokines, chemokines, and matrix-degrading enzymes, which explains its role in regulating catabolic events in osteoarthritis [26]. The NF-κB signaling pathway begins with the activation of IκB kinase (IKK), resulting in phosphorylation and degradation of IκBα by the proteasome. Subsequently, p65 protein is released, phosphorylated, and translocated from the cytoplasm to the nucleus. These events activate the expression of several genes, such as MMP-13 and IL-6 [89]. Therefore, the phosphorylated p65 (p-p65) level was found to increase in osteoarthritis whereas the IκBα level was decreased [22,23,31,35,84].
Enhanced p-p38, p-JNK, and p-ERK in osteoarthritis indicate the involvement of the mitogen-activated protein kinase (MAPK) signaling pathway [76]. MAPK is a mediator which regulates downstream expression of proinflammatory cytokines and MMPs [90]. It also acts as a pain mediator [90]. This pathway could be a potential avenue for new drug discovery to halt the progression of osteoarthritis. It begins when proinflammatory cytokines and growth factors bind to their respective receptors on the cell membrane. These act as upstream activators and cause intracellular MAP kinases (MKKs) to phosphorylate specific MAP kinases. MKK1 and 2 will activate ERK1 and 2 and MKK3 and 6 responsible for p38 phosphorylation while MKK4 and 7 phosphorylate JNK1 and 2. Activated MAP kinases in turn activate other protein kinases and transcriptional regulatory proteins which lead to the upregulation of certain inflammatory genes such as MMPs, IL-1, and TNF-α. These cytokines can then maintain JNK activation and cause more cytokine and MMP production [90].
PI3K/AKT signaling is known to be activated by cytokines like IL-1β when it binds to its cell surface receptor. Upon stimulation, membrane protein PI3K induces phosphorylation of AKT which has shown to have a synergistic effect on NF-κB signaling. Activation of the PI3K/AKT pathway will enhance production of MMPs by cells, for example, chondrocytes [91].
The expression of karyopherin alpha 2 (KPNA2), which regulates delivery of p65 to the nucleus, also increased in osteoarthritis [96]. Bromodomain-containing protein 4 (BRD4) played a role in the NF-κB signaling pathway [28]. Inhibition of BRD4 will suppress IL-1β-induced expression of proinflammatory cytokines and phosphorylation of p65. The BRD4 level was increased in a C57BL/6 mouse model of osteoarthritis [28]. Nerve growth factor (NGF) had been shown by Blaney Davidson et al. to increase in bovine chondrocytes treated with TGF-β1 and IL-1β [97]. TGF-β1-induced NGF expression was found to be dependent on an activin receptor-like kinase 5-Smad2/3 (ALK5-Smad2/3) signaling pathway as blockage of this pathway can prevent the expression. ALK5 is a type of transmembrane receptor of TGF-β. Once it activates, it will trigger downstream signaling cascades via the Smad-dependent pathway [98]. In a recent study using cartilage explants cultured with osteoarthritic synovium-CM or IL-1β, the TGF-β/Smad2/3P pathway that is protective against mechanical loading was diminished [99], which could lead to further destruction of the cartilage. Moreover, the complement system components [100] and soluble macrophage biomarkers (CD163 and CD14) [101] are also shown to be upregulated in osteoarthritis.
Overall, the integrated regulation of cytokines, chemokines, MMPs, and the signaling pathway is summarized in Figure 1.

The Role of miRNA in Osteoarthritis
MicroRNAs (miRNAs) are short, endogenous noncoding RNAs functioning to regulate posttranscriptional gene expression by binding to the 3 ′ untranslated region (3 ′ UTR) of target genes [31,35]. Once bound to target genes, they can block the translation process or decrease the stability of the messenger RNAs (mRNAs) [102]. Several microarray and database analyses have shown the involvement of miRNAs in osteoarthritis ( Figure 2) [103].
Recent human studies highlighted that miR-381a-3p [31] and miR-454 [51] were upregulated in osteoarthritis. Xia et al. discovered that nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor alpha (IκBα) is a target gene of miR-381a-3p [31]. Hence, upregulation of miR-381a-3p will decrease IκBα expression and further enhance the activation of NF-κB. Wu et al. showed that miR-454 overexpression in osteoarthritis promoted the proliferation of synovial fibroblasts and increased inflammatory factors [51].
On the other hand, Chen et al. showed that miR-149 bound to transforming growth factor-(TGF-) 1-activating kinase 1 (TAK1) 3 ′ UTR to regulate its expression [84]. 12 Mediators of Inflammation TAK1 expression increased when miR-149 decreased in osteoarthritis. NF-κB is the downstream signaling pathway of TAK1; thus, upregulated TAK1 causes increased activation of NF-κB in osteoarthritis [104]. Zhang et al. demonstrated that miR-373 decreased in osteoarthritis and it targeted P2X7 receptor (P2X7R) [49]. P2X7R expression was enhanced in osteoarthritis; this led to increased chondrocyte proliferation and release of inflammatory factors such as IL-6 and IL-8. In addition, P2X7R is adenosine triphosphate-(ATP-) gated plasma membrane ion channel which is involved in IL-1β maturation and its release from activated immune cells [105]. Damaged cells released ATP which led to activation of P2X7R and further release of inflammatory cytokines [106]. Mao     13 Mediators of Inflammation of osteoarthritis can be targeted to prevent further degradation of the joint tissue. IL-1 receptor antagonist has been developed, and it showed promising results in animal studies. However, its effects on humans require further studies [108]. Besides, IL-6 is well known for its contribution to progression of osteoarthritis. IL-6 or its receptor such as soluble IL-6 receptor can be a target for drug development. The recently discovered functional role of miRNAs in regulating joint homeostasis should also be explored, and they are another avenue for intervention. Circular RNAs (circRNAs), a single-stranded, noncoding regulatory RNA, can function as miRNA sponges and inhibit miRNA activity. It provides a possibility to be targeted as a therapeutic strategy.
The use of inflammatory markers to predict disease progression and treatment efficacy should also be explored. Currently, the evaluation of patients' conditions and improvements is based on subjective instruments like Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and radiographic findings. Future studies should attempt to validate the correlation of these instruments with inflammatory markers to provide a more objective measurement of disease progression. Established biomarkers of osteoarthritis progression such as urinary type II collagen degradation (uCTX-II) are shown to associate with incidence and progression of radiographic osteoarthritis [109].

Conclusion
Inflammation plays an integral role in the pathogenesis of osteoarthritis. Various molecules released by the chondrocytes and synoviocytes and infiltrating immune cells, such as cytokines, chemokines, and MMPs, are involved in regulating the joint anabolism and catabolism process. Their expressions are in turn governed by NFκB, MAPK, PI3K/AKT, prostacyclin, and nitric oxide pathways. The involvement of these molecules and signaling pathways is well-established in cellular, animal, and human studies. However, more studies are required to link and explore the connection between all these molecules involved. This review article collates the latest evidence on the relationship between inflammation and osteoarthritis to provide a better understanding on the pathogenesis of osteoarthritis. These pathways should be exploited as potential targets for drug intervention as currently pharmacotherapies targeting the underlying mechanism of osteoarthritis are still lacking in the market.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.