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Posted: January 31st, 2023

Medical Sciences (Anatomy, Physiology, Pharmacology etc.)

Medical Sciences (Anatomy, Physiology, Pharmacology etc.)
CHAPTER 1: INTRODUCTION

1.1 Rheumatoid arthritis
Rheumatoid arthritis (RA) is a painful crippling chronic autoimmune inflammatory disease, affecting 1:100 of the global population with a female preponderance (3:1 female: males) (Edwards, et al., 1999). It is a significant socio-economic burden on society as it increase the risk of other potential diseases such as cardiovascular diseases, depression and fatigue (Dayer & Choy, 2010 and Pollard et al., 2005) whilst transformative, the mortality rate of RA patients is higher (Buch and Emery 2002, Wolfe et al., 1994).
Characteristically the small joints of the hands, wrists and feet are affected first in a symmetrical fashion but any synovial joint can be involved. There are three forms of clinical presentation into which most cases of RA can broadly be recognised. A chronic progressive form in which the disease begins with minimal joint involvement and then progresses slowly over a period of years to multiple joint disease with severe functional limitation (Falconer et al.,2018). This is the most common pattern of arthritis seen. Second form is an intermittent course which is punctuated by acute episodes of arthritis with periods of remission in between (Aletaha & Smolen, 2018). The third form is an explosive onset with multiple joint involvement and acute synovitis which may go into partial remission after three years or so (Aletaha & Smolen, 2018). This pattern of RA is more commonly seen when RA begins in the elderly patient.
Cells of the myelomonocytic lineage can differentiate into numerous cell types that are involved in disease for instance monocytes, macrophages, osteoclasts and dendritic cells and activation of these cells leads to the production of cytokines and mediators responsible for inflammation. Monocytes are central to the RA pathology as they accumulate in the blood and continuously migrate into the inflamed joints where they acquire an activated phenotype and can differentiate into inflammatory macrophages, dendritic cells and osteoclasts (Ammari et al., 2018 and Goudot et al., 2017). As a consequence of their marked plasticity, the differentiation pathways can be influenced by excess or imbalance of particular pathophysiological stimuli such as cytokines or growth factors, resulting in altered differentiation or maturation if regulatory mechanisms fail. Different immune modulators such as cytokines and effector cells and signalling pathways are known to involve in the pathophysiology of RA (Smolen and Steiner, 2003). The complex interaction of cytokines and effector cells are accountable for the joint damage that begins at the synovial membrane (Smolen and Steiner, 2003).
RA is represented as pannus formation, synovitis, bone erosions and joint destruction. The joint is contained within a joint capsule which surrounds joints such as hand, wrist elbow, ankle and foot. Synovial inflammation is a defensive cellular response against invading pathogens but defects give rise to RA (Fig.1 and Fig.2). The major characteristic of RA is symmetric polyarticular inflammation of the synovial membrane which is a connective tissue that lines the inner surface of joint capsules (Kunisch et al., 2004).

Figure 1. Schematic view of normal joint (a) and a RA affected joint (b) (Adapted from, Smolen and Steiner, 2003).

Figure 2. Inflammation of synovium membrane in RA.
A) Normal synovium membrane compared with
B) RA synovium membrane (Adapted from, Pitzalis et al., 2013).

The synovial fluid provides lubrication and nourishment for joint movement. The synovial fluid is produced by macrophage-like synoviocytic cells termed as type A synoviocytes, which contain 25% of synovial lining cluster of differentiation (CD) cells that include CD14, CD18 and CD68 macrophage. The type B, fibroflast-like cells which express high levels of uridine diphosphoglucose dehrydogenase, play an important role in the synthesis of hyaluronan, a glycosaminoglycan (Edwards and Willoughby 1982). The synovial CD68 macrophages express major histocompatibility complex II (MHC II) and antigen presenting cells (APC) CD4+ T- helper (Th) on their cell surface. CD4+ T cells are stimulated by interleukin-4 (IL-4) to develop into Th1/Th17 cells (control-cell mediated responses) and Th2 cells (control antibody-mediated responses) and Treg cells (regulate immune reactions) (Kinne et al., 2002).
In RA, the synovial membrane is invaded by undifferentiated peripheral blood mononuclear cells including plasma cells, neutrophils, mast cells, dendritic cells, CD4+ T-cells (Th1 cells, Th17 cells and Th2 cells), B cells, immunoregulatory Treg cells, synovial fibroblast-like cells and resident synovial macrophages (Smolen and Steiner, 2003). Cascade induces monocytes adhesion to extracellular matrix molecules (ECM) such as intercellular adhesion molecule-1 (ICAM-1 or CD54) (Dustin et al; 1986) and their expression can be up-regulated by IL-1 and Tumor necrosis factor-α (TNF-α) (Aboot et al., 1992), CD102 (ICAM-2) (de Fougerolles et al; 1991) and CD50 (ICAM-3) (Fawcett et al; 1992, de Fougerolles et al; 1992), CD62e endothelial-leucocyte adhesion molecule-1 (ELAM-1, E-selectin) (Carlos et al;1991) and CD106 vascular cell adhesion molecule-1 (VACM-1) (Elices et al; 1990) to initiate migration of circulating monocytes to promote synovial inflammation. Several immuno-histochemical staining studies have shown that ICAM-1, VCAM-1 and ELAM-1 are all highly expressed by rheumatoid synovial vascular endothelial cells and cells in the lining layer (Koch et al., 1991; Morales-Ducret et al., 1992; Wilkinson et al., 1993). Monocytes express β2-integrin complex CD11/CD18 (CD11a, CD11b, CD11c) on their surface, which is the receptor for the endothelial ligands CD54, CD102 (deFougerolles et al; 1991 & 1992 and Marlin, 1987) and CD50 (Fawcett et al; 1992, Campanero et al; 1993 and El- Gabalawy et al; 1994) and this binding facilitate monocytes adhesion to the endothelium.
Subsequently, monocytes differentiate into macrophages as a result synovial lining becoming hyperplastic leading to inflammation of synovium membrane (Smolen and Steiner, 2003). It is generally accepted that increased number of macrophages in the synovial tissue mostly arises from the infiltration of circulating monocytes and initiate the acute inflammatory arthritic response (Udalova et al., 2016). However, like macrophages, monocytes also display phenotypical and functional heterogeneity. The increase of soluble CD14 in RA relates to monocyte-macrophage activation (Bas et al., 2004 and Yu et al., 1998).
The initial event leading to RA is a breakdown of immune tolerance, resulting in autoantibody production via antigen-specific T and B cell activation. B lymphocytes express cell surface proteins, including immunoglobulin and differentiation antigens such as CD20 and CD22. The autoantibodies can form larger immune complexes that can further stimulate the production of pro-inflammatory cytokines, including TNF-α, through complement and Fc-receptor activation (Smolen et al., 2007). T-cell and B-cell activation result in increased production of cytokines and chemokines, leading to a feedback loop for additional T-cell, macrophage and B-cell interactions (Smolen and Steiner, 2003; Smolen et al., 2007). TNF-α and IL-6 play a dominant role in the pathogenesis of RA (McInnes and Schett, 2007; Firestein, 2003). IL-1 which increase synovial fibroblast cytokines and chemokines and involved in endothelial cell adhesion molecule expression (McInnes and Schett, 2007). VEGF which cause angiogenesis and contribute to pannus formation (Paleolog, 2002) and IL-17 which recruit monocytes and neutrophils by increasing local chemokine production, facilitate T-cell infiltration and activation and amplify immune response by inducing IL-6 production (Nalbandian and Crispin, 2009), also have a significant impact on the RA disease process. However, it appears that monocytes and tissue-resident macrophages have an essential role mediating and promoting the onset of RA pathogenesis due to their antigen-triggered responses (Edilova et al., 2020).
Macrophages play an important role in RA because they are abundant in the inflamed synovial membrane and at the cartilage-pannus junction (Mulherin et al., 1996). The degree of macrophage infiltration and activation correlates not only with the joint pain and inflammatory status of the patient (Tak et al., 1997), however also with the radiological progression of permanent joint damage (Mulherin et al., 1996) and the disease feature that eventually defines quality of life. The expression of CD14 and CD68 is predominant on synovial macrophages compared peripheral blood monocytes (Yoon et al., 2004). High expression of CD68 on synovial macrophages have been found to be pre-dominantly accountable for production of increased amount of TNF- α, IL-1 at the cartilage–pannus junction (Chu et al., 1991). Cartilage damage takes place and chondrocytes express receptors for the joint disease severity and response to treatment in RA (Firestein et al., 1990, Zamani, et al., 2013) suggest TNF- α and IL-1 direct involvement in cartilage destruction. Cytokines produced by monocytes and macrophages are pro-inflammatory and induce tissue destruction. TNF-α, IL-1 and IL-8 have all been shown to cause synovitis (Pettipher et al., 1986; Henderson and Pettipher, 1989; O’ Bryne et al., 1990; Endo et al., 1991) and TNF-α and IL-1 can also cause cartilage degradation when injected intra-articularly in rabbit knee joints. Transgenic mice bearing a human TNF-α transgene modified in the 3’-region express higher levels of TNF-α and develop a chronic arthritis resembling RA which is prevented by anti- TNF-α treatment (Keffer et al., 1991). In human RA, TNF-α and IL-1 are likely to be important cytokines that are responsible for cartilage destruction. Both TNF-α and IL-1 can stimulate synovial cells and chondrocytes to produce metalloproteinases which destroy the extracellular matrix components such as collagen and proteoglycan (reviwed by Firestein, 1992; Dinarello, 1992). They also inhibit matrix synthesis.
Furthermore, the induction of TNF-α production can be completely reversed by addition of IL-10. Aarvak et al., 1999 reported that IL-17, which is present in T cell-rich areas of RA synovial samples, is exclusively produced by T-helper (Th)0 or Th1 clones that are derived from the synovial membrane or synovial fluid of RA patients. In addition, IL-17 indirectly induces the formation of osteoclasts from progenitor cells (Kotake et al., 1999) and enhances the production of nitric oxide (NO) in articular chondrocytes (Shalom-Barak et al., 1998), thus potentially contributing to cartilage and bone destruction.
A study by Gracie et al., 1999 reported that in the RA synovial membrane IL-18, a cytokine of the IL-1 family (Dinarello, 1999), is expressed most prominently in CD68+ macrophages that are contained in lymphoid aggregates. CD14+ macrophages of the RA synovial fluid also express the IL-18 receptor and IL-18, either alone or in concert with IL-12 and IL-15, strongly enhances the production of IFN-γ, TNF-α, GM-CSF and NO by cultured synovial cells. Treatment with recombinant murine IL-18 markedly aggravates experimental arthritis (Gracie et al., 1999), indicating that IL-18 has pro-inflammatory effects in this disorder.
These inflammatory responses act as the major orchestrator of the synovial membrane inflammation and accumulation of an excessive synovial fluid. This leads to establishment of pannus in the joint capsule, morning stiffness and pain of joints with progressive bone erosion resulting in deformity and loss of mobility of the joint (reviewed in: Pitzalis et al., 2014). The test-tube experiments show that synovial fluid monocyte cells have round-shaped adherent CD68+ cells and could differentiated into macrophages that produce TNF-α (Panayi et al., 1974). TNF- α has a central role in regulating the action of downstream pro-inflammatory cytokine signalling cascades (Brennan et al., 1989).
These studies suggest that RA arises from differentiation of monocyte cells into macrophages and postulate that TNF-α is a pivotal cytokine in the pathogenesis of RA based on the fact that TNF-α is a pro-inflammatory cytokine causing arthritis and it is required for other inflammatory cytokine for example; IL-1 and GM-CSF production by RA synovial cells (Brennan et al., 1989) and present in abundance in rheumatoid joints (di Giovine et al., 1988). Macrophages resulting from monocyte precursors differentiate into distinct functional phenotypes depending on the local tissue environment.
1.2 Macrophage polarisation
The differentiated naïve macrophages (M0) can further mature and activate into pro-inflammatory (M1) and anti-inflammatory (M2) macrophages (Mills et al., 2000). Interferon regulatory factor (IRF), signal transducers and activators of transcription (STAT) pathway and suppressor of cytokine signalling (SOCS) proteins (Sica and Bronte, 2007). M1 and M2 macrophages are characterised based on their functional properties such as cytokine production, gene expression and phenotypic such as surface markers properties (Gordon and Martinez et al., 2010, Mantovani et al., 2007).
The IRF-STAT pathways are activated (Fig.3) by interferon gamma-γ (IFNγ), lipopolysaccharide, Interluekin-4 (IL-4) and IL-13 (Mills et al., 2000, Nathan et al., 1983 and Stein et al., 1992). IFNγ Toll-Like Receptor binding signal activates IRF, signal transducers and activators of transcription pathway to polarise M0 macrophage to M1 macrophage via STAT1 (Sica and Bronte, 2007). LPS and TLR4 signalling pathway can also polarise M0 macrophages to M1 macrophages by activating STAT1-α and STAT1-β in a Myeloid differentiation primary response 88 (MyD88) independent manner (Toshchakov et al.,2002). In addition, SOCS3 proteins activates nuclear factor kappa B (NF-κB)-phosphatidyl inositol 3 kinase (PI3) pathways to produce nitric oxide via G-protein coupled receptor P2Y(2)R nitric oxide synthase-2 (NOS2) (Eun et al., 2014), cell growth and cell differentiation factor Activin A (Arnold et al., 2014) to promote M1 markers expression and down-regulate IL-10 production (Sierra-Filardi et., 2011). Bruton’s tyrosine Kinase (Btk) has been indicated as critical for M1 polarisation as absence of Btk polarised M1 macrophages to M2 macrophages in response to LPS stimulation (Ni et al., 2014). The hypoxia inducible factor HIF-1α regulates M1 polarization by regulating NOS2 expression and the M1 state (Takeda et al., 2010).

Figure 3. Signaling molecules involved in M1 polarization. STAT, signal transducers and activators of transcription; IRF, interferon regulatory factor; SOCS, suppressor of cytokine signaling 3; Btk, Bruton’s tyrosine kinase; HIF-1, hypoxia inducible factor 1; TNF-alpha, tumor necrosis factor; iNOS, inducible nitric oxide synthase; NOS2, nitric oxide synthase 2; NF-κB, nuclear factor-kappa B; NO, nitric oxide; PI3K, phosphatidyl inositol 3 kinase; TLR4, Toll-like receptor 4; LPS, lipopolysaccharide.

In contrast, M0 macrophages are polarised toward M2 macrophage by IL-4 and IL-13 by binding to theirsurface receptors via STAT 6 (Sica and Bronte, 2007). This signalling activates STAT6 (Fig. 4) to activate anti-inflammatory gene expression such as Arginase-1, CD206 (reviewed in: Lawrence and Natoli, 2011, Tugal et al., 2013). Kruppel-like factor 4 (KLF-4) synchronises with STAT6 to induce Arginase-1, Peroxisome proliferator-activated receptor- (PPAR-γ) and inhibit TNF-α, COX-2 and NOS2 by sequestering essential co-activators of NF-κB (Liao et al., 2011). IRF4 and NF-κB p50 subunit has also shown to play a role in M2 polarisation (Satoh et al., 2013, Porta et al., 2009). The hypoxia inducible factor HIF-2α regulates M2 polarization by arginase 1 expression and the M2 state (Takeda et al., 2010). Additionally, the cytokine IL-21 mediates M2 polarization by decreasing NOS2 expression and increasing STAT3 phosphorylation and BMP-7 induces M2 polarization in vitro via activation of the SMAD-PI3K-Akt-mTOR pathway (Li et al., 2013).

Figure 4. Signaling molecules involved in M2 polarization. STAT, signal transducers and activators of transcription; IRF, interferon regulatory factor; HIF-2, hypoxia inducible factor 2; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-kappa B; PI3K, phosphatidyl inositol 3 kinase; TLR4, Toll-like receptor 4; Arg-1; arginase 1; KLF-4; Krüppel-like factor 4; FIZZ1, resistin-like molecule-alpha (Relm-alpha); BMP-7, bone morphogenetic protein 7; PPARγ, peroxisome proliferator-activated receptor γ; FABP4, fatty acid binding protein 4; LXRα; liver X receptor alpha.

M1 macrophages express high levels of MHC II, CD68, CD86, CD80 surface markers and secrete high levels of TNF-α, IL-6, IL-12, IL-18 (Martinez et al., 2008; Mosser and Edwards, 2008). In disease context, M1 macrophages initiate and sustain inflammation and cause joint erosion and therefore can be harmful to health. M2 macrophages express high levels of CD200R, CD206, CD163, Arginase-1 surface markers and produce high levels of IL-10 and low level of IL-12 (Duluc et al., 2007 and Roszer et al., 2015) and contribute to vasculogenesis, tissue remodeling and repair.
Some studies suggest that M2 macrophages can be further classified into M2a, M2b, M2c and M2d subsets based on the applied stimuli and the induced transcriptional changes (Mantovani et al., 2004; Martinez and Gordon, 2014; Murray et al., 2014, Colin et al., 2014; Ferrante and Leibovich, 2012). M2a subtype is induced by IL-4 and IL-13 and expresses high levels of CD206, IL-1R, CD163, IL-6 and chemokine ligand (CCL)-17 (Martinez et al., 2008; Mosser and Edwards, 2008). M2b subtype activation is elicited by immune complexes, IL-1 receptor ligands and bacterial LPS and expresses IL-1, IL-10, TNF-α, CD86, IL-6, CCL-1 (Martinez et al., 2008; Mosser and Edwards, 2008), and the M2c subtype activation is initiated in response to IL-10, glucocorticoids and TGF-β and express CD206, C163, IL-10, TGF-β, CXCL13, CCL-2 (Martinez et al., 2008). The M2d subtype activation is caused in response to IL-6 and adenosines and expresses VEGF, IL-10, IL-12, TGF-β, CCL-5 and CXCL-16 (Wang et al., 2010 and Ferrante et al., 2013; Martinez et al., 2008).
Study by Miossec et al., 1990 suggests that the anti-inflammatory cytokine IL-4 plays a protective role in arthritis, although its virtual absence from synovial samples points to the lack of protective mechanisms, rather than to active regulation. This Th2-like cytokine down regulates monocyte-macrophage cytotoxicity and cytokine production (Isomaki, et al., 1996) including that of TNF-α (Hart et al., 1996). Furthermore, Allen et al., 1993 reported that IL-4 decreases IL-1β production while increasing IL-1 receptor antagonist production, thus suggesting a synchronised anti-inflammatory approach. IL-10 is a macrophage-derived cytokine (Abbas et al., 1996) which reduces HLA-DR expression and antigen presentation in monocytes and inhibits the production of pro-inflammatory cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and Fcγ receptors by synovial macrophages (Isomaki, et al., 1996). Consistently with cytokine and chemokine down-regulation, IL-10 suppressed experimental arthritis (Abbas et al., 1996). Study by Bessis et al., 1996 suggested that IL-13 exerts suppressive effects in experimental arthritis, probably through a selective effect on monocytes-macrophages. In RA, IL-13 is produced by synovial fluid mononuclear cells, which, when exposed to exogenous IL-13, reduce their own production of IL-1 and TNF-α (Isomaki et al., 1996).
Targeting monocyte-macrophage differentiation should be a powerful way of inhibiting inflammation and bone erosion in arthritis. Their plasticity is a major property that helps the switch from M1 phenotype to M2 phenotype (Mantovani et al., 2004). The polarity balance between M1 and M2 is essential for adequate immune function as dysfunction causes excessive production of pro-inflammatory cytokines. Fukui et al., (2017) and Zhu et al., (2015) confirmed that M1/M2 macrophage subset ratios disequilibrium being higher in RA patients’ synovial fluid compared to patients with osteoclast.
The synovial CD68 macrophages play a central role in the pathology of RA (Yanni et al., 1994). In a double-immunofluorescence staining studies by Ambarus et al., (2012) and Wiktor-Jedrzejczak and Gordon (1996), the CD68+ marker was found to be co-localised with the IFN-γ dependent polarisation markers, IL-4 dependent polarised markers and IL-10 dependent polarised markers. Kennedy et al., (2011) suggest that depletion of macrophages from RA synovial cell cultures can significantly reduce TNF- α levels.
Soler et al., (2015) suggests that the degree of joint erosion and the contribution to hyperplasia of the intimal synovial lining layer could also be linked to the increased number in the synovial tissue. For example, Fukui et al., (2017) and Zhu et al., (2015) suggested that intimal lining layer contain mainly mature resident macrophage markers CD163 and CD32 co-localised with CD68+, whereas the synovial sub-lining contain more mixed phenotypes CD68+ co-localized with CD163 and CD32 and CD64, and the CD200R and CD14+, proposing that it is actively infiltrated with immature monocytes derived macrophages. Furthermore, Ambarus et al., (2012) concluded that disease activity in RA seemed associated with the number of synovial sub-lining macrophages, but not with intimal lining layer macrophages.
Some studies suggest that surface markers for both M1 and M2 phenotypes may coexists on the same cell (Trombetta et al., 2018 and Cutolo et al., 2018). For example, to evaluate ex-vivo and in vitro polarisation markers of M1 and M2 macrophage cellular compartments Ambarus et al., (2012) purified CD14+ monocytes from the peripheral blood of RA patients and compared with the same cells from healthy donors. Conversely, CD64, CD200R and CD16 labelling did not show a significant difference between the two phenotypes. A study by Quero et al., (2017) did not show any specific difference in M1 or M2 marker expressions where GM-CSF M1 macrophage expressed CD163 and CD206 which should be M2 markers. Similarly Zhao et al., (2017) analysed M1 (CD68+CD192+) and M2 (CX3CR1+CD163+) but no significant difference was found, and concluded that RA peripheral blood seem to be composed by mixed M1 and M2 monocyte sub-populations. However, contrary to these studies, a gene expression study by Hofkens et al., (2013) from rodents during the course of antigen-induced arthritis, indicated, the up-regulation of M1 markers (IL-1β, IL-6, FcγRI and CD86) even though M2 markers (Arg1 and Ym1), remained high and constant throughout the disease period.
Literature review indicates that disequilibrium of M1 and M2 markers is present in RA patients. Therefore, agents with a potential to inhibit pro-inflammatory cytokines production may be useful in treating arthritis and other inflammatory diseases. For that reason, monocyte-macrophage differentiation has a great potential as a new model of inflammatory diseases and that alteration of this pathway may reduce distortion of synovial membrane. To understand the early events that lead to monocyte-macrophage differentiation in RA, it is important to understand the molecular signalling pathways involved in monocyte-macrophage differentiation.

1.3 Intracellular signalling pathways
The intracellular signalling pathway is considered to play an important role in induction and maintenance of chronic inflammation. The protein kinase C (PKC) is family of proline-directed serine-theronine kianses which is activated by diacylglycerol (DAG) and calcium ions for translocation from the cytosol to the plasma membrane. PKC signalling pathway activates network of extracellular signal regulated protein kinases I and 2 (ERK1/2) Mitogen-activated protein kinases (MAPKs), c-Jun-N-terminal kinases (JNKs) and p38 MAPKs comprising p38-α (MAPK14), -β (MAPK11), -γ (MAPK12 / ERK6), and -δ (MAPK13 / SAPK4 participate in the transduction of signals from the cell surface to inside the cell to regulate cell cycle progression, cell migration, cell proliferation, cell survival, cell differentiation and apoptosis (Tanaka and Nishizuka, 1994; Newton, 2001 and reviewed in: Seger et al., 1995 and Kyriakis et al., 1996). These pathways are approximately 60-70 % identical to each other but differ in sequence, size and their activation in response to stimuli. ERK1/2 MAPKs are activated by growth related stimuli (Cobb et al., 1991 and Sugden and Clerk, 1997) whereas the JNKs and p38 MAPKs are activated in response to cellular stresses (Ip and Davis, 1998). Some studies suggest that the MAPK signal is an important modulator of M2b macrophage polarisation as the activation of p38, ERK1/2, and JNK is enhanced in M2b macrophages induced by granulin or administration of activated lymphocyte-derived DNA (Chen et al., 2013 and Zhang et al., 2010).
Protein phosphorylation plays a central role for controlling cell cycle processes and once MAPKs signalling pathways are activated in response to stimuli, they activate cell cycle regulating Cyclin D1 and p21WAF1 genes expression for cell proliferation and differentiation. These cell cycle regulating genes plays a critical role in cell cycle progression and cell differentiation where cyclin D1 gene bind to cyclin dependent kinase (ckd)-4 and cdk6 to promote cell cycle progression from growth (G1) phase to S phase (DNA replication) by Rb protein phosphorylation for cell cycle progression. On the other hand, p21WAF1 gene binds to cdk-2-cyclin complex to inhibit G1 phase transition to S phase by inhibiting phosphorylation of Rb to promote monocyte-macrophage differentiation (Matsumoto et al., 2006).
ERK1/2 MAPK signalling pathway normally participates in cell proliferation and cell survival (Xia et al., 1995) whereas p38 MAPK signalling promotes cell differentiation. However, recent studies suggest that in addition to ERK1/2 effect on cell proliferation and cell survival, ERK1/2 signalling pathway can also regulate cell differentiation (Miranda et al., 2001, Kawamura et al., 1999 and Tokuda et al., 1999) through cross talk with p38 MAPK (Shimo et al., 2007). In addition, there are some reports which suggest that p21WAF1 is regulated by ERK1/2 MAPK and ERK1/2 specific inhibitor PD98059 reported to inhibit expression of p21WAF1 in the leukemic and cancer cell lines (Agadir et al., 1999, Das et al., 2000, Dufourny et al., 1997, Sato et al., 2000, Sugibayashi et al., 2001 and Miranda et al., 2001).
The ERK1/2 and p38 signalling pathways are activated by phosphorylation at specific sites and their activation can be monitored in vitro using phosphorylated-specific antibody (Miranda et al., 2001). ERK1/2 consists of 44 kDa ERK1 and 42 kDa ERK2, which share approximately 84% sequence homology. The two phosphorylation events, first on tyrosine residue and a second on a proximal threonine residue are required for ERK1/2 full activation. However, ERK1/2 can even enter and exit the nucleus of cell in the absence of activated signalling pathway due to its interaction with nuclear pore proteins. ERK is involved in the regulation of IL-6, IL-12, IL-23 and TNF-α synthesis (Goodridge et al., 2003; Feng et al., 1999). ERK1/2 activity is inhibited by its specific PD98059 inhibitor (Kosako et al., 2009 and Whitehust et al., 2002).
A study by Schett et at., 2000 suggest that p38 MAPK, JNK and ERK, are expressed in the rheumatoid synovium and have been implicated in the pathogenesis of RA. ERK MAPK inhibitors (for example; PD098059) have been found to reduce nociceptive responses in an adjuvant-induced monoarthritis in rats (Cruz et al., 2005) and inflammation in an ear oedema model in mice and in an experimental osteoarthritis model in rabbits (Jaffee et al., 2000; Pelletier et al., 2003), suggesting that ERK plays an important role in chronic inflammation. In Schett et al., (2000) ERK MAPK has been shown to be activated in synovial fibroblasts following stimulation with IL-1, TNF-α and fibroblast growth factor and also found to be activated in mononuclear cell infiltrates and synovial fibroblasts in synovial tissue from RA patients, suggesting involvement of ERK in joint damage associated with pro-inflammatory cytokine production by macrophages. p38 MAPK signalling pathway is involved in human inflammatory disease and is activated in the rheumatoid synovium (Feldmann et al., 2001). Due to presence of different stress factors and increased pro-inflammatory cytokines in the synovium, the activation of the p38MAPK signalling pathway in RA joints is conceivable. p38α is a 38 kDa protein that regulates LPS-induced TNF-α and IL-1β from monocyte cells (Han et al., 1993, Lee et al., 1994) and it has pro-inflammatory properties in RA synovial fluid (Korb et al., 2006). In another study Schett et al., 2008 reported that Phospho-p38α (p-p38α) is localised to the RA synovial intimal lining, which includes fibroblast-like synoviocytes and monocytes that produce IL-6 and a variety of other pro-inflammatory mediators. Study by Zwerina et al., (2006) suggest that activation of p38 MAPK is an important step in TNF-α mediated inflammatory bone destruction and inhibition of p38MAPK in animal models leads to reduced inflammation, which correlates with reduced expression of IL1, IL6 and RANKL cytokines. p38α activity can be inhibited by its specific inhibitor SB203580 which acts as a specific competitive ATP binding inhibitor (Davies et., 2000) and reduce pro-inflammatory cytokine TNF-α expression in RA animal models (Sweeney, Firestein, 2006; Hammaker, Firestein, 2010; Kumar et al., 2003).

1.4 Anti-rheumatic drugs
Several existing drugs are available to treat inflammatory rheumatic disease to reduce pain, inflammation in the joints and to slow disease progression. The National Institute for Health and Care Excellence (NICE, 2020) guidelines recommend use of cyclooxygenase inhibitors (for example; naproxen, ibuprofen) to reduce RA symptoms like pain and inflammation and recommend using DMARDs (for example; methotrexate, leflunomide, sulfasalazine) as first-line treatments within 3 months of onset of persistent RA symptoms to slow the progression of RA.
The NICE guidelines recommend combination of DMARDs and corticosteroids (prednisolone or azathioprine) for patients who are unresponsive to DMARDs monotherapy (Rathinam et al., 2019). The glucocorticoids slow the disease progression by inhibiting transcription of cytokines gene expression.
Biologics such as anti-IL-6 ( sarilumab, tocilizumab), anti-IL-12 anti-IL-23 ( ustekinumab) and Janus associated kinase inhibitors ( tofacitinib, baricitinib, cytokine modulators (adalimumab, etanercept, infliximab, certolizumab pegol, golimumab) are used in combination with methotrexate as options for unresponsive patients to DMARDs and corticosteroids combination therapy (NICE, 2020).
According to NICE (2020) the long term side effects of DMARDs include nausea, hair loss, liver toxicity and prolonged use of corticosteroids can induce immunosuppression against infection, osteoporosis, hyperglycaemia and hypertension. Similarly, biologics, Janus associated kinase inhibitors and cytokine modulators also have unwanted side effects such as hypersensitivity, gastrointestinal symptoms, headache, upper respiratory infections, common cold, congested nose sore throat and injection site reactions.
In spite of the dramatic improvements seen with the biologics, 40% of patients remain unresponsive to available treatmentsSome patients have to undergo regular change to therapeutics. For these reasons, there is intense global effort to develop more potent and effective orally active anti-inflammatory drugs which can cure disorders like RA from its first diagnosis (Scott et al., 2010). Therefore, sulphated disaccharides may pose as potential new anti-inflammatory therapeutic for the treatment of RA.

Table – Pharmacologic therapies for rheumatoid arthritis.
Classification Name Mechanism of action Potential mechanisms Reference
Conventional synthetic DMARDs Methotrexate Analog of folic acid Folate-dependent processes; Adenosine signaling; Methyl-donor production; Reactive oxygen species; Adhesion-molecule expression; Cytokine profiles Eicosanoids and MMPs. (Brown et al., 2016)
Leflunomide/ Teriflunomide Pyrimidine synthesis inhibitor DHODH-dependent pathway; Leukocyte adhesion; Rapidly dividing cells; NF-kB; Kinases; Interleukins; TGF-β. (Kasarello et al., 2017)
Sulfasalazine Anti-inflammatory and immunosuppression Cyclooxygenase and PGE2; Leukotriene production and chemotaxis; Inflammatory cytokines (IL-1, IL-6, TNF-α); Adenosine signaling; NF-kB activation. (Linares et al., 2011)
Chloroquine /Hydroxychloroquine Immunomodulatory effects Toll-like receptors; Lysosomotropic action; Monocyte-derived pro-inflammatory cytokines; Anti-inflammatory effects; Cellular immune reactions; T cell responses; Neutrophils; Cartilage metabolism and degradation. (Rainsford et al., 2015)
Biological DMARDs
Antibody-based therapies
Classification Name Mechanism of action Potential mechanisms Reference
TNF-α targeted therapy Infliximab TNF-α inhibitor Phagocytosis and pro-inflammatory cytokines; Chemoattractant; Adhesion molecules and chemokines; Treg cell function; Function of osteoclasts, leukocytes, endothelial and synovial fibroblasts. (Kim and Moudgil, 2017)
Adalimumab
Etanercept
Golimumab
Certolizumab pegol
B-cell targeted therapy Rituximab B cell depleting Fc receptor gamma-mediated antibody-dependent cytotoxicity and phagocytosis; Complement-mediated cell lysis; antigen presentation; B cell apoptosis; Depletion of CD4+ T cells. (Mota et al., 2017)
Ofatumumab
Belimumab Inhibitors of B cell function
Atacicept
Tabalumab
T-cell targeted therapy Abatacept CD28/CTLA4 system Autoantigen recognition; Immune cell infiltrate; T cells activation. (Mellado et al., 2015)
Belatacept CD80/CD86
Interleukin targeted therapy Tocilizumab IL-6 inhibition Innate and the adaptive immune system perturbation; Acute-phase proteins. (Raimondo et al., 2017)
Anakinra IL-1 inhibition Inflammatory responses; Matrixenzyme. (Cavalli and Dinarello, 2015)
Canakinumab
Rilonacept
Secukinumab IL-17 inhibition Mitochondrial function; Autophagosome formation. (Kim et al., 2017)
Ixekizumab
Growth and differentiation factors Denosumab RANKL inhibitor Maturation and activation of osteoclast. (Fassio et al., 2017)
Mavrilimumab GM-CSF inhibitor Activation, differentiation, and survival of macrophages, dendritic cells, and neutrophils; T helper 1/17 cell; modulation of pain pathways. (Burmester et al., 2017)
Small molecules
Classification Name Mechanism of action Potential mechanisms Reference
JAK pathway Tofacitinib JAK1 and JAK3 inhibitor T-cell activation, pro-inflammatory cytokine production, synovial inflammation, and structural joint damage. (Yamaoka, 2016; Winthrop et al., 2017)
Baricitinib JAK1 and JAK2 inhibitor
Filgotinib JAK1 inhibitor

1.5 Sulphated disaccharides
It has been reported that large sulphated polysaccharides such as calcium pentosan polysulphate found in chemically sulfated beechwood xylosan are mildly anti-inflammatory but not anti-rheumatic. However, glycosaminoglycan polysaccharides with repeat disaccharide subunits possessing 2-amino and 6′-carboxylate groups were found to possess anti-inflammatory and anti-rheumatic activity (Smith et al., 1994). For example, chondroitin sulphate has been shown to have anti-arthritic activity when administered orally in rats and human (Rona et al., 1998). Similarly, heparin an anti-coagulant is a polyanionic sulfated polysaccharide (Young, 2008), which is generally found in mast cells and is co-released with histamine into the vasculature during infection at injured tissues. At sites of tissue injury it dissociates from its protein core to exist as free glycosaminoglycan chains (Nader et al., 1999). It has been found to possess weak anti-asthma activity when given locally by inhalation (Lever et al., 2001). However, heparin physiological analogs, heparan sulphate have been found to inhibit T cell-mediated immune responses effectively in adjuvant-induced arthritis and delayed-type hypersensitivity in rodents when given orally in a nanogram amounts (Cahalon et al; 1997 and Lider et al., 1995).
Heparan sulphates are composed of glycosaminoglycan chains that are negatively charged under physiological conditions through the presence of sulphate and uronic moieties. They are present throughout at the cellular surface and reside in most cell membranes and are prominent in extracellular matrix. Heparan-sulphate glycosaminoglycan chains tend to exist as proteoglycan components that are tethered to a protein core. Therefore, they are expressed on the surface of cells, giving a net negative charge to these surfaces. During inflammation, heparan sulphate disaccharides can be generated by mammalian heparanase or heparinase I enzyme activity which results in the disruption of the extracellular matrix. The heparan sulphate disaccharides then negatively downregulate inflammatory responses by inhibition of macrophage TNF-α, IL-8 and IL-1β synthesis (Cahalon et al; 1997 and Chowers et al., 2001) and T cell function (Hecht et al., 2004). Intracellular signalling in neutrophils has been shown to be affected by heparin (Lever and Page, 2001) and heparan sulphated disaccharides which inhibit the transcription factorNF-κB. This in turn regulates the synthesis of TNF-α and leukocyte adhesion to the endothelium (Hershkoviz et al., 2000).
Cahalon and colleagues (1997) produced heparin tri-sulphated disaccharides, di-sulphated disaccharides and non-sulphated disaccharides by the action of bacteria (Flavobacterium heparium) heparinase I on porcine intestinal mucosa heparin sodium salt using high-performance liquid chromatography. They studied the impact of the sulphated disaccharide molecules on macrophage TNF-α production in vitro by incubating mouse peritoneal macrophages with saline or with different concentrations of tri-sulphated disaccharides, di-sulphated disaccharides and non-sulphated disaccharides and stimulated the mouse peritoneal macrophages with LPS. The study shows that tri-sulphated disaccharide and di-sulphated disaccharide produce a bell-shaped inhibition curve where the degree of inhibition of these compounds increased with increasing concentrations. The non-sulphated disaccharides did not inhibit macrophage TNF-α production.
Furthermore, Cahalon et al., (1997) used an in vivo model to study the effects of sulphated disaccharides on cell-mediated inflammation in mice and rats. They showed that inhibitory effect of di-sulphated disaccharide was bell-shaped where higher concentrations of tri-sulphated disaccharide and di-sulphated disaccharide were found be less effective. The non-sulphated disaccharide was not effective compared to tri-sulphated disaccharide and di-sulphated disaccharide on cell-mediated inflammation in mice and rats.
In addition, Cahalon et al., (1997) tested sulphated disaccharides impact on adjuvant arthritis by inducing adjuvant arthritis in female Lewis rats with M.tuberculosis antigen and treated the rats 12 days after the development of limbs swelling. The di-sulphated disaccharides demonstrated bell-shaped inhibition dose-response curves. The non-sulaphted disaccharide was less effective. The tri-sulphated disaccharides and di-sulphated disaccharides inhibited adjuvant arthritis more effectively when administered orally in nanogram amounts.
The extracellular matrix heparan derived sulphate disaccharides differ from the heparin derived sulfate disaccharides due to their backbone composition. As shown in Figure 1.02, The heparin-derived sulphated disaccharides backbone is composed of iduronic acid and glucosamine moieties whereas, heparan sulphate disaccharides backbone is composed of glucuronic acid and glucosamine moieties having either a glycosidic -O- linkage or -N-linkage (Salmivirta et al., 1996). Various degrees of sulfation occur on each monosaccharide unit, ranging from zero to tri-sulfation (Salmivirta et al., 1996, Cahalon et al., 1997 and Chowers et al., 2001). Cahalon et al., (1997) suggesting that sulphate group of sulphated disaccharides may be functionally important for their inhibitory action.

Figure 1.0 2 Backbone composition of heparan and hepain.
Heparan and heparin glycosaminoglycan consist of heterogeneous mixtures of repeating units of D- glucosamine and L-iduronic acids or D-glucuronicacids, sulfation at each residue may vary (from Salmivirta et al., 1996).

1.6 Rational for this study
Inflammation is a defensive cellular mechanism in response to harmful stimuli but uncontrolled inflammation leads to inflammatory diseases such as RA. The synovial CD68 macrophages are accountable for excessive pro-inflammatory TNF-α release. In spite of available therapies there is still a need for orally active anti-inflammatory drugs. Hence, compounds with potential to polarise CD68 pro-inflammatory macrophages to anti-inflammatory macrophages to inhibit TNF-α production may be useful in treating chronic inflammatory diseases.
Heparin derived sulphated disaccharide molecules has been reported to inhibit macrophage TNF-α production, delayed-type hypersensitivity, adjuvant arthritis, rat collagen-induced arthritis with unknown mechanisms of action. This suggests that heparin derived sulphated disaccharides which are released during inflammation can be used as molecular regulators of inflammation.
Additionally, preliminary reports (Bajwa and Seed, 2015) indicated that these compounds have a distinct effect on the monocyte-macrophage differentiation in vitro and formed the basis of this study. These preliminary findings led to the researchaims and objectives listed below as further investigations on the the mechanisms of anti-inflammatory actions of the sulphated disaccharide compounds could pave the way for development of these compounds as potent therapeutics for RA.
The studies directed at specific molecules will contribute to understanding of the pathogenesis of RA and the development of more effective therapies and preventive measures. Therefore, in this study four sulphated disaccharides with different structural patterns were used to investigate the unknown cellular and molecular mechanisms of action of sulphated disaccharides in macrophage function. This study relates to sulphated disaccharide compounds containing one to eight sulphates at concentrations of 10-11 M to 10-4 M. This study relates to methods of testing the impact of sulphated disaccharides to investigate cell surface markers, cellular signalling pathways by which macrophage polarisation may be affected by sulphated disaccharides for a deeper understanding of the mechanism of action of these compounds.

References
Aletaha, D., & Smolen, J. S. (2018). Diagnosis and management of rheumatoid arthritis: a review. Jama, 320(13), 1360-1372.
Falconer, J., Murphy, A. N., Young, S. P., Clark, A. R., Tiziani, S., Guma, M., & Buckley, C. D. (2018). Synovial cell metabolism and chronic inflammation in rheumatoid arthritis. Arthritis & Rheumatology, 70(7), 984-999.
Rathinam, S. R., Gonzales, J. A., Thundikandy, R., Kanakath, A., Murugan, S. B., Vedhanayaki, R., … & FAST Research Group. (2019). Effect of corticosteroid-sparing treatment with mycophenolate mofetil vs methotrexate on inflammation in patients with uveitis: a randomized clinical trial. Jama, 322(10), 936-945.

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