**1. Introduction**

40 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

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Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic joint inflammation. The precise aetiology of this autoimmune process remains unclear. Soluble factors produced by infiltrating synovial cells play an important role in driving the inflammatory process that leads to inflammatory cell migration and proliferation in the synovial tissue. These soluble factors consist mainly of cytokines that either promote or suppress inflammation.

A number of cytokines have been identified in synovial fluid and the synovial membrane. Cytokines such as TNF, IL-1 and IL-6 stimulate T-cells and induce subsequent cartilage and bone erosion (Kang et al., 2009). Along with IL-18, these cytokines are produced by synovial macrophages and synovial fibroblasts. IL-18 causes joint inflammation and subsequent bone destruction by facilitating T-cell activation and stimulating B-cell production of autoantibodies. Deletion of the IL-18 gene in mice has been shown to result in a significant reduction in the incidence of joint inflammation and bone destruction (Wei et al., 2001).

CD4+ T-cells proliferate in inflamed synovial joints through stimulation of IL-15. Inhibition IL-15 results in a significantly lower production of TNF and IL-1. As such, IL-15 blockade abolishes severe joint inflammation in collagen induced-arthritis mouse models (Ruchatz et al., 1998). In the CD4+ T-cell population, Th17 has been demonstrated as a pathogenic T-cell that produces IL-17 to induce neutrophil migration (Shibata et al., 2009). IL-17 is known to stimulate receptor-activator of nuclear factor kappa-B ligand (RANKL) production by osteoblast cells to promote osteoclastogenesis in RA bone erosion (Joosten et al., 2003). Th17 development is governed by TGF, IL-1 and IL-23 (Paradowska-Gorycka et al., 2010; Santarlasci et al., 2009). Higher concentrations of IL-23 (a member of the IL-12 family cytokines) are detected in the serum and synovial fluid of patients with greater severity of RA (Melis et al., 2010). IL-23 can also be produced by osteoblast cells after stimulation with TNF (unpublished data).

 It is possible that remission of joint inflammation in RA patients can occur spontaneously. The fluctuation in inflammation within the joint results from auto-inhibition through the production of anti-inflammatory cytokines via regulatory T (Treg) cells (Raghavan et al.,

IL-12 Family Cytokines in Inflammation and Bone Erosion of Rheumatoid Arthritis 43

Fig. 1. Schematic representation of structure of members of the IL-12 family cytokines.

The cytokines involved in mediating T cell development belong to the IL-12 family cytokines. This family of cytokines are heterodimer secreted glycoproteins. One subunit is an IL-6 like protein and the other an IL-6 soluble receptor like protein. They are therefore also named IL-6/IL-12 family cytokines. IL-12, IL-23, IL-27 and IL-35 all belong to this IL-12

IL-12 consists of a p40 subunit and a p35 subunit. IL-23 consists of the same p40 subunit but couples with a p19 subunit. IL-12 was the first cytokine to be identified that had the capability of driving Th1 development (Hsieh et al., 1993; Murphy et al., 1994). IL-23 was discovered almost a decade later and was initially seen as a novel cytokine with the ability to aid Th1 development at the later stages of cell differentiation (Oppmann et al., 2000). Recent evidences suggest that IL-23 is in fact functionally very different from IL-12. Aside from its effects on Th1 cell development, IL-23 also stimulates production of IL-17 by Th17 cells (Horai et al., 2000; Zelante et al., 2007). Osteoclast formation has been shown to be upregulated by IL-23 through its effects on macrophages (Chen et al., 2008). Unlike IL-12, IL-23

The cytokine IL-27 is composed of a p40-related protein called EBi3 (Epstein-Barr virus induced gene 3) and a p35-related protein known as p28. EBi3 was first identified in 1996 following its expression during infection of B-lymphocyte with the Epstein Barr Virus. The gene produced was a secreted glycoprotein related to p40 (Devergne et al., 1996). p28 was discovered some years later in a research investigation of IL-6 homologous proteins using bioinformatics. The p28 protein was found only to be efficiently secreted when coupled with EBi3. Following this discovery, the heterodimeric protein of EBi3 and p28 was named as IL-27 (Pflanz et al., 2002). The function of IL-27 was again, initially, thought to be similar to IL-12 in driving the early stages of Th1 cell development. Further study of IL-27 has shown it to be capable of inducing IL-10 production from Treg cells and subsequently inhibiting Th17 responses (Murugaiyan et al., 2009). In inflammatory mouse disease models, such as collagen induced arthritis (CIA) mouse model, IL-27 is able to suppress inflammation

EBi3 can be coupled with the p35 subunit of IL-12 to form a heterodimeric protein (Devergne, Birkenbach and Kieff, 1997). The function of EBi3/p35 remained a mystery until relatively recently when studies used a recombinant EBi3/p35 protein in a rheumatoid arthritis mouse model. In these experiments, joint inflammation in CIA mice was effectively

plays more of a pathological role in inflammatory and autoimmune diseases.

**2.2 IL-12 family cytokines in inflammation** 

(Niedbala et al., 2008).

family cytokines and share certain protein subunits (Fig.1).

2009) and other regulatory mechanisms. Treg cells encompasse CD4+ T-cells and produce anti-inflammatory cytokines – such as IL-10, TGF and IL-35 – to suppress Th17 and other T effector cells (Sabat et al., 2010). Recent studies have identified IL-35 (a member of the IL-12 family cytokines) as a potent suppressor of Th17 cells and promoter of Treg cell expansion (Collison et al., 2007; Niedbala et al., 2007). Injection of recombinant IL-35 into mice with collagen induced-arthritis has demonstrated effective suppression of the onset of joint inflammation (Niedbala et al., 2007). The mechanism of this model is thought to be through the promotion of Treg cell activity and suppression of Th17 function (Chaturvedi et al., 2011). Despite strong evidence from *in vivo* animal models demonstrating the therapeutic properties of IL-35 in the treatment of joint inflammation, there continues to be a lack of evidence from *in vitro* human bone inflammatory models. The efficaciousness of IL-35 as an immunomodulatory therapy against RA therefore remains to be proven.

In this chapter, we will concentrate on the role of the IL-12 family cytokines – namely IL-12, IL-23, IL-27 and IL-35 in the development of rheumatoid arthritis. We will present, analyse and summarise the most recent work in our field of research. We aim to provide an up-todate and comprehensive overview of the compelling evidence and novel ideas paving way for a new generation of medical therapies against RA.

#### **2. T cell activation and IL-12 family cytokines**

#### **2.1 T cell activation in rheumatoid arthritis**

The main infiltrative inflammatory T cell in the synovial joint is the CD4+ T cell. Dependent on cytokine production and cell linage control cell signalling, CD4+ T cells can be divided into 4 sub-populations – Th1, Th2, Th17 and immune regulatory T (Treg) cells. These sub-types differ in function and activity. Th1, Th2 and Th17 cells act mainly as T effector cells whereas Treg cells display an immune suppressing role. T helper (Th) cell differentiation and expansion is controlled by maturated dendritic cells (DC) via three different means of signalling. The first is antigen presentation where MHC II (present on the surface of DCs) relay antigens to T cell receptors (TCR) found on T cells. In RA, pathological auto-antigen signalling mediated by DCs and other antigen presenting cells lead to auto-antigen induced T cell responses. The exact cause of this remains unclear, however, recent evidence has identified connective tissue protein and citrullinated vementin as likely mediators. Both have been shown to trigger an auto-immune T cell response following presentation by MHC II in RA patient (Snir et al., ; van Lierop et al., 2007). The second type of signalling is co-stimulation and is mediated via co-stimulation molecules found on DCs. An example of this is CD40 when it binds to T cell costimulation receptor (CD40L). The final means of signalling is mediated by cytokines or a group of cytokines and is the most important form of signalling generated by DCs. Cytokines produced by activated DCs bind to receptors on naïve T cells and drive T cell differentiation into Th1, Th2 or Th17. In some instances, it induces differentiation into Treg cells and suppresses over-reactive T cells. In patients with RA, this complex system can result in joint inflammation that is self-limiting and accounts for the fluctuating nature of the disease. Despite vast amounts of research dedicated to investigating the precise mechanism of T cell development, in health and in disease, it remains a mystery.

Fig. 1. Schematic representation of structure of members of the IL-12 family cytokines.

#### **2.2 IL-12 family cytokines in inflammation**

42 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

2009) and other regulatory mechanisms. Treg cells encompasse CD4+ T-cells and produce anti-inflammatory cytokines – such as IL-10, TGF and IL-35 – to suppress Th17 and other T effector cells (Sabat et al., 2010). Recent studies have identified IL-35 (a member of the IL-12 family cytokines) as a potent suppressor of Th17 cells and promoter of Treg cell expansion (Collison et al., 2007; Niedbala et al., 2007). Injection of recombinant IL-35 into mice with collagen induced-arthritis has demonstrated effective suppression of the onset of joint inflammation (Niedbala et al., 2007). The mechanism of this model is thought to be through the promotion of Treg cell activity and suppression of Th17 function (Chaturvedi et al., 2011). Despite strong evidence from *in vivo* animal models demonstrating the therapeutic properties of IL-35 in the treatment of joint inflammation, there continues to be a lack of evidence from *in vitro* human bone inflammatory models. The efficaciousness of IL-35 as an

In this chapter, we will concentrate on the role of the IL-12 family cytokines – namely IL-12, IL-23, IL-27 and IL-35 in the development of rheumatoid arthritis. We will present, analyse and summarise the most recent work in our field of research. We aim to provide an up-todate and comprehensive overview of the compelling evidence and novel ideas paving way

The main infiltrative inflammatory T cell in the synovial joint is the CD4+ T cell. Dependent on cytokine production and cell linage control cell signalling, CD4+ T cells can be divided into 4 sub-populations – Th1, Th2, Th17 and immune regulatory T (Treg) cells. These sub-types differ in function and activity. Th1, Th2 and Th17 cells act mainly as T effector cells whereas Treg cells display an immune suppressing role. T helper (Th) cell differentiation and expansion is controlled by maturated dendritic cells (DC) via three different means of signalling. The first is antigen presentation where MHC II (present on the surface of DCs) relay antigens to T cell receptors (TCR) found on T cells. In RA, pathological auto-antigen signalling mediated by DCs and other antigen presenting cells lead to auto-antigen induced T cell responses. The exact cause of this remains unclear, however, recent evidence has identified connective tissue protein and citrullinated vementin as likely mediators. Both have been shown to trigger an auto-immune T cell response following presentation by MHC II in RA patient (Snir et al., ; van Lierop et al., 2007). The second type of signalling is co-stimulation and is mediated via co-stimulation molecules found on DCs. An example of this is CD40 when it binds to T cell costimulation receptor (CD40L). The final means of signalling is mediated by cytokines or a group of cytokines and is the most important form of signalling generated by DCs. Cytokines produced by activated DCs bind to receptors on naïve T cells and drive T cell differentiation into Th1, Th2 or Th17. In some instances, it induces differentiation into Treg cells and suppresses over-reactive T cells. In patients with RA, this complex system can result in joint inflammation that is self-limiting and accounts for the fluctuating nature of the disease. Despite vast amounts of research dedicated to investigating the precise mechanism of T cell development, in health and in disease, it remains a

immunomodulatory therapy against RA therefore remains to be proven.

for a new generation of medical therapies against RA.

**2. T cell activation and IL-12 family cytokines** 

**2.1 T cell activation in rheumatoid arthritis** 

mystery.

The cytokines involved in mediating T cell development belong to the IL-12 family cytokines. This family of cytokines are heterodimer secreted glycoproteins. One subunit is an IL-6 like protein and the other an IL-6 soluble receptor like protein. They are therefore also named IL-6/IL-12 family cytokines. IL-12, IL-23, IL-27 and IL-35 all belong to this IL-12 family cytokines and share certain protein subunits (Fig.1).

IL-12 consists of a p40 subunit and a p35 subunit. IL-23 consists of the same p40 subunit but couples with a p19 subunit. IL-12 was the first cytokine to be identified that had the capability of driving Th1 development (Hsieh et al., 1993; Murphy et al., 1994). IL-23 was discovered almost a decade later and was initially seen as a novel cytokine with the ability to aid Th1 development at the later stages of cell differentiation (Oppmann et al., 2000). Recent evidences suggest that IL-23 is in fact functionally very different from IL-12. Aside from its effects on Th1 cell development, IL-23 also stimulates production of IL-17 by Th17 cells (Horai et al., 2000; Zelante et al., 2007). Osteoclast formation has been shown to be upregulated by IL-23 through its effects on macrophages (Chen et al., 2008). Unlike IL-12, IL-23 plays more of a pathological role in inflammatory and autoimmune diseases.

The cytokine IL-27 is composed of a p40-related protein called EBi3 (Epstein-Barr virus induced gene 3) and a p35-related protein known as p28. EBi3 was first identified in 1996 following its expression during infection of B-lymphocyte with the Epstein Barr Virus. The gene produced was a secreted glycoprotein related to p40 (Devergne et al., 1996). p28 was discovered some years later in a research investigation of IL-6 homologous proteins using bioinformatics. The p28 protein was found only to be efficiently secreted when coupled with EBi3. Following this discovery, the heterodimeric protein of EBi3 and p28 was named as IL-27 (Pflanz et al., 2002). The function of IL-27 was again, initially, thought to be similar to IL-12 in driving the early stages of Th1 cell development. Further study of IL-27 has shown it to be capable of inducing IL-10 production from Treg cells and subsequently inhibiting Th17 responses (Murugaiyan et al., 2009). In inflammatory mouse disease models, such as collagen induced arthritis (CIA) mouse model, IL-27 is able to suppress inflammation (Niedbala et al., 2008).

EBi3 can be coupled with the p35 subunit of IL-12 to form a heterodimeric protein (Devergne, Birkenbach and Kieff, 1997). The function of EBi3/p35 remained a mystery until relatively recently when studies used a recombinant EBi3/p35 protein in a rheumatoid arthritis mouse model. In these experiments, joint inflammation in CIA mice was effectively

IL-12 Family Cytokines in Inflammation and Bone Erosion of Rheumatoid Arthritis 45

osteoarthritic patients and even healthy individuals but in significantly lower instances (Kim et al., 2000). The number of collagen type II (CII) reactive T cell in the peripheral blood mononuclear cell (PBMC) and synovial fluid monocytes (SFMCs) from RA patients was significant higher than those from osteoarthritis and health control individuals. The higher IFN concentration in the culture supernatant was also associated with higher IL-12 production. This result indicated that CII reactive Th1 response is dominant in RA patients

Study of TNF, TNF-RI/II and IL-12 serum level in RA patients concluded that IL-12p40 levels have a stronger association with disease than TNFlevels (Ebrahimi et al., 2009). IL-12 binds to IL-12 receptor1 and 2 to trigger STAT4 phosphorylation in T cells. RA patients not only produced more IL-12p40, but are also more sensitive to IL-12 stimulation, which based on the fact of T cells from RA patients showed earlier onset and higher levels of STAT4 phosphorylation in comparison with osteoarthritis and health controls (Sun et al., 2011). To date, anti-TNFtherapy has been proven as the most effective therapy in treatment of rheumatoid arthritis (Radovits et al., 2009;Salliot et al., 2011). CD4+ T cells in peripheral blood mononuclear cells (PBMC) of RA patient have higher frequency of IL-12 receptor and lower of IL-4 receptor. The frequency of IL-12R+ T cell was remarkably reduced with both methothotrexate (MTX) therapy and anti-TNF therapies (Herman et al., 2011). However another clinical study showed STAT4 phosphorylation was not induced in CD4+ T cells from untreated RA patients by anti-CD3 and anti-CD28, but STAT4 activation was induced in healthy individuals and anti-TNF treated patients (Aerts et al., 2010). This result disagreed with previous concepts of the role of IL-12 induced Th1 responses in RA. Since STAT4 phosphorylation was not induced by IL-12, but anti-CD3 and CD28 in this experimental setting, this result may indicate anti-TNF altered T cell response in RA patients. Since IL-12 is the only cytokine that strongly drives Th1 cell development and subsequent production of IFNγ, the role of IL-12 in RA inflammation was challenged by the studies in Collagen induced arthritis (CIA) mouse model with IFNγ receptor gene knockout or IFNγ blockade (Boissier et al., 1995; Manoury-Schwartz et al., 1997). Mice with IFN gene deficiency and IFN blocker did not show reduced joint inflammation but rather enhanced inflammation. These may suggest that IL-12 stimulates other cytokines to induce autoimmune joint inflammation in disease. IFN production from Th1 responses is rather suppressing the joint inflammation which was seen in IL-27 suppressing joint inflammation

Comprehensive studies by using autoimmune disease mouse models with gene knockout mice have demonstrated the complexity of IL-12p40, p35 and its receptors 1 and 2 (Airoldi et al., 2005; Gran et al., 2002; Kikawada, Lenda and Kelley, 2003; Yoshida et al., 2009). IL-12p40 and IL-12 receptor1 gene knockout mice showed resistance to joint inflammation in CIA mice while IL-12p35 and IL-12RII gene knockout mice are significantly more susceptible to joint inflammation induced by collagen type II (Hoeve et al., 2006; Murphy et al., 2003). Discovery of IL-23 provided a fully explanation for the confounding phenotype in these gene knockout mice (Ooi et al., 2009). Knockout of the IL-23p19 subunit in mice showed similar levels of resistance to joint inflammation as IL-12p40 knockout (Hoeve et al., 2006). Given that IL-23 shares the same protein subunit of IL-12p40, it can be concluded that IL-23 is the key pathological cytokine in joint inflammation. To support this theory is the discovery of significantly higher levels of IL-23 in both serum and synovial

(Park et al., 2001).

(see section 3.2).

fluid of RA patients (Melis et al., 2010).

Fig. 2. Diagram showing CD4+ T cell differentiation resulting from production of IL-12 family cytokines produced by DCs. The presentation of auto-antigens by DCs and cytokine production result in T cell differentiation. The type of T cell response depends on the type of cytokine production in the inflammatory environment. Both DCs and iTr(35), a type of Treg cell, produce IL-35 to suppress Th1, Th17 and Th2 responses.

resolved by the EBi3/p35 recombinant protein. It was suggested that the therapeutic mechanism of this recombinant protein was through induction of Treg cell development and IL-10 expression leading to suppression of Th1 and Th17 responses (Niedbala et al., 2007).

EBi3 and p35 are also highly expressed in Treg cells thus seemingly a key component of the immune regulating function of mouse Treg cells. Deficiency in either EBi3 or p35 gene in Treg cells will result in a reduced ability to suppress effector T-cell proliferation. Without EBi3 or p35, Treg cells are unable to resolve gut inflammation in mouse inflammatory bowel disease (IBD). The EBi3/p35 heterodimeric protein has since been named IL-35 (Collison et al., 2007). The functional effects of IL-35 in mouse models have been clearly demonstrated by a number of research groups. The role of this particularly cytokine in immune regulation of humans has now been confirmed (Chaturvedi et al., 2011).
