**2. The cellular basis of RA and current therapies**

RA is a systemic, chronic, progressive autoimmune inflammatory disease which affects approximately 1% of the population worldwide. The disease is polyarticular, and is characterised by synovitis, pannus formation, neovascularisation and hyperplasia caused by the infiltration of leukocytes. This in turn leads to the destruction of cartilage, tendon and bone, and the associated joint stiffness, swelling and pain that is the hallmark of this disease. Systemic symptoms also include inflammation in distil areas of the body, including the lungs, pericardium and pleura. Vasculitis, atherosclerosis, myocardial infarction and stroke are consequently commonly linked to RA.

The cells infiltrating the affected joint space are central to the pathology of RA, and include B cells, T cells, and cells of the monocyte/macrophage lineage as well as fibroblast-like synoviocytes. In their activated state within the affected joint these cells produce autoantibodies such as those against citrullinated proteins (CCP) and immunoglobulin (Rheumatoid factor (RF)), tissue degrading enzymes such as the matrix metalloproteaes (MMPs), as well as pro-inflammatory molecules such as TNF, Interleukin (IL)-6 and IL-1 that are central to tissue destruction, disease chronicity, and the maintenance of the inflammatory state. The ability of some cell types (B cells and RA synovial fibroblasts) to migrate to other sites has also been proposed as a precipitating event in the spread of disease to other joints (Lefevre et al., 2009).


Table 1. Current RA therapies

RA is a systemic, chronic, progressive autoimmune inflammatory disease which affects approximately 1% of the population worldwide. The disease is polyarticular, and is characterised by synovitis, pannus formation, neovascularisation and hyperplasia caused by the infiltration of leukocytes. This in turn leads to the destruction of cartilage, tendon and bone, and the associated joint stiffness, swelling and pain that is the hallmark of this disease. Systemic symptoms also include inflammation in distil areas of the body, including the lungs, pericardium and pleura. Vasculitis, atherosclerosis, myocardial infarction and stroke

The cells infiltrating the affected joint space are central to the pathology of RA, and include B cells, T cells, and cells of the monocyte/macrophage lineage as well as fibroblast-like synoviocytes. In their activated state within the affected joint these cells produce autoantibodies such as those against citrullinated proteins (CCP) and immunoglobulin (Rheumatoid factor (RF)), tissue degrading enzymes such as the matrix metalloproteaes (MMPs), as well as pro-inflammatory molecules such as TNF, Interleukin (IL)-6 and IL-1 that are central to tissue destruction, disease chronicity, and the maintenance of the inflammatory state. The ability of some cell types (B cells and RA synovial fibroblasts) to migrate to other sites has also been proposed as a precipitating event in the spread of

TARGET NAME FORM STATUS

*T cells* Abatacept (CTLA4) Fusion protein Clinical use

Drugs (antimetabolites, antiinflammatory)

1, 2, 5Humanised Ig, 2human nonglycosylated Ig,

1Fusion protein. 2,4,5,6Human Ig, 3chimeric

1R antagonist

Small molecule inhibitors

mouse/human Ig, 7IL-

Clinical use

1Clinical use 2,3,4 Pre-clinical 5Approved for human use – not

Clinical use

Pre-clinical

RA

**2. The cellular basis of RA and current therapies** 

are consequently commonly linked to RA.

disease to other joints (Lefevre et al., 2009).

*B cells* 1Rituximab (CD 20),

Methotrexate Other DMARDs eg Sulphalazine, Cyclosporin, Corticosteriods

2MDX-1342 (CD19), 3anti-BAFF, 4anti-CD16, 5Eculizumab (complement C5),

1Etanercept, 2Adalimumab, 3Infliximab, 4Golimumab, 5Certolizumab, 6Tocilizumab, 7Anakinra, 8Bevacizumab

1, 2,3 Multiple,

(anti-Syk)

Table 1. Current RA therapies

4CG11746, 4PC132765, 6CP-690550, 5R788

*All rapidly dividing cells* 

*Cytokines*  1-5TNF, 6IL-6R, 7IL-1, 8VEGF

*Signalling molecules*  1IKK2, 2PDE4, 3p38, 4Btk, 5Syk, 6JAK3

Knowledge of the processes responsible for disease activity and progression has lead to significant advances in the treatment of RA in the last 30 years. Early treatment, within months of the onset of persistent symptoms, is recommended, and at the present time usually takes the form of disease modifying anti-rheumatic drugs (DMARD) such as methotrexate. In more recent years however, the choice of treatment for RA has expanded significantly, and importantly now utilises agents that are less globally immunosuppressive than methotrexate (Weinblatt et al., 1985) (Table 1). These newer therapies target either specific cell types, such as B and T cells that present within the inflamed synovium, or their products (Genovese et al., 2008; Tedder, 2009; Townsend et al., 2010; Buch et al., 2011). Indeed, anti-cytokine therapies have revolutionised the treatment of RA in recent years. In particular, the use of anti-TNF biologicals such as Adalimumab, Etanercept and Infliximab have become the treatment of choice in those who do not respond to conventional DMARDs (Taylor et al., 2009), although biologicals that target other pro-inflammatory cytokines are also approved for use. These include Tocilizumab (anti-IL-6R) (Fleischmann et al., 2006; Jones et al., 2010) and Anakinra, a recombinant IL-1 receptor antagonist, as well as Bevacizumab, an antibody that targets vascular endothelial growth factor (VEGF) and hence may reduce neovascularisation that pannus formation depends upon. A variety of small molecule inhibitors designed to target critical elements of the B cell receptor, T cell receptor or cytokine signalling pathways such as inhibitors of IKK2, PDE4 and Btk (Bruton's tyrosine kinase), have shown interesting results in some animal models of arthritis, as have clinical trials with the syk inhibitor R788 (Podolin et al., 2005; Lindstrom et al., 2010; Di Paolo et al., 2011). The p38 inhibitors however, which showed such promise in animal models have not lived up to expectations in clinical trials and have not progressed beyond phase II (Genovese, 2009).

Despite the significant advances made with this arsenal of therapies, the goal of achieving sustained remission of RA has remained elusive and even with long term DMARD and biologic therapy is relatively uncommon. The efficacy of treatments is also unpredictable. Thus, a significant proportion of patients do not respond adequately to first line DMARD treatment and are then moved on to biologics. Even here, approximately 40% of patients do not respond to anti-TNF therapy for example. Moreover, many of these treatments are accompanied by significant side-effects, ranging from injection site reactions, increased infection rates and neutropenia to the potential for an increased risk of malignancies (van Vollenhoven, 2009).

When taking a global view of all the therapies for RA, either in use in the clinic, in early trials, in animal models, or in *in vitro* studies, it becomes clear that all are designed to target the ongoing process of inflammation. Namely the cells present in the joint during the inflammatory process, or their soluble products (cytokines), rather than targeting a causative agent for RA. However, for RA sufferers a single causative agent has not, and probably will never, be defined. Rather, RA is a complex disease with a multi-factorial etiology. It's prevalence in women (3:1 female to male ratio) suggests a hormonal contribution, and there are clear links to environmental factors such as smoking as well as a predisposition to RA with certain HLA haplotypes (Bax et al., 2011). Genetic and twin studies also suggest a strong environmental influence as well as a genetic link. As a tractable causative agent or single predisposing gene is therefore unlikely to be identified, a therapy that will treat disease in the early stages, that will prevent progression to a chronic state and thereby allow the inflammatory state to resolve, thereby preventing tissue damage, bone and cartilage destruction and progression, remains the holy grail of many researchers.

Targeting DAMP Activation of

**TLR4** Lipopolysaccharide

(LPS)

**TLR6** Diacyl lipoprotein Bacteria,

**TLR7/8** ssRNA Bacteria,

**TLR9** CpG-DNA Bacteria,

molecule

DAMPs in red have been reported in the RA joint.

**TLR11** Profilin-like

Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis? 215

**TLR3** dsRNA Viruses mRNA Released from

**TLR5** Flagellin Bacteria Unknown Unknown

**TLR10** Unknown Unknown Unknown Unknown

Viruses

Viruses

Viruses, Protazoa

Table 2. Exogenous and endogenous activators of human TLRs.

Bacteria, Viruses

D, eosinophil derived neurotoxin, antiphospholipid antibodies, serum amyloid A,cardiac myosin, PAUF, CEP, monosodium urate crystals Biglycan, versican

Hyaluronic acid fragments

HMGB1, surfactant proteins A and D, defensin 2, HSP60, 70, 72, 22, Gp96, S100A8, S100A9, neutrophil elastase, antiphospholipid antibodies, lactoferrin, serum amyloid A, oxidised LDL, saturated fatty acids, resistin, PAUF, monosodium urate crystals Biglycan, fibronectin EDA, fibrinogen, tenascin-C Heparin sulphate fragments, hyaluronic acid fragments

Antiphospholipid antibodies, ssRNA, cardiac myosin

IgG-chromatin complexes, mitochondrial DNA

Protazoa Unknown Unknown

Induced upon tissue damage Degradation of tissue

activated/necrotic cells

Released from activated/necrotic cells

Induced upon tissue damage

> Degradation of tissue

Released from activated/necrotic cells

Released from activated/necrotic cells

Unknown Unknown

Given that RA is an inflammatory condition it is likely that a precipitating event initiates a state of inflammation. In the normal individual, inflammation is invariably initiated in response to danger signals sensed by a series of cellular receptors known as pattern recognition receptors (PRRs). PRRs were originally defined by their ability to recognise and respond to invading pathogens (bacterial, viral, fungal) but are now increasingly linked to the detection of damaged 'self' molecules known as DAMPs. A large body of evidence has emerged in the last decade implicating one particular family of PRRs, the TLRs, in driving inflammation during RA.
