**3.1 TLRs and RA pathology**

TLRs are expressed on a variety of different cell types, many of which are found within the inflamed rheumatoid joint, including myeloid cells, fibroblasts, epithelial and endothelial cells. In humans the first evidence linking the presence of TLRs with RA pathology arose from the comparison of TLR expression between normal or non-inflamed joints and RA joints. Significant up-regulation of a number of TLRs was observed in both synovial tissue and circulating immune cells isolated from RA patients. Table 3 depicts the specific pattern of expression of these TLRs in RA.


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

TLRs are a highly conserved family of PRRs. With the most recent addition of murine TLR13 (Shi et al., 2011), 14 mammalian TLRs have been reported to date, with 10 human subtypes. All TLRs are type I transmembrane proteins comprising an extracellular domain of multiple leucine rich repeats (LRRs), a single membrane spanning -helix and a cytoplasmic Toll/IL-

TLRs can be classified according to their subcellular localization: the endosomal TLRs 3, 7, 8 and 9 reside in intracellular compartments, whilst the others are found at the plasma membrane. This distribution also reflects the ligand specificity of TLRs; the cell surface receptors predominantly recognize pathogenic and self surface elements, whereas endosomal receptors primarily sense nucleic acids. Recognition of ligand triggers receptor dimerization which in turn triggers a multitude of signalling cascades leading to the expression of pro-inflammatory mediators such as cytokines and chemokines, which are designed to combat the perceived danger. In this way the body mounts an effective immune response (reviewed in (Piccinini et al., 2010). The TLR ligands that induce such a response include both PAMPs and DAMPs, and a more detailed list of them, with particular reference to those found in RA, can be found in Table 2. Thus, TLRs are critical for both the

TLRs are expressed on a variety of different cell types, many of which are found within the inflamed rheumatoid joint, including myeloid cells, fibroblasts, epithelial and endothelial cells. In humans the first evidence linking the presence of TLRs with RA pathology arose from the comparison of TLR expression between normal or non-inflamed joints and RA joints. Significant up-regulation of a number of TLRs was observed in both synovial tissue and circulating immune cells isolated from RA patients. Table 3 depicts the specific pattern

**TLR PAMP SOURCE DAMP SOURCE TLR1** Triacyl lipoprotein Bacteria -defensin 3 Released from

> HSP60, 70, Gp96, HMGB1, HMGB1-nucleosome complexes, -defensin 3, surfactant proteins A and

Viruses, Parasites activated/necrotic cells

Released from activated/necrotic cells

response to invading pathogens and the response to 'sterile' tissue damage.

inflammation during RA.

**3. The toll-like receptors** 

**3.1 TLRs and RA pathology** 

of expression of these TLRs in RA.

**TLR2** Lipoprotein Bacteria,

1 receptor (TIR) homology signalling domain.


DAMPs in red have been reported in the RA joint.

Table 2. Exogenous and endogenous activators of human TLRs.

Targeting DAMP Activation of

**3.2 Which TLRs are important in RA?** 

increase cytokine production.

8 in human disease.

pathogenesis *in vivo*.

**3.3 The role of TLRs in animal models of RA** 

Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis? 217

Further studies using *ex vivo* human disease models have provided evidence of a functional role for TLRs in driving inflammation in RA. Adenoviral over expression of dominant negative Myd88, an adaptor molecule required for signalling by all TLRs except TLR3, inhibited cytokine synthesis in RA synovial cells (Sacre et al., 2007). The naturally occurring TIR signalling antagonist single-immunoglobulin interleukin-1 receptor-related (SIGIRR) is also efficacious in suppressing cytokine synthesis in these cells (Drexler et al., 2010). Together these data suggest that TLRs may contribute to synovial inflammation but do not rule out the possibility that IL-1-mediated signalling, which shares the TIR-myd88 derived

framework, is responsible for these findings, nor do they pinpoint any specific TLR.

Evidence of a role in RA for both cell surface TLRs and endosomal TLRs in human disease is accumulating. In particular, over expression of dominant negative Mal, an adaptor protein required exclusively by TLR2 and 4, has been shown to inhibit cytokine and protease synthesis in RA synovial cells, supporting the contribution of these two family members to the synthesis of pro-inflammatory mediators in the RA joint (Sacre et al., 2007). Blockade of the function of TLR2 and 4 using neutralizing antibodies has also been reported, and while commercially available antibodies to either TLR2 or TLR4 had no effect on cytokine production in isolated RA synovial cells at 10 g/ml (Sacre et al., 2007), 1 g/ml of an anti-TLR2 antibody (OPN301) inhibited spontaneous cytokine release in RA tissue explants as effectively as anti-TNF antibodies (Nic An Ultaigh et al., 2011). Inhibition of TLR4 by the naturally occurring antagonist LPS isolated from *Bartonella Quintana*, also partially inhibited cytokine release in RA synovial biopsies (Abdollahi-Roodsaz et al., 2008). Stimulation of TLRs 2, and 4 has also been shown to induce cytokine synthesis in cell cultures isolated from RA synovia (Sacre et al., 2008). While the same workers found that TLRs 7 and 9 were not responsive to their respective ligands in RA cultures, stimulation of TLRs 3 and 8 did

The contribution of endosomal TLRs to cytokine synthesis in RA is also supported by other studies; chloroquine, which prevents intracellular TLR function by inhibiting endosomal acidification, reduces cytokine release in synovial cells (Sacre et al., 2008). The selective serotonin reuptake inhibitors, antidepressant drugs fluoxetine and citalopram and the antidepressant small molecule mianserin are also efficacious in inhibiting synovial cell cytokine release (Sacre et al., 2010). These drugs also inhibit TLR3, -7, -8 and -9 activity, by mechanisms which are yet unknown. More specifically, the small molecule imiquimod, which targets TLR8, also inhibited the production of TNF from human RA synovial membranes (Sacre et al., 2008). There have also been anecdotal reports of improved symptoms in RA patients taking anti-depressants (Krishnadas et al., 2011). Taken together these studies suggest a significant role for TLR2 and 4 as well as the endosomal TLRs 3 and

In addition to studies in human tissue, the contribution of TLRs to inflammation and joint destruction has been examined in rodent models of arthritis. Mice with targeted deletions in TLRs have demonstrated that specific family members are important in driving disease


nd = not determined

F= function = the ability of the TLR to respond to its cognate ligand in each cell/tissue type

Table 3. Distribution of TLR expression in the RA joint

Further studies using *ex vivo* human disease models have provided evidence of a functional role for TLRs in driving inflammation in RA. Adenoviral over expression of dominant negative Myd88, an adaptor molecule required for signalling by all TLRs except TLR3, inhibited cytokine synthesis in RA synovial cells (Sacre et al., 2007). The naturally occurring TIR signalling antagonist single-immunoglobulin interleukin-1 receptor-related (SIGIRR) is also efficacious in suppressing cytokine synthesis in these cells (Drexler et al., 2010). Together these data suggest that TLRs may contribute to synovial inflammation but do not rule out the possibility that IL-1-mediated signalling, which shares the TIR-myd88 derived framework, is responsible for these findings, nor do they pinpoint any specific TLR.
