**4. Which TLR activators drive chronic inflammation in RA?**

Infection has long been purported to be a key underlying factor in RA pathogenesis. However, whilst pathogenic stimuli may trigger inflammation in RA, a causative infectious agent for RA has not been found and there is little evidence to suggest that PAMPs generate sustained joint inflammation (Schumacher et al., 1999; Chen et al., 2003). In contrast, data implicating DAMPs in RA pathogenesis have emerged from a number of independent studies in which factors derived from the serum, synovial fluid or synovial cells of RA patients can activate TLR mediated signalling pathways (Brentano et al., 2005; Roelofs et al., 2005; Sacre et al., 2007).

DAMPs are endogenous molecules that are immunologically silent in healthy tissues but become active upon tissue injury. They include intracellular molecules released from necrotic cells or secreted from activated cells, extracellular matrix molecule fragments created by tissue damage or proteolysis and extracellular matrix molecules that are specifically expressed upon tissue injury. In normal circumstances they act as danger signals that alert the organism to tissue damage and initiate the process of tissue repair. In addition to this physiological role however, there is evidence which indicates that DAMPS also contribute to the pathogenesis of many inflammatory and autoimmune diseases characterized by aberrant TLR activation including RA.

High levels of some DAMPs are detected in the destructive milieu of the RA joint (Table 2) (reviewed in (Piccinini et al., 2010), where they are hypothesized to drive chronic inflammation by invoking a perpetual destructive cycle where inflammation leads to the creation of new stimulators of inflammation (Roelofs et al., 2008). A number of approaches have been taken to examine the effect of DAMP administration, deletion or blockade in animal models of arthritis and data supporting the role of specific molecules in such models are summarized in Table 4.

In particular, the administration of the fibronectin EDA domain (FNEDA), fibrinogen, HMGB-1 and tenascin-C intra-articularly to mice provokes pathological inflammation *in vivo,* (Pullerits et al., 2003; Gondokaryono et al., 2007; Midwood et al., 2009). Moreover, targeted deletion of tenascin-C protects mice from experimental disease; synovial inflammation is induced but is transient and little tissue destruction occurs in contrast to wild type mice (Midwood et al., 2009) suggesting that tenascin-C plays a crucial role in disease chronicity.

Targeting DAMP Activation of

understanding of both.

show no significant association with RA.

Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis? 221

pathologically post translationally modified in this and other ways may reveal the antigens

 In summary therefore, the presence of DAMPs within the RA synovia or their elevated levels within the peripheral circulation of patients, implicates their involvement in disease pathology. This hypothesis is now underscored by evidence in animal models of RA that includes the effects on disease after targeted deletion of DAMPs, the induction of disease by administration of DAMPS as well as the manipulation of DAMP function / expression. However, whilst targeted deletion of a particular DAMP is possible in the mouse it is clearly not a viable therapeutic option in the clinic. A more achievable goal is to target the receptors or signalling pathways involved in DAMP activity, an approach that requires a detailed

**6. Distinct mechanisms of DAMP versus PAMP-mediated TLR activation** 

Whilst there is now clear evidence for a role for endosomal TLRs -3 and -8, their ligands are still to be defined; all DAMPS that have been implicated in RA to date mediate their effects via either TLR2 or 4 (Tables 2 and 4). However, despite considerable evidence implicating TLRs -2 and -4 in RA, there is also conflicting evidence. In particular, the inability of some function blocking antibodies to prevent spontaneous cytokine production in isolated RA synovial cells (Sacre et al., 2007), and the protective effect of TLR2 deletion in murine arthritis (Abdollahi-Roodsaz et al., 2008) might suggest that these TLRs are not important in RA. Moreover, SNPs of TLR2 and TLR4 or polymorphisms in human TLR4 that prevent LPS responsiveness do not correlate with RA disease susceptibility. For example, the Asp299Gly mutation in TLR4 (Kilding et al., 2003; Radstake et al., 2004) or Arg677Trp and Arg753Gln polymorphisms in TLR2 (Sanchez et al., 2004) that prevent PAMP induced activation of cells

However, the apparent discordance with SNP data and some antibody studies may be accounted for by the idea that the mechanism of TLR activation by DAMPs is unlikely to be the same as that used by the pathogenic activation of TLRs. LPS-relevant SNPs and antibodies that prevent LPS-TLR4 association may therefore not be applicable to TLR4:DAMP association. Indeed, studies with HMGB1, HSPs and tenascin-C, all DAMPS that have been implicated in RA, have revealed differences in the gene expression profiles and cytokines produced by these DAMPs when compared to LPS. This disparity was despite the uniform use of the TLR4 receptor. Thus, whilst HMGB1 and LPS induce many of the same genes in neutrophils from septic patients, there are also distinct differences, in particular in the expression of IL-8 (Silva et al., 2007). HSP60 is able to induce IFN alpha production in peritoneal macrophages and bone marrow derived DCs where LPS cannot (Osterloh et al., 2007), and tenascin-C exhibits a different profile of cytokine induction in RA synovial fibroblasts to LPS, being unable to induce IL-8 in these cells (Midwood et al., 2009). The induction of different gene patterns implies that the DAMPS are using TLR4 in a different way from the PAMPs. This is perhaps not surprising when we consider that TLR4 recognises a wide variety of ligands, ranging from HSPs to lipids to breakdown products of the extracellular matrix (Piccinini et al., 2010) and it is unlikely that the TLR4 molecule would be able to recognise such a diverse repertoire of molecules in the same way. This is borne out by findings from crystallography studies which have revealed three basic mechanisms for TLR:PAMP association. Thus, the crystal structure of the extracellular domain of TLR3 complexed with ds RNA reveals that this molecule interacts directly with

that drive autoimmunity; thereby shedding light on RA disease pathogenesis.


Table 4. Evidence supporting the role of specific DAMPs in driving inflammation in RA
