**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 show no significant association with RA.

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

Targeting DAMP Activation of

**7. Conclusion** 

yet to be tested in RA.

during inflammation.

chronicity rather than its consequences.

Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis? 223

Examples of other molecules able to modify TLR signalling pathways, and consequently TLR-induced cytokine production are already established in the literature, and in particular a number of molecules that contain ITAM motifs have been shown to modulate TLR signalling pathways (Ivashkiv, 2008). Some such as TREM1 appear to cooperate with TLR molecules, amplifying the production of pro-inflammatory cytokines (Bleharski et al., 2003) whilst others such as the FcR and the CD300F molecule inhibit TLR signalling (Wang et al., 2010). Other cell surface molecules able to modulate TLR activity include the TAM (Tyro3, Axl, Mer) receptors and SIGIRR (Rothlin et al., 2007; Drexler et al., 2010). The mechanisms responsible for these activities are varied and include ITAM mediated changes in IL-10 production and the induction of inhibitory signalling molecules. Conversely, recent data has emerged detailing a requirement for TLR4 in CD16 signalling revealing that TLRs in their

In conclusion, it is clear that whilst the advent of specific biological therapies for the treatment of RA has significantly improved treatment of this disease in the last 10 years, there is still a significant un-met clinical need for therapies that target the cause of disease

 Increasing evidence from *in vitro* studies, murine models of disease, and human studies, suggests that the TLRs play a significant role in RA. In particular, TLRs 2 and 4 and the intracellular TLRs 3 and 8 are increasingly regarded as key to the pathogenesis of this disease. However, PAMPs derived from infectious agents are not found in RA joints and are unlikely to be the causative TLR ligands in RA. Rather, there is now increasing evidence that endogenously derived DAMPs, either in their native form or in a citrullinated state, are able to drive the chronicity of RA. DAMPs comprise an enormously diverse subset of molecules and targeting them as a form of RA therapy could be achieved in a number of different ways. Many of these approaches have already found some success in other fields, but have

For example, for DAMPs whose expression is specifically up-regulated during inflammation it may be possible to manipulate this induction of expression at the genetic level. This approach has been taken in a murine lung carcinoma model where knockdown of versican expression in Lewis lung carcinoma cell lines (LLC) ablated their tumorigenic capability, promoting mouse survival and reduced metastasis. Conversely, over expression of versican in LLC lines with

The use of micro RNAs (miRNAs) to manipulate gene expression is also attracting considerable attention. MicroRNAs are endogenous RNAs that post-transcriptionally modulate gene expression (reviewed in (Guo et al., 2010). Not surprisingly, regulation of gene expression by microRNAs has also been extended to the TLR signalling paradigm (reviewed in (O'Neill et al., 2011)) where they impose several levels of regulation on the TLR signalling axis. For example, miR-155, miR-21 and miR-147 regulate the expression of TLRs 2-4, downstream signalling mediators such as MyD88 and TRIF, as well as transcription factors NF-B and IRF3 (reviewed in (O'Neill et al., 2011)). Recent studies have reported that miR-155, miR-146a and miR-203 are upregulated in RA synovial fibroblasts, resulting in altered cytokine and MMP synthesis (Stanczyk et al., 2008; Li et al., 2010; Stanczyk et al., 2011). These insights may create a novel approach to limiting excessive TLR activation

low innate metastatic potential increased lung metastasis (Kim et al., 2009).

turn can modulate other signalling pathways (Rittirsch et al., 2009).

residues on the external surface of the TLR3 homodimer (Liu et al., 2008). More recent modelling of TLRs 7 and 9 also suggests direct ligand binding to the TLR molecule (Kubarenko et al., 2010). In contrast, TLR1:TLR2 hetero-dimerisation results in the formation of a hydrophobic pocket into which the lipopeptide PAM3Cys fits (Jin et al., 2007). Lastly, the structure of the TLR4:MD2:LPS complex reveals that LPS does not initially make direct contact with TLR4, but rather first binds to MD2, altering its conformation and allowing it to bind to and cause homodimerisation of TLR4 (Park et al., 2009). In this case, TLR4 residues important for LPS activity include those required for MD2 binding and those required for receptor homodimerisation. TLR4 responses to LPS also require the presence of CD14 which facilitates the transfer of LPS to MD2.

Our knowledge of the receptor complexes used by DAMPs is far from complete, but it is already clear that these molecules have a further level of complexity in their receptor organisation. Thus, of those that require TLR4, some, such as HSP70, biglycan and s100 also require both CD14 and MD2 for activity. Others, such as Gp96, HMGB1 and fibronectin EDA require only MD2, whilst another group that includes surfactant protein A and lactoferrin require only CD14 (Piccinini et al., 2010). The last, and probably the most diverse group of DAMPs use co-receptors or accessory molecules that are distinct from CD14 and MD2. Immune complexes of citrullinated fibrinogen for example have recently been shown to use a combination of Fcgamma receptors and TLR4 (Sokolove et al., 2011), and may also use CD11b/ CD18 (Barrera et al., 2011). A-SAA, which has been associated with RA and uses the TLR2 receptor has been shown to also use both CD36 and FPRL-1 as co-receptors, while low molecular weight hyaluronan forms complexes with TLR2 and CD44, and biglycan, which may use both TLR2 and TLR4, uses a variety of molecules including CD14, MD2, P2x4 and NLRP3 (Babelova et al., 2009). Other DAMPs such as tenascin-C do not use CD14 or MD2 (Midwood et al., 2009). Whether they bind directly to TLR4 or use an as yet undefined co-receptor molecule(s) is unclear at present.

Because of their alternative use of co-receptor molecules, DAMPs are therefore likely to use different residues on the TLR4 molecule than those used by LPS, so it may not be surprising that SNPs that affect LPS binding and antibodies designed to prevent LPS activation of TLR4 are inactive in RA where DAMP mediated activation of TLR4 may be critical to disease pathology. This hypothesis is confirmed by studies of the D299G and T399I mutations in TLR4. These have been shown to prevent LPS activation, but to enhance the ability of TLR4 to respond to fibrinogen. (Hodgkinson et al, 2008)

The signalling mechanisms used by DAMPs are also not well defined, and there is very little data available at present to suggest therapeutic targets for DAMPs. However, the use of TLR molecules suggests that many of the same pathways activated by PAMPs may be relevant. This has been confirmed by recent studies of oxidised LDL signalling (a TLR4 DAMP), which shows activation of many familiar pathways such as those involving IKK and the MAP kinases. Studies with tenascin-C also show that MyD88 signalling is important in response to this molecule (Midwood et al., 2009). Any differences between DAMP and LPS mediated signalling pathways are likely to come from the DAMP use of alternative co-receptors. Molecules such as CD36, CD44 and integrins already have defined signalling pathways. Whether DAMP signalling will prove to be simply a combination of TLR:MyD88 driven pathways and those emanating from any co-receptor molecules remains to be defined. It is more likely however, that the signal transduction mechanism of DAMPs will be a result of a combination of both pathways, where each is able to modulate / modify the other.

Examples of other molecules able to modify TLR signalling pathways, and consequently TLR-induced cytokine production are already established in the literature, and in particular a number of molecules that contain ITAM motifs have been shown to modulate TLR signalling pathways (Ivashkiv, 2008). Some such as TREM1 appear to cooperate with TLR molecules, amplifying the production of pro-inflammatory cytokines (Bleharski et al., 2003) whilst others such as the FcR and the CD300F molecule inhibit TLR signalling (Wang et al., 2010). Other cell surface molecules able to modulate TLR activity include the TAM (Tyro3, Axl, Mer) receptors and SIGIRR (Rothlin et al., 2007; Drexler et al., 2010). The mechanisms responsible for these activities are varied and include ITAM mediated changes in IL-10 production and the induction of inhibitory signalling molecules. Conversely, recent data has emerged detailing a requirement for TLR4 in CD16 signalling revealing that TLRs in their turn can modulate other signalling pathways (Rittirsch et al., 2009).
