Damage-associated molecular patterns (DAMPs) are chemically heterogeneous endogenous host molecules rapidly released from damaged or dying cells that incite a sterile inflammatory response mediated via pattern recognition receptors (PRRs). The sources of DAMPs are dead or dying cells or the extracellular matrix and can signal through the PRRs, the Toll-like receptors or cytosolic Nod-like receptors, culminating in nuclear factor κB (NF-κB) activation and pro-inflammatory cytokine secretion. Together, these molecules are involved in sterile inflammation and many are associated with rheumatic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythromatosus, psoriatic arthritis and systemic sclerosis. These diseases are associated with inflammation and many danger signals are found in sites of sterile inflammation and mediate inflammation. The present review examines the role of DAMPs in rheumatic conditions and suggests avenues for their therapeutic modulation.

INTRODUCTION

Polly Matzinger first proposed the ‘danger theory’ that is that the immune system is concerned with ‘danger’ rather than ‘self’ [1]. This ‘danger theory’ explains why we have transplant rejection based on activation of the immune system, immune activation against tumours, tissue damage and autoimmune disease, all of which do not involve microbial pathogens. It is proposed that otherwise internal innocuous signals are released from cells upon damage, ‘stress’ or trauma and that these damage-associated molecules evoke an immune response. The self/non-self model explains the inflammation associated with foreign organisms but it fails to explain the inflammation that is associated with tissue damage and autoimmunity. The ‘danger model’ as proposed by Matzinger could unify danger and tissue damage with autoimmunity. Inflammation following tissue damage or stress is a co-ordinated response orchestrated by an engagement of a variety of receptors and molecules leading to enhanced pro-inflammatory cytokines and direct adaptive immunity.

Toll-like receptors (TLRs) are a germline-encoded group of pattern recognition receptors (PRRs) that share high homology with the Toll gene in fruit flies and are found in plants and vertebrates [2]. TLRs are a critical component of the innate immune response system and have evolved to protect us from pathogenic microbes through recognition of conserved microbial and viral patterns [3]. Although PRRs sense microbial patterns to elicit an immune response facilitated via multiple adaptor proteins, they also recognize ‘endogenous’ signals to elicit such an immune response [2]. Although pivotal as the first line of defence against invading micro-organisms, TLRs also are associated with a variety of rheumatic autoimmune conditions including rheumatoid arthritis (RA), systemic lupus erythromatous (SLE), psoriatic arthritis and systemic sclerosis (SSc). Nod-like receptors (NLRs) are intracellular molecular sensors for pathogens and damage-associated molecular patterns (DAMPs), and activation of these receptors are also associated with rheumatic diseases [2]. Identification of danger signals has increased in recent years and include a broad base of molecules that seem not to share any common features and are widely implicated in the initiation and propagation of many diseases. It is likely that the list of DAMPs grows larger in the future emphasizing the importance of danger in immune homoeostasis. The present review examines the roles of DAMPs in autoimmune rheumatic diseases and suggests ways in which therapeutic blockade may be employed.

DAMPs AND SENSING DANGER

DAMPs/alarmins are the endogenous molecules released from necrotic/stressed cells to elicit an immune response via distinct PRRs that include TLRs and cytosolic NLRs that culminates in nuclear factor κB (NF-κB) activation and pro-inflammatory cytokine and chemokine expression and are therefore important in the initiation and propagation of inflammation. Inflammatory responses seen after DAMP release following trauma closely resemble the response to microbial pathogens as they share the same receptors. There are ten human TLRs and they are highly evolutionarily conserved. As our knowledge of PRRs has increased so too has the number of DAMPs and a paradigm shift in conventional thinking has occurred. Cell death and damage are not a ‘normal event’ and are hazardous to the organism and we have therefore evolved mechanisms to alert the host to this and limit the damage via an inflammatory response and maturation of dendritic cells to orchestrate the immune response. These DAMPs include intracellular transcription factors such as high-mobility group box protein-1 (HMGB-1), calcium regulators S100s, extracellular heat-shock proteins (HSPs) or extracellular matrix (ECM) proteins such as hyaluronic acid and metabolic waste products like uric acid. Despite being chemically heterogenous, DAMPs share a common feature: they are intracellular and often ‘hidden’ from the immune system upon cell damage they are released for the immune system to ‘recognize’ and respond and they normally serve a housekeeper function within the cell. These potent mediators of inflammation released from dying or stressed cells may represent a therapeutic target in rheumatic diseases where chronic inflammation is present and provide a link between ‘damage’ and inflammation. Table 1 lists DAMPs associated with rheumatic diseases. These autoimmune diseases are associated with a loss of tolerance and an antigenic response to autoantigens with autoantibodies against self antigens. Given that many of these DAMPs are extracellular, they may be readily measured in serum and these could be used for biomarker studies in diseases in which they play a key role.

Table 1
DAMPs associated with rheumatic diseases
DAMPPutative receptor
HMGB-1 TLR2/4 RAGE 
Nucleic acid TLR3/8 and 9 
S100A8 RAGE/TLR4 
S100A9 RAGE/TLR4 
S100A12 RAGE 
Uric acid/MSU TLR2/4 NALP3 
Hyaluaronic acid TLR2/4 
Tenascin-C TLR4 
Fibrinogen TLR4 
HSP60 TLR4 
HSP70 TLR4 
SAA TLR2 
DAMPPutative receptor
HMGB-1 TLR2/4 RAGE 
Nucleic acid TLR3/8 and 9 
S100A8 RAGE/TLR4 
S100A9 RAGE/TLR4 
S100A12 RAGE 
Uric acid/MSU TLR2/4 NALP3 
Hyaluaronic acid TLR2/4 
Tenascin-C TLR4 
Fibrinogen TLR4 
HSP60 TLR4 
HSP70 TLR4 
SAA TLR2 

HMGB-1

HMGB-1 is the prototypical DAMP. It is a highly conserved non-histone protein that is mainly located in the nucleus where it functions as a DNA-binding protein, stabilizes nucleosomes and facilitates gene transcription. HMGB-1 can be released passively by necrotic cells after tissue damage to elicit signalling [4]; however, it can also be actively secreted from the cell even though it lacks a leader sequence, through an unconventional mechanism, resulting in NF-κB activation [5]. This active secretion is facilitated by hyperacetylation of the molecule that allows this to be released into the extracellular environment [6]. The redox status of HMGB-1 is also critical in its biological activity, through different redox forms of the cysteine residues within the protein. HMGB-1 has been found to activate three different receptors TLR2 and TLR4 and receptor for advanced glycation end products (RAGE). RAGE is a member of the immunoglobulin superfamily that binds structurally diverse ligands. Soluble RAGE is a negative regulator of signalling. The fact that it binds and signals through TLRs suggests that this may need to be associated with other molecules for optimal signalling, such as pathogen-derived molecules. HMGB-1 serum levels in RA patients correlate with disease activity. TLR3 and TLR4 are overexpressed in RA synovial tissue as compared with osteoarthritis tissue [7]. HMGB-1 is found elevated in RA synovial tissue and the pro-inflammatory cytokine tumour necrosis factor (TNF)-α was found to translocate HMGB-1 from the nucleus [8]; furthermore, HMGB-1 directly induces arthritis in naïve mice but this is independent of TNF-α [9], suggesting it is interleukin (IL)-1 mediated. HMBG-1 has also been shown to promote angiogenesis, which facilitates the ingress of lymphocytes into the joint. Importantly targeting HMGB-1 via a neutralizing antibody in the collagen-induced arthritis model of RA significantly attenuated disease [10] and anti-HMGB-1 treatment ameliorates barrier dysfunction and improves survival after haemorrhagic shock [11]. Interestingly, research has demonstrated that hypoxia couples extracellular release of HMGB-1 which was much more prominent than the release simply by cell necrosis [12]. It is well established that the RA joint microenvironment is profoundly hypoxic and the mechanism through which hypoxia regulates HMGB-1 release may include reactive oxygen species (ROS) as certain cysteine residues regulate the physiological activity of HMGB-1 [13]. It is well established in RA that there is an increase in synovial macrophages in the synovial tissue expressing TLR2 and TLR4 [14] and activation of TLRs can itself activate hypoxia-regulated genes. Interestingly TLR4 knockout mice are protected from spontaneous RA in the IL-1Ra mouse model possibly mediated via a reduction in Th17 cell frequency [15]. Synergy may also occur in this system between DAMPs and lipopolysaccharide (LPS) from microbial origin [16].

In SLE, HMGB-1–nucleosome complexes have been found in the serum and that these complexes activated resident dendritic cells and anti-double-stranded DNA antibodies. This is important as anti-DNA antibodies are a hallmark of SLE and there is impaired apoptotic clearance of cells in this condition [17]. Impaired apoptotic clearance would lead to elevated levels of extracellular DNA, normally ‘hidden’ from the immune system, with a resultant activation of TLRs and dendritic cell maturation. Furthermore, DNA–chromatin complexes in SLE were found to contain HMGB-1 that was necessary to activate dendritic cells and B-cells via TLR9-dependent signalling contributing to disease pathogenesis and an type I interferon signature [18]. SLE is associated with glomerulonephritis, arthritis and vasculitis and high levels of interferon. It may be that, alongside its pathogenic role in SLE, HMGB-1 may be a useful biomarker if it was found to be able to predict disease activity, for instance.

SSc is an idiopathic autoimmune disease characterized by specific autoantibodies, vascular abnormalities, inflammation and fibrosis. The fibrosis is associated with an immune infiltrate (Figure 1). These autoantibodies are against DNA/RNA components and are diagnostic. The disease has a high mortality rate due to interstitial lung disease as a consequence of the disease and there is currently no effective treatment. We have previously demonstrated that TLR8-mediated RNA containing immune complexes stimulate CD14+ monocytes to secrete tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in a MyD88-dependent fashion [19]. Serum of SSc patients that induces TIMP-1 via intracellular TLR8-mediated pathways was abrogated via enzyme-mediated cleavage of RNA and also was reduced in monocytes in which IL-1-receptor-associated kinase 4 (IRAK4) was deleted [19]. Thus serum contains an RNA/DNA containing autoantibody that mediates TIMP-1 release. It is suggested that damaged vascular cells occur early in SSc development and the release of DNA/RNA from these cells may initiate an autoimmune response via TLR stimulation. TIMP-1 is part of the family of proteins that inhibit multiple matrix metalloproteinases (MMPs) in a one to one fashion and thus the balance of MMPs to TIMPs regulates the deposition of ECM. A balance favouring TIMPs increases the expression of ECM promoting fibrosis. Interestingly serum and skin levels of HMGB-1 were found to be elevated in (SSc) and correlated positively with the Rodnan skin score [20]. HMGB-1 has itself been found to directly up-regulate α-smooth muscle actin [20], a marker of myofibroblasts, the stromal cell responsible for excessive collagen production in fibrotic diseases.

CD3 T-cells in SSc skin biopsy

Figure 1
CD3 T-cells in SSc skin biopsy

CD3+ T-cell in a skin biopsy from a diffuse SSc patient stained with antibodies to CD3 and developed using DAB.

Figure 1
CD3 T-cells in SSc skin biopsy

CD3+ T-cell in a skin biopsy from a diffuse SSc patient stained with antibodies to CD3 and developed using DAB.

S100s

The S100 proteins are calcium-binding proteins expressed in cells of myeloid origin with two calcium-binding EF-hand motifs. They have a role in cell migration and cytoskeletal regulation and are low-molecular-mass proteins. The receptor first identified for mediating the effects of S100 DAMPs was RAGE [21] and blockade of RAGE reduced inflammation in a variety of disease models; however, TLR4 is also a receptor for S100 proteins. RAGE is the receptor of protein and lipids that have become glycated or oxidized. S100A8 (MRP8), S100A9 (MRP9) and S100A12 are found in abundance at sites of inflammation. S100A8 and S100A9 can form heterodimers. Both S100A8 and S100A9 have been demonstrated in synovial membranes from RA patients [22] and serum levels have been suggested to correlate with disease activity and appears superior to C-reactive protein (CRP) as a biomarker [23]. S100A12 has been demonstrated in the synovial and serum of RA and psoriatic arthritis patients [24]. Interestingly the soluble receptor RAGE is decreased in RA patients suggesting a propensity toward inflammation [25] as soluble RAGE acts as a decoy receptor. In an animal model of human RA S100A8 mediated the breakdown of the chondrocyte matrix via activation and up-regulation of the matrix-degrading enzymes MMPs. S1008 levels are increased in SLE and stimulation of CD8+ T-cells with S100 resulted in IL-17 secretion in lupus patients suggesting they play a pathogenic role through IL-17 promotion, which is an important inflammatory cytokine [26]. In SSc, S100A4 has recently been shown to amplify transforming growth factor (TGF)-β-induced activation of dermal fibroblasts [27]. This is interesting as it appears to synergize with TGF-β in up-regulating collagen expression. Furthermore, genetic deletion of S100A4 in the TSk-1 mouse, a mouse model of fibrosis, ameliorated skin fibrosis, indicting the crucial role of S100A4 in the fibrosis at least in this model [27]. It appears that S100 DAMPs play different pathogenic roles in different rheumatic diseases as, in RA, the elevated levels of S100A8 and S100A9 [22] appear to facilitate the breakdown of ECM mediated via MMPs [25]; in contrast, in SSc, it has been demonstrated that ECM deposition is increased and there is an apparent synergy with TGF-β [27]. The differential effects on different fibroblasts from different anatomical locations maybe due to epigenetic imprinting in the cells as epigenetics is modified by the environment.

Uric acid

Uric acid is a waste product of purine catabolism derived from DNA and RNA via the enzyme xanthine oxidase. Uric acid in the form of monosodium urate (MSU) crystals precipitate incites an inflammatory response that is clinically gout often manifest in the big toe. We can convert uric acid into allantoin. The first paper to describe uric acid as a danger signal was published in 2003 [28] and this showed clearly that dead or dying cells release uric acid into the microenvironment and this induces activation of dendritic cells; the sentinels of the immune system [28]. Furthermore, uric acid in vivo was found to promote inflammation from necrotic cells in sterile inflammation underscoring its role as a DAMP. MSU crystals were shown to induce inflammation and require TLR2 and TLR4 for this response in vivo and MSU crystals injected into wild-type mice induced a rapid inflammatory cell influx that was attenuated in TLR2- and TLR4-deleted mice [29]. Although it could be suggested that MSU crystals themselves simply damage the cell with resulting release of DAMPs; these are the ‘real’ danger signals.

NLRs are intracellular sensors of endogenous DAMPs and pathogen-associated molecular patterns (PAMPs) that gain access intracellularly. Activation of NLRs are associated with RA in animal models [30]. NLRs are cytosolic sensors that comprise many proteins that acts as scaffolds to form a large multimeric structure with the adaptor protein apoptosis associated speck-like protein containing a caspase recruitment domain (ASC), termed the ‘inflammasome’, and this leads to activation of the enzyme caspase-1 and secretion of IL-1β and IL-18 from the cell [31]. To date 22 human NLRs have been described [31]. They contain a C-terminal region with leucine-rich repeats and a nucleotide-binding domain. MSU crystals have been demonstrated to activate NACHT, LRR and PYD domain-containing protein 3 (NALP3)/cryopyrin resulting in both IL-1β and IL-18 secretion [32]. Mice deficient in specific components of the inflammasome are protected from MSU-induced inflammation [32]. Exactly how MSU activates the inflammasome is not entirely clear but it is suggested that this is due to destabilization of the phagolysosome [33] and redox status may also play a role in this ‘destabilization’. This may be an acute mechanism to phagolysosomal expulsion; however, further research is required to decipher the precise mechanism. Furthermore, single nucleotide polymorphisms (SNPs) in components of the inflammasome may influence the risk of gout.

ECM

Tissue injury and remodelling occur in multiple settings. Tissue remodelling occurs through the action of proteolytic enzymes that cleave ECM molecules, in the case of tissue damage certain molecules such as hyaluaron are cleaved and others such as tenascin-C are up-regulated to provoke an immune response leading to tissue regeneration and restore tissue homoeostasis.

Hyaluaron is a non-sulfated glycosaminoglycan that is a critical component of the ECM. Hyaluaron can exist in two molecular masses. High-molecular-mass polymer of hyaluaron exist (>1000 kDa) during normal tissue homoeostasis, however, when cleaved to a lower-molecular mass hyaluaron a different response occurs. Low-molecular-mass hyaluaron has been shown to elicit an inflammatory response mediated via TLR4 [34]. Interestingly, lung-specific overexpression of high-molecular-mass hyaluron was protective against lung injury [34]. The mechanism is not entirely clear but appears to involve NF-κB activation [33]. Hyaluaron fragments has also been shown to induce inflammatory cytokine expression in mouse macrophages [35] and that this is mediated via NF-κB activation. Thus, the low-molecular-mass form of hyaluaron is pro-inflammatory, whereas the high-molecular-mass form is protective. Elevated levels of hyaluaron has been demonstrated in animal models of RA and correlated with disease severity [36] and low-molecular-mass hyaluaron was found to alter chondrocyte function and is facilitated through TLR2 engagement and the downstream adaptor protein MyD88 [37]. Interestingly in an animal model of RA intra-articular injection of high-molecular-mass hyaluronic acid significantly reduced the inflammation and structural cartilage damage. Thus, high-molecular-mass hyaluaron is chondroprotective. Blockade of the enzymes that degrade high-molecular-mass hyaluaron to its smaller fragments reduced pro-inflammatory cytokine secretion in RA synovial fibroblasts, suggesting that this could be an amenable therapeutic target [38]. Indeed low-molecular-mass hyaluaron has been found to promote the activation of the inflammasome leading to increased IL-1 and is NLRP3-dependent [39]. In RA, it is conceivable of a vicious cycle of inflammation and damage leading to more inflammation and unremitting damage perpetuating as the ECM is cleaved and cellular debris is released.

Tenascin-C

Tenascin-C is a glycoprotein that has a restricted expression postnatally. It is an essential part of the ECM, its expression is restricted to sites of damage and has been shown to ligate TLR4 and is necessary for inflammation in a mouse model of inflammatory arthritis [40]. Furthermore, synovial fibroblasts from RA patients stimulated with tenascin-C induces pro-inflammatory cytokine expression [40]. Tenascin-C is also found in higher levels in the synovia of RA patients which is both inflamed and tissue damage is occurring. Tenascin-C also correlates with disease markers in RA and is expressed by myeloid cells and is transcriptionally regulated upon stimulation with pro-inflammatory stimuli such as LPS [41]. It has been shown in an animal model of RA that the ECM molecule tenascin-C mediates its effects through the binding of the integrin α9 and this leads (possibly with other DAMPs) to pro-inflammatory cytokine secretion, which leads to polarization of naïve T-cells to Th17 cells that migrate to the joint leading to joint destruction via IL-17. The primary cytokine responsible for this was IL-6 and inhibition of integrin α9 by antibody blockade reduced the secretion of IL-6 (but not TGF-β levels) and also destructive Th17 cells [42]. It has recently been demonstrated that tenascin-C is required for a robust pro-inflammatory response in a model of sepsis by LPS; in other words, tenascin-C is a target gene of LPS and regulates levels of the classic pro-inflammatory cytokine TNF-α post-transcriptionally by modulating miR155 levels [43]. Tenascin-C's role in other rheumatic conditions is as yet unexplored.

Fibrinogen

Fibrinogen has also been demonstrated to be a DAMP and activate macrophage chemokine secretion via TLR4 [44]. Interestingly, citrullinated fibrinogen was found to induce pro-inflammatory cytokine production in macrophages and was mediated through TLR4 and the downstream adaptor MyD88 [45]. This is of major interest as many RA patients have autoantibodies to citrullinated proteins; citrullination is the post-translational modification of arginine to citrulline mediated via the enzyme peptidyl arginine deiminase (PAD), although these antibodies may target many citrullinated proteins, many anti-citrullinated peptide antibody (ACPA)-positive patients have circulating autoimmune complexes containing citrullinated fibrinogen [46]. Thus it could be that only citrullinated fibrinogen mediates an inflammatory response not ‘native’ fibrinogen, due to the modification that elicits both antibodies to the protein but also T-cells as a recent study has described a citrullinated-fibrinogen-specific T-cell line that results in pro-inflammatory cytokine secretion in response to citrullinated fibrinogen antigen and amplifies arthritis in the collagen-induced arthritis model of RA [47]. Furthermore, PAD2-mediated citrullinated fibrinogen induced high pro-inflammatory mediators in RA synovial fibroblasts mediated via TLR4 ligation [48].

HSPs

HSPs are a group of evolutionarily conserved proteins that are molecular chaperones functioning to help fold and transport a variety of proteins and are highly induced by a variety of stressors including heat, hypoxia and acidity. HSPs gain their name from the fact they are highly up-regulated at high temperatures, they share high sequence conservation between mammalian and microbial species and are dominant immunogens. The first HSP to be described as a DAMP was HSP70, and extracellular HSP70 induced a pro-inflammatory response in monocytes via CD14 and downstream NF-κB activation [49]. Although released HSP lack any normal protein leader sequence and hence lack the ability to be secreted in the canonical way, therefore release of HSPs occurs passively through cell necrosis but also through exosomes. In RA synovial tissue and isolated cells elevated levels of HSP70 and its transcriptional activator heat-stock factor (HSF)-1 have been described as well as other HSPs. However, in RA it appears that HSPs may play a protective role as immunization with a conserved residue of mycobacterium Hsp70 prevented adjuvant-induced arthritis in rats [50] and this was due to T-cells that recognized a conserved residue within the HSP. Wieten et al. [51] demonstrated that the protection conferred by immunization with the Hsp70 from the mycobacterium in mice with proteoglycan-induced arthritis, a progressive T-cell-dependent arthritis in mice that are transgenic for aggrecan, was due to induction of IL-10 T-cells secreting the anti-inflammatory cytokine. Thus, the HSPs appear to be immunoregulatory in nature in RA pointing towards a ‘DAMPening’ effect. Thus, immunization with Hsp70 or peptide could reduce RA inflammation via promotion of regulatory T-cells. In a mouse model of fibrosis, mimicking SSc, it was shown that genetic overexpression of Hsp70 ameliorated the fibrosis and inflammation, as did oral administration of geranylgeranylacetone, an HSP-inducing drug, indicating that Hsp70 is immunoregulatory. Although HSPs have been reported to be pro-inflammatory or in the case of RA anti-inflammatory, does not take away from the notion that they are DAMPs, regardless of the end outcome.

Serum amyloid A

Serum amyloid A (SAA) is an acute-phase response protein that is rapidly induced upon infection from the liver by hepatocytes and can be elevated by many orders of magnitude. SAA's role, though, has remained unclear. However, SAA has been proposed to be a danger signal based on the fact that it binds TLR2 and induces inflammatory signals [52]. We have also recently demonstrated that SAA binds TLR2, but not TLR4, in primary dermal fibroblasts and that this culminates in pro-inflammatory mediators being released in an NF-κB-dependent manner [53]. We clearly showed that this was reduced by the addition of a TLR2-neutralizing antibody but not TLR4, thereby showing this is TLR2-mediated. Importantly dermal fibroblasts from SSc patients had higher basal levels of TLR2 as compared with healthy control dermal fibroblasts and were more responsive to SAA (Figure 2) [53]. This is all suggestive of SAA mediating a pathogenic role in SSc via TLR2-mediated induction of cytokines. Figure 3 illustrates the possible role of SAA in SSc via TLR2 activation. Interestingly there is a polymorphism in the TLR2 gene associated with a specific subtype of SSc [54]. In RA also, SAA is elevated and has been shown to induce proteolytic enzymes in RA synovial fibroblasts that mediate breakdown of the joint and enhanced cell migrational effects facilitated through surface integrins [55]. Furthermore, SAA has been demonstrated to induce leucocyte recruitment and stimulate angiogenesis [56]: both key events in RA development. It would be interesting to see if SAA levels can predict treatment response in RA.

Expression of TLR2 on SSc dermal fibroblasts

Figure 2
Expression of TLR2 on SSc dermal fibroblasts

Histogram demonstrates higher basal levels of TLR2 from SSc dermal fibroblast compared with control dermal fibroblast cultures or antibody alone. This Figure was reproduced with permission from O’Reilly, S., Cant, R., Ciechomska, M., Finnegan, J., Oakley, F., Hambleton, S. and, van Laar, J. M. (2014) Serum amyloid A (SAA) induces IL-6 in dermal fibroblasts via TLR2, IRAK4 and NF-κB, Immunology, 143, pp. 331–340. © 2014 John Wiley & Sons Ltd.

Figure 2
Expression of TLR2 on SSc dermal fibroblasts

Histogram demonstrates higher basal levels of TLR2 from SSc dermal fibroblast compared with control dermal fibroblast cultures or antibody alone. This Figure was reproduced with permission from O’Reilly, S., Cant, R., Ciechomska, M., Finnegan, J., Oakley, F., Hambleton, S. and, van Laar, J. M. (2014) Serum amyloid A (SAA) induces IL-6 in dermal fibroblasts via TLR2, IRAK4 and NF-κB, Immunology, 143, pp. 331–340. © 2014 John Wiley & Sons Ltd.

SAA mediates fibrosis in SSc via TLR2 ligation

Figure 3
SAA mediates fibrosis in SSc via TLR2 ligation

Endothelial cell (EC) damage through hypoxia, ROS, virus or necrosis leads to the release into the extracellular environment of SAA, this binds TLR2 on dermal fibroblasts, thus leading to activation of NF-κB after dissociation of inhibitor of NF-κB (IκB), NF-κB translocates to the nucleus to activate the transcription of IL-6 and TIMP-1. Both of these molecules are profibrotic mediating collagen deposition and thus ECM accumulation. Inhibition of TLR2 with OPN-301 blocks this effect [53].

Figure 3
SAA mediates fibrosis in SSc via TLR2 ligation

Endothelial cell (EC) damage through hypoxia, ROS, virus or necrosis leads to the release into the extracellular environment of SAA, this binds TLR2 on dermal fibroblasts, thus leading to activation of NF-κB after dissociation of inhibitor of NF-κB (IκB), NF-κB translocates to the nucleus to activate the transcription of IL-6 and TIMP-1. Both of these molecules are profibrotic mediating collagen deposition and thus ECM accumulation. Inhibition of TLR2 with OPN-301 blocks this effect [53].

THERAPEUTIC TARGETING

The evidence for the role of DAMPs in rheumatic disease is now strong and as our knowledge of DAMPs and their signalling mechanisms has expanded so too has the possibility of selective blockade of such compounds in rheumatic diseases. This can occur on multiple levels and include blockade of the ligands (DAMPs), their putative receptors or several components of the downstream signalling cascade. Although in RA the use of anti-TNF-α therapeutic antibodies have been hugely effective and have heralded a revolution in rheumatology there are a significant proportion of patients in whom these biologics are ineffective and few achieve sustained remission. Furthermore, biologics can have unwanted side effects, including risk of infections and hypersensitivity. Thus targeting DAMPs may be more beneficial in these patients.

Targeting the DAMP itself may be one therapeutic option. In the case of HMGB-1 in rheumatic diseases the use of anti-HMGB-1 antibodies has shown striking benefit in animal models of RA, with a reduction in damage and MMP expression, and demonstrates the clinical utility of anti-HMBG-1 therapy. However, animal models of RA do not reflect human disease completely and the ultimate test is in a clinical trial.

Also small molecules that cause HMGB-1 nuclear sequestration may also yield beneficial results, as HMGB-1 has to shuttle from the nucleus to the cytoplasm to direct it for release from the cell [5]. Oxaliplatin, a platinum-based antineoplastic drug, was shown to trap HMGB-1 in the nucleus, therefore attenuating its pro-fibrotic effects and release from macrophages and reduced the intensity and severity of arthritis in the collagen-induced arthritis model. A multimodal RAGE-specific blocker has been developed, termed FPS-ZM1, for use in amyloid-β-mediated brain pathology in an animal model of Alzheimer's disease. This may be a RAGE blocker that restricts binding of other non-amyloid RAGE ligands. IMO-3100, manufactured by Idera, is a TLR7/TLR9 antagonist that has shown therapeutic benefit in plaque psoriasis after 4 weeks of treatment in a Phase II clinical trial. This drug has shown benefit here and further trials will examine its role in larger cohorts. This may also be useful in SLE where self-RNA is prominent. Dynavax also have a TLR7/TLR9 inhibitor in development for SLE. In general endosomal TLR antagonists are less advanced (Table 2). Opsona therapeutics (Dublin) have a therapeutic monoclonal antibody targeting TLR2 and are pursuing a clinical trial in delayed graft function for renal transplant recipients. Targeting TLR2 in RA and SSc could be possible as we demonstrated in SSc that anti-TLR2 antibody treatment in dermal fibroblasts reduced SAA-mediated NF-κB activation and pro-fibrotic molecule secretion [53]. In SSc, there is currently no treatment that modifies the disease and is therefore a huge unmet clinical need.

Table 2
Endosomal TLR antagonists under development
CompoundManufacturerTargetIndication
IMO-3100 Idera TLR7/TLR9 Plaque psoriasis (Phase II) 
DV056 Dynavax Technologies TLR7/TLR9 SLE 
CompoundManufacturerTargetIndication
IMO-3100 Idera TLR7/TLR9 Plaque psoriasis (Phase II) 
DV056 Dynavax Technologies TLR7/TLR9 SLE 

The TLR adaptor proteins MyD88 and Mal have been inhibited in RA synovial explants in culture conditions and this leads to reductions in pro-inflammatory mediators but also MMPs, the destructive enzymes in RA [57]. Targeting these may be much more specific than targeting the upstream receptor as microbial products share the TLRs and thus may increase the risk of infection. Lipoplexes are lipid-based molecules that can be used to deliver siRNA and could be used to target adapter molecules.

An interesting study demonstrated that mianserin was an inhibitor of endosomal TLRs and reduced the endosomal TLR-mediated IL-6 and TNF-α from RA patient synovial explants [58]. Mianserin is a serotonin antagonist used for depression. It is of major interest that two licenced selective serotonin inhibitors fluoxetine and citalopram ameliorated the severity of disease in the collagen-induced arthritis model and that these antidepressants also reduced IL-6 and TNF-α levels in RA synovial explants. Their mechanism of action was via inhibition of TLR signalling [59]. Enzyme blockade so that hyaluron is not cleaved to its inflammatory mediator low-molecular-mass hyaluron is also a therapeutic option [38]. S100A9 has been suggested to be a target of laquinimod, a drug under evaluation in multiple sclerosis (MS).

Thus therapeutic modulation of TLR signalling through the use of serotonin antagonists may be one therapeutic in DAMP-mediated RA.

The adjuvant potential of all DAMPs has a broader significance in general and may be harnessed in development of new vaccines against microbial pathogens due to their rather potent adjuvant effects.

DAMPs AS BIOMARKERS

Clinically useful biomarkers can be diagnostic, prognostic or predict response to treatment and many studies have sought to identify them in various rheumatic diseases. Given the fact that autoantibodies, such as ACPA, can be present years before onset of disease symptoms in RA and systemic autoimmunity precedes synovitis this suggest other factors may be driving the pathogenesis [60]. DAMPs can be readily measured in serum with conventional methods and this could be ‘scaled up’ for larger laboratories. HMGB-1 has been shown to be an important DAMP in RA and SLE and has been shown to be associated with disease activity [12]. This indicates that HMGB-1 could be a biomarker that may be prognostic and predict response to treatment; however, this needs further clarification. S100 proteins also appear to be specific biomarkers in RA and psoriatic arthritis [23]. Early treatment in RA is critical in halting disease progression and hence identifying a specific biomarker before synovitis would be of great importance. In diseases such as SSc, a robust biomarker would be of importance especially where it could diagnose clinically silent pulmonary hypertension enabling early intervention. Although many biomarkers have been suggested in autoimmune rheumatic diseases very few are used routinely and few have been extensively validated. Further validation of DAMPs as biomarkers in rheumatic diseases is necessary before these can be used to aid clinical management.

CONCLUSIONS

DAMPs are associated with danger and facilitate their effects through PRRs such as the TLRs and NLRs [2]. They are found in a variety of autoimmune rheumatic conditions including RA, SLE, gout and SSc. These are underscored by excessive inflammation with the cost of which is further cell damage and thus perpetuation of the cycle. DAMPs function through a myriad of receptors in a variety of cells and targeting the DAMP itself or downstream signalling may be a possibility to modulate the disease. A growing appreciation of the role of DAMPs in rheumatic diseases will aid the development of compounds that block these molecules.

In SSc, it has been demonstrated that SAA is mediating pro-inflammatory mediators via TLR2, thus targeting TLR2 may be a possibility. OPN-305 is a monoclonal antibody produced by Opsona Therapeutics targeting TLR2 specifically and has recently entered a clinical trial for delayed graft function in renal transplants. Advances in technologies are enhancing our understanding of DAMPs and their association with autoimmune rheumatic diseases. However, little is known of the functional interaction of DAMPs and cross-talk between their signalling pathways or if there is a hierarchy among DAMPs. An understanding of the local microenvironment that governs the release of the DAMPs or regulates their intracellular sequestration will help us design more targeted drugs to these molecules. For example, the redox status of HMGB-1 clearly regulates its bioactivity, with HMGB-1 primarily reduced within the cell, and thus factors that can alter the redox status of HMGB-1 may be useful.

Abbreviations

     
  • ACPA

    anti-citrullinated peptide antibody

  •  
  • CRP

    C-reactive protein

  •  
  • DAMP

    damage-associated molecular pattern

  •  
  • ECM

    extracellular matrix

  •  
  • HMGB-1

    high-mobility group box protein-1

  •  
  • HSP

    heat-shock protein

  •  
  • IL

    interleukin

  •  
  • LPS

    lipopolysaccharide

  •  
  • MSU

    monosodium urate

  •  
  • MMP

    matrix metalloproteinase

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NLR

    Nod-like receptor

  •  
  • PAD

    peptidyl arginine deiminase

  •  
  • PRR

    pattern recognition receptor

  •  
  • RA

    rheumatoid arthritis

  •  
  • RAGE

    receptor for advanced glycation end products

  •  
  • ROS

    reactive oxygen species

  •  
  • SAA

    serum amyloid A

  •  
  • SLE

    systemic lupus erythromatous

  •  
  • SSc

    systemic sclerosis

  •  
  • TGF

    transforming growth factor

  •  
  • TIMP

    tissue inhibitor of matrix metalloprotease

  •  
  • TLR

    Toll-like receptor

  •  
  • TNF

    tumour necrosis factor

References

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