Semin Immunopathol DOI 10.1007/s00281-015-0503-7

REVIEW

Role of innate immune system in systemic sclerosis Nicola Fullard 1 & Steven O’Reilly 1

Received: 6 April 2015 / Accepted: 16 June 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Recognition of microbial or viral compounds is crucial to elicit an immune response and pattern recognition receptors (PRRs) form the first line of defence. An important family of PRRs are the Toll-like receptors (TLRs) with numerous evidences indicating their crucial role in identifying microbial or viral compounds. However, the danger theory, where the innate immune system responds to danger signals such as proteins released during damage or necrosis rather than only non-self is gaining ground. Indeed, TLRs are able to recognise endogenous molecules and have been implicated as key players in numerous autoimmune diseases including systemic sclerosis (SSc). TLR2 is known to be upregulated in SSc and has been shown to respond to the endogenous ligand amyloid A resulting in increased IL-6 secretion. TLR4 is now known to respond to a variety of endogenous ligands including fibronectin, containing alternatively spliced exons encoding type III repeat extra domain (EDA). EDA is only expressed upon tissue damage, and elevated levels can be found in SSc patients, idiopathic pulmonary fibrosis and cardiac allograft fibrosis, while deletion of EDA or TLR4 in mice reduces their fibrotic response. Further, stimulation of TLR8 with single-stranded RNA leads to increased expression of TIMP-1. This has been shown to require both IRAK4 and NF-κB with evidence suggesting autoantibodies bind to RNA to stimulate TIMP-1 production in monocytes. Therefore, TLR-mediated signalling provides numerous potential This article is a contribution to the Special Issue on Immunopathology of Systemic Sclerosis - Guest Editors: Jacob M. van Laar and John Varga * Steven O’Reilly [email protected] 1

School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH3, UK

therapeutic targets for development of therapies for the treatment of multi-systemic autoimmune diseases. Keywords Systemic sclerosis . Innate immunity . TLRs . NODs . Inflammasomes

Introduction The innate immune system is critical in responding to conserved patterns in microbial species and endogenous signals to elicit an immune response that is non-specific and without memory. There have been several pivotal developments in understanding the innate immune system, and these have led to major shifts in thinking about innate immunity. Toll-like receptors (TLRs) are a germline-encoded group of pattern recognition receptors that share high homology with the Toll gene in fruit flies and are found in eukaryotes and plants [1]. Recognition of microbial or internal signals is essential in mounting an effective immune response, and the pattern recognition receptors (PRRs) are critical to this response, acting as the first line of defence. PRRs developed early in evolution for the detection of pathogens and TLRs are the best characterised PRRs to date. However, there are also other important PRRs involved in innate immune recognition, and these include the cytosolic NOD-like receptors and the RIG-Ilike receptors [2]. The RIG-I-like receptors are a family of helicases that function to sense and respond to viral RNA intracellularly [2]. Systemic sclerosis (SSc) is an idiopathic autoimmune connective tissue disease characterised by vascular abnormalities, chronic inflammation, cytokine disturbances and fibrosis. The fibrosis primarily affects the skin and internal organs, and currently, there is no effective disease-modifying therapy. Recent data has suggested that the disease may have an infectious trigger-activating innate

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immunity, and abnormalities in the TLR-signalling system have been frequently observed [3]. Although TLRs trigger signalling cascades, resulting in a release of pro-inflammatory mediators, a variety of other effects can be elicited upon stimulation. Inflammation and tissue fibrosis is coupled, and the innate immune system plays a pivotal role. Although activated fibroblasts are the driving force behind fibrosis secreting excess extracellular matrix (ECM), immune cells are critical and their activation and contribution to SSc are now being appreciated. The aim of this review is to give a current overview of the role of the innate immune system and TLRs in SSc and suggest where therapeutic modulation can occur.

TLR signalling TLRs are transmembrane proteins that contain an extracellular leucine-rich repeat and a cytosolic Toll-IL-1 receptor domain (TIR) that is responsible for downstream signalling [4]. This culminates in the activation of NF-κB and interferon families leading to induction of pro-inflammatory cytokine expression. NF-κB is a family of five transcription factors, acting as homo- and heterodimers, to regulate immunity. The TLRs also use an assortment of adaptor proteins to help elicit their signalling, and chief among these is myeloid differentiation response gene 88 (MyD88). MyD88 is utilised by all the TLRs with the exception of TLR3. Other adaptors include MyD88adaptor-like (MAL), TIR-domain-containing adaptor proteininducing β (TRIF), TRIF-related adaptor molecule (TRAM) and sterile α-and HEAT/armadillo motif containing protein (SARM) [5]. Interestingly, as opposed to the other adaptor molecules, SARM is a negative regulator of TLR signalling [5]. It functions to negatively regulate the TLR system through direct interactions with TRIF [5]. It appears that SARM is also involved in neuronal stress-induced responses and is preferentially expressed in neuronal tissue [6]. Mal only signals downstream of TLR4 and TLR2. Mal is often referred to as the bridging adaptor due to its role in bringing MyD88 to the TIR domains of TLR4. TLR3 utilises TRIF to induce antiviral responses [7]. The use of the adaptor proteins eventually leads to a release of pro-inflammatory cytokines and instructions to adaptive immunity. Negative feedback is crucial to such responses so that collateral damage does not occur and includes the TLR-induced increase in microRNA146 and miR147, resulting in a feedback loop to tame the immune response thus limiting inflammation via targeting of the signalling cascade components [8, 9].

Role of danger signals and TLRs in SSc The danger theory proposed by Matzinger states that the immune system responds to danger rather than just non-self [10].

It is suggested that otherwise innocuous proteins are released from cells upon damage, necrosis or stress and that these elicit signalling via TLRs to induce inflammation and direct immunity. Such molecules would normally be sequestered intracellularly and upon damage or stress either released passively or actively secreted. The molecular makeup of danger molecules shares no similarity, and they are a diverse group of molecules ranging widely in size. The fact that they are released from damaged cells and elicit a response indicates the organism is responding to ‘danger’. These molecules represent an important host defence against damage and danger and serve to restore homeostasis. One of the first insults in SSc is damage to the vasculature; it could be that early vascular endothelial damage could lead to the release of danger signals provoking an innate immune response. A fascinating recent observation was that the metabolic intermediate succinate is a ‘danger signal’ that induces IL-1β secretion [11]. Succinate achieves this increase in IL-1β through stabilisation of hypoxiainducible factor 1α [11]. Succinate is an intermediate in the TCA cycle and can be generated in monocytes by a variety of factors including hypoxia itself as there is a metabolic shift to glycolysis. It is likely in the future that the palette of damage associated molecular patterns (DAMPs) will increase. Although danger signals are now universally accepted and the early issues with LPS contamination of protein preparations have now been clarified, a question still remains as to whether the cell recognises a ‘danger signal’ differently to a pathogenic signal even when binding the same receptor? In other words, is there a distinct downstream response from exogenous (PAMPs) and endogenous DAMPs? Is this effect mediated through PRR accessory receptors? Maybe the epigenetic architecture of the cells stimulated is different in response to the ligand? Is there a functional hierarchy among danger molecules?

TLR2 in fibrosis To date, 13 TLRs have been identified, with 10 in humans, and each respond to distinct microbial or endogenous ligands [7]. Their expression is either membrane-bound or intracellularly in endocytic vesicles. TLR2 is a membrane-bound TLR that generally responds to motifs in bacteria to elicit a proinflammatory response. However, endogenous ligands such as serum amyloid A have also been identified for TLR2 [12, 13]. Serum amyloid A is an acute-phase reactant that is elevated highly after microbial infection and mainly synthesised by hepatocytes in the liver; however, synthesis extrahepatically can also occur. Serum amyloid A has long been known to be associated with acute-phase responses and inflammation; it is also elevated in SSc [14]. We were the first to demonstrate that in dermal fibroblasts that serum amyloid A induces IL-6

Semin Immunopathol TLRs and their known endogenous ligands

secretion via a TLR2-dependant pathway, but using cells deficient in TLR4, we could find no effect of amyloid A [15]. Further transfection of dominant negative NF-κB mutants into cells demonstrated that this induction of IL-6 was NF-κBdependant with chemical inhibition of NF-κB also diminishing IL-6 induction. We could further show that TLR2 was elevated on SSc patient fibroblasts, and use of a TLR2-specific neutralising antibody reduced serum-amyloidA-induced IL-6 [15]. The induction of IL-6 is important as IL6 is an essential pro-fibrotic molecule that mediates pleiotropic effects [16]. A recent study has found similar effects in lung fibroblasts of SSc patients and that patients with elevated amyloid A displayed an increased incidence of pulmonary hypertension, a severe complication of SSc [17], therefore implicating SAA as a biomarker. Interestingly, a rare variant is associated with SSc and higher levels of IL-6 in dendritic cells, the sentinels of the immune system [18], which is involved in the fibrotic-signalling cascade and is associated with pulmonary hypertension. A rare variant in A20, a negative regulator of TLR signalling, is also associated with SSc [19].

Table 1

TLR4

Incubation of fibroblasts with exogenous fibronectin EDA leads to ECM deposition and alpha-smooth muscle actin expression, while animal models of fibrosis in mice, with a genetic deletion of fibronectin EDA or TLR4 blockade, results in reduced fibrosis compared to WT. It is proposed that an initial insult such as tissue damage leads to enhanced fibronectin EDA release stimulating TLR4 signalling [27] (Fig. 1). Because the skin and the constituent cells are the interface between the external world and the body, any damage to this organ would result in danger signals being released. Fibronectin EDA has also been demonstrated to be elevated in idiopathic pulmonary fibrosis [28], and fibronectin EDA is also associated with cardiac allograft fibrosis [29]. Stimulation of TLR4 induces fibrosis by augmenting TGF-β signalling [30]. In the bleomycin model of fibrosis, it was demonstrated that mice without TLR4 compared to wild type mice had significantly less fibrosis, and this was associated with reduced antibodies and IL-6 [31]. This is suggestive of IL-6 mediating a role in fibrosis [32]. Furthermore, in a kidney fibrosis model, TLR4 deletion significantly reduced interstitial fibrosis [33]. A recent study has found that the chemokine CXCL4, a 70 amino acid that is secreted by platelet and dendritic cells, is a biomarker in SSc and that elevation of CXCL4 potentiates the response to TLRs [34]. Indeed, the endogenous TLR4 ligand S100A8 is elevated in SSc sera [35].

TLR4 is the best characterised TLR to date and is the receptor for LPS (endotoxin), a component of all gram-negative bacteria. Indeed, mice with no TLR4 signalling are resistant to endotoxic shock, a serious systemic disorder with high mortality [20]. TLR4 forms a complex with MD2 which is critical in signalling. Although the main ligand of TLR4 is LPS, it is now well established that other endogenous ligands can activate TLR4 and cause a pro-inflammatory state; these endogenous ligands are released from dead or dying cells as a signal to initiate an inflammatory response to arrest ‘danger’ [21]. The list of endogenous ligands for TLR4 is now growing rapidly and include heat shock proteins, high mobility-group box protein 1 (HMGB-1) [22, 23], hyaluronan [24], fibronectin and tenascin-C (Table 1). It is interesting that a group of these molecules are ECM-related proteins such as fibronectin. It was shown that for instance tenascin-C, which is an important glycoprotein of the ECM, induces pro-inflammatory signalling in synovial fibroblasts and that tenascin-C-null mice are protected from arthritis [25]. Fibronectin has also been identified as binding to TLR4 but only fibronectin that contains alternatively spliced exons encoding type III repeat extra domain (EDA) [26]. This fibronectin-spliced form is only expressed upon tissue damage and helps adopt a reparative phenotype. In adult tissues, fibronectin EDA expression is negligible and is markedly upregulated in response to damage and wound healing. Thus, it could be suggested that only certain forms of the ECM molecules provoke an immune response. A recent elegant study found elevated levels of fibronectin EDA in SSc serum samples and also skin biopsies [27].

TLR

Endogenous ligand

TLR2

HMGB-1 Serum amyloid-A Snapin A mRNA HMGB-1 HSP-20, HSP-60, HSP-70, HSP-96 Fibrinogen Extra domain A of fibronectin Tenascin-C Surfactant protein A S100A8/9 Soluble tuberculosis factor HSP-60, HSP-70, HSP-96 ssRNA (immune complexes) ssRNA (immune complexes) DNA (immune complexes)

TLR3 TLR4

TLR6/2 TLR7 TLR8 TLR9

Intracellular TLRs Intracellular TLRs include TLR3, 7 and 8. These intracellular TLRs are cytosolic sensors for nucleic acids often of viral

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Fig. 1 Fibronectin EDA mediates fibrosis. Fibronectin EDA is released upon damage and is never normally expressed; this resulting fibronectin EDA is recognised by TLR4 on the surface of dermal fibroblasts; this activates adaptor proteins MyD88 and TRAF that can activate the central transcription factor NF-κB and inflammatory cytokines as well as reducing the microRNA miR29a, because the bona fide target of

miR29a is collagen; and this suppression results in upregulation of collagen. At the same time, activation of this pathway can result in activator protein-1 (AP-1) activation and upregulation of TIMP-1. Negative feedback systems exist and include SOCS1, TOLLIP-1 and IRAK-M

origin. TLRs 7/8 bind single-stranded RNA sequences that are rich in uracil or uracil and guanosine bases which are found in many RNA viruses [36]. TLR3 activates the TRIF-dependant pathway to activate interferons, and the other TLRs utilise MyD88. TLR3 stimulation with poly IC in monocytes leads to upregulated expression of siglec-1, an adhesive protein known to be regulated by interferons as blocking interferon reduces siglec-1 expression [37]. Siglec-1 is important in mediating the binding of macrophages to other cells. TLR3 stimulation has also been shown to upregulate endothelin-1 mRNA, a potent pro-fibrotic molecule, in dermal fibroblasts. Blockade with bafilomycin has been shown to reduce this TLR3-mediated upregulation of endothelin-1. Agarwal et al. demonstrated that SSc fibroblasts upregulated TLR3 in response to interferon-α and that this results in enhanced production of IL-6 and monocyte chemoattractant protein-1 (MCP-1) [14], ultimately leading to enhanced recruitment of monocytes in the tissue. We have previously found that stimulation of TLR8 with single-stranded RNA in monocytes leads to increased TIMP-1 secretion [38] (Fig. 2). Further to this, using a patient with a genetic lesion in IRAK4, we could

show that this was both IRAK4 and NF-κB-dependant through incubation of the patient’s serum with benzonase, an enzyme that facilitates the cleavage of RNA, which leads to diminished induction of TIMP-1 [38]. It is suggested that the RNA species in the SSc patient’s serum is complexed with autoantibodies and that this is stimulating the monocytes to produce TIMP-1. Such changes in self-RNA/DNA being recognised by the immune system are common in other autoimmune diseases such as systemic lupus erythematosus (SLE) [39], as are interferon genes. We have also recently shown that this was mediated by epigenetic modifications through alterations in histone methylation. Single-stranded RNA was also found to be linked to cardiac fibrosis implicating singlestranded RNA as a key component of fibrotic mechanisms. Interferon regulatory factors (IRF) are transcriptional regulators of interferons and their target genes. IRF5 is one of the multiple IRFs, and SNPs in this gene have been associated with SSc [40]. Interestingly, autoantibodies to topoisomerase containing sera was found to induce IFN-α in mononuclear cells and showed an association with lung fibrosis [41]. Interestingly, gadolinium, a contrast agent that can cause

Semin Immunopathol Fig. 2 TLR8 mediates fibrosis in SSc. RNA released from dead or damaged cells is complexed with autoantibodies; this stimulates monocytes TLR8 to elicit signalling that activates IRAK4 and NF-KB which leads to secretion of TIMP-1 from these cells; this TIMP-1 inhibits MMP1 activity and ultimately leads to increased deposition of excess ECM. We have used a patient with no IRAK4 to demonstrate its requirement in this signalling cascade

nephrogenic systemic fibrosis, has been shown to activate TLR7 in monocytes ultimately resulting in the release of pro-inflammatory and pro-fibrotic mediators [42]. It also appears that high mobility group box (HMGB) proteins are required for nucleic acid sensing [43] by TLRs; it could be that different levels of HMGBs determine responsiveness to DAMPs. Epstein-Barr virus (EBV) is a herpes virus and has recently been shown to activate TLR signalling, and the EBV genes are found in SSc skin biopsies [44]. Furthermore, infection of normal dermal fibroblasts or endothelial cells with EBV induced interferon regulatory factors, interferon genes and TGF-β, ultimately leading to expression of ECM proteins and alpha-smooth muscle actin [44]. Thus, EBV may be the initial trigger of SSc. EBV has also been associated with idiopathic pulmonary fibrosis [45].

NLRs, the inflammasome and SSc Nod-like receptors (NLRs) are cytoplasmic intracellular sensors for pathogens. When activated, NLRs lead to activation of the ‘inflammasome’, a multi-molecular signalling complex that includes apoptosis-associated speck-like protein (ASC) and results in the activation of caspase 1 and processing and secretion of cytokines IL-1β and IL-18. Indeed, a SNP in NLRP1 gene has been demonstrated in SSc [46]. NLRP1 is one of the most investigated inflammasomes and can sense a

variety of molecular triggers. The role of the inflammasome in SSc is only recently being delineated with elevated caspase 1 levels being demonstrated in SSc dermal fibroblasts. Using an in vivo model of fibrosis, the bleomycin model, it was found that mice with a deletion of NLRP3, and thus the inflammasome, were resistant to fibrosis [47]. It has also been shown that the inflammasome is critical and necessary for the fibrosis associated with silicosis (silica particles), which is mediated through NALP3. Furthermore, mice with liver fibrosis had attenuated liver fibrosis when components of the inflammasome were reduced [48]. Recent evidence has suggested that the inflammasome is important in myofibroblast generation and that this is enhanced by TGF-β signalling and a direct association with NLRP1 and SMAD signalling via enhanced reactive oxygen species [49, 50]. Interestingly, the ‘danger signal’ uric acid, a metabolite of purine metabolism, has been found to promote activation of the inflammasome and lung fibrosis in lung disease. The exact ligands that are activating the NLRs and thus the inflammasome in SSc though are as yet unidentified. Silica is related to SSc although it is more likely that endogenous triggers are activating the inflammasome. Interestingly, cholesterol crystals have been shown to be danger signals activating the inflammasome [51] and also histones too mediated via TLR9 [52]. Although activation of NLRP3 is mediated through a variety of diverse stimuli, there is no evidence of direct binding of these activators to NLRP3. It is suggested activation, at least in the case of particulates such as silica, occurs through phagolysosome

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destabilisation [53]; however, the list of activators of NLRP3 is likely to grow.

Targeting the innate immune system therapeutically Because of the recent resurgence in interest in the innate immune system, a great deal of knowledge has been accrued and possible therapeutic targets identified. Many compounds have now been developed that can inhibit TLR signalling, and these can be used to delineate the contribution of TLRs to disease. This can happen on many levels and include blockade of the ligand (DAMP), blockade of the TLR or inhibition of the downstream-signalling adaptors. In terms of TLR blockade, Opsona Therapeutics (Dublin) has a TLR2-specific antibody, and this is being evaluated in delayed graft function in renal transplant patients [54]. This may be of interest in SSc where TLR2 activation leads to the acquisition of a myofibroblast phenotype in fibroblasts [55]. Eritoran is a specific TLR4 inhibitor and has shown promise in in vitro assays and also in animal models of TLR4-mediated disease. However, in a recent clinical trial, there was no reduction in 28-day mortality in patients with sepsis [56]. The TLR adaptor proteins MyD88 and Mal could also be therapeutic targets, and these have been inhibited in rheumatoid arthritis synovial explants in vitro [57]. It appears the intracellular TLRs are of major importance in SSc, and targeting these may be the most fruitful. Idera Pharmaceuticals have a TLR7/9 antagonist in clinical trial for plaque psoriasis. With the inflammasome also being associated with fibrosis, small molecule inhibitors of the inflammasome would be of interest in SSc. It was recently published that MCC950 is a specific inhibitor of NLRP3, and this was shown to be effective in animal models of inflammasome-dependant diseases and ex vivo samples from patients with Muckle-Wells disease, a familial cold autoinflammatory syndrome caused by gain-of-function mutations in NLRP3 [58]. Glyburide has also been shown to inhibit the inflammasome which is a sulfonylurea [59]. An increasing amount of effort is focused on the development of inflammasome and in general innate immunity inhibitors.

Conclusion

is notoriously heterogeneous, and the clinical variation may be underpinned by SNPs in TLRs. Functional SNPs have been described in TLR4 and are associated with RA [60]. Small molecule inhibitors of the inflammasome have also shown promise in animal models [58] and could prove useful in SSc for which there is currently no effective medical therapy.

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