Toll-like receptor triggering in systemic sclerosis: time to target

Abstract

SSc is an autoimmune disease that has features of vascular abnormalities, inflammation and skin and lung fibrosis. Toll-like receptors (TLRs) are sentinel receptors that serve to recognize pathogens or internal danger signals leading to downstream signalling pathways that ultimately lead to inflammation and modification of adaptive immunity. Inflammation and fibrosis appear intricately connected in this disease and TLR ligation on fibroblasts can directly activate these cells to produce copious amounts of collagen, a hallmark of disease. The presence of damage-associated molecular patterns in association with fibrosis has been highlighted. Given their prominent role in disease, this review discusses the evidence of their expression and role in disease pathogenesis and possible therapeutic intervention to mitigate fibrosis.

Introduction

While for many years the focus of inflammatory disease was on adaptive immunity, recently there has been huge and unprecedented progress in our understanding of innate immunity and inflammation. This has led to a deeper understanding of the interplay of the innate immune system, inflammation, cytokine regulation and specific diseases. We now appreciate that Toll-like receptors (TLRs) are a family of germline-encoded pattern recognition receptors that recognize both microbial and endogenous ligands, eliciting downstream signalling responses that culminate in activation of specific transcription factors and inflammation and, depending on the context, can eliminate pathogens or be associated with inflammatory disease [1].

SSc is an autoimmune multisystem connective tissue disease in which there are vascular abnormalities, capillary rarefication, inflammation and fibrosis [2]. It is characterized by the presence of specific autoantibodies and fibrosis and can be defined as limited (limited to areas of the skin) and diffuse, based on progressive fibrosis of the skin [3]. Although recent studies have unveiled multiple pathways associated with disease pathogenesis, treatment is still lacking due to an unpredictable clinical course and lack of detailed pathological mechanisms [4]. Diffuse SSc is particularly concerning, with a 5-year survival rate of 70% [5]. Excessive deposition of extracellular matrix (ECM), particularly fibrillar collagens, are central to fibrosis. Activation of quiescent fibroblasts to the myofibroblast that secretes excessive ECM, is contractile and is apoptosis resistant are also key effector cells. In the past few years, innate immunity, including monocytes and macrophages, has been found to be intricately linked to disease pathogenesis and more broadly the wound healing response [6]. Therefore the aim of this review was to evaluate the evidence of the role of innate immunity in SSc and possible targeting for therapeutic benefit.

TLRs

Since the first description of the founding member TOLL in the 1990s, multiple TLRs have been described [7]. TLRs are pattern recognition receptors (PRRs) that are evolutionary conserved from Caenorhabditis elegans to humans, indicating their importance to host defence [8]. There are currently 12 known TLRs and specific TLRs are associated with specific subcellular locations (Fig. 1). Other PRRs exist, such as nucleotide-binding, leucine-rich repeat receptors and cGAS, but these are not discussed here.

TLRs recognize conserved structures on microbial species or host-derived endogenous molecules. Cell surface TLRs include TLR1, 2, 4, 5 and 6. These primarily recognize bacterial and fungal structures [9]. Endosome localized TLRs include 3, 7, 8 and 9. These primarily recognize nucleic acid or CpG-rich DNA [10, 11].

Given that these receptors have such an integral role in innate immune responses, it is unsurprising that they have been associated with a variety of autoinflammatory diseases, including RA, PsA, SLE and SSc [6, 12–15]. This review examines the role of TLRs in SSc, evaluating the evidence for pathogenic roles and therapeutic targeting.

Membrane-bound TLRs

Cell surface TLRs include 1, 2, 4, 5, 6 and 11. In the main, they detect and respond to bacterial and fungal structures that are highly conserved, termed pattern-associated molecular patterns (PAMPs) [8]. However, they also respond to what are termed damage-associated molecular patterns (DAMPs), found on diverse molecules. DAMPs are released from intracellular stores upon damage or cellular stress and are normally sequestered within the cell; as such, they are regarded as endogenous danger signals. Because DAMP-mediated inflammation is independent of pathogen infection, they are referred to as ‘sterile inflammation’ [16]. Because there is only a restricted number of receptors, the immune system recognizes highly conserved regions of the pathogens to elicit a response. In comparison, the molecules described as DAMPs share little similarity and are defined more contextually in terms of ‘danger’. The classic PAMP for TLR4 is lipopolysaccharide (LPS) found in Gram-negative bacteria [17]. However, TLR4 can also be activated by DAMPs such as high-mobility group box protein 1 (HMGB1) [18], heat shock proteins [19], S100 family proteins [20], hyaluronan and tenascin-C [14]. How molecules that share no structural similarity can bind to the same single receptor and trigger an inflammatory response remains an enigma.

TLR4 is actively expressed on monocytes, but also fibroblasts, in SSc patients. The expression of TLR4 is significantly elevated in patient-derived fibroblasts in culture [21]. Indeed, stimulation of these cells with the classic TLR4 ligand LPS led to significant upregulation of ECM components such as collagen [21]. Mechanistically, the stimulation of TLR4 with LPS led to an amplified TGF-β1 response and a significant downregulation of miRNA29a [21], which is a negative regulator of collagen [22]. Furthermore TLR4 knock-out mice are protected from experimentally induced fibrosis [23] and stimulation of fibroblasts led to TGF-β gene signatures [24]. While this proved the role of TLR4 in fibrosis generation, it is not LPS that is driving this response in patients. Tenascin-C is a large multimodular glycoprotein that is highly expressed in the developing embryo but is hardly detectable in adult tissues [25, 26]. This has been described as a danger signal expressed at sites of inflammation and tissue damage [14]. Therefore, based on its danger signal and that tenascin-C is a TLR4 ligand, it was hypothesized that it may be involved in SSc. Bhattacharyya et al. [27] found significantly elevated levels of tenascin-C in skin biopsies from SSc patients, isolated dermal fibroblasts and also serum. Tenascin-C was positively regulated by the master profibrotic molecule TGF-β1, leading to enhanced ECM deposition and was dependent on TLR4 for its profibrotic effects. Remarkably, in an animal model of fibrosis, the bleomycin model, mice deficient in tenascin-C were protected from both skin and lung fibrosis, both key features of SSc [27]. This demonstrates that tenascin-C in a restricted expression pattern can mediate fibrosis and a lack of resolution of fibrosis is associated with a persistent ‘danger signal’ [27, 28]. Removal or inhibition of the danger signal would be a viable therapeutic target.

It was also demonstrated that fibronectin extra domain A (EDA) is elevated in SSc [29]. Interestingly, although both LPS and tenascin-C activate TLR4, it appears they elicit divergent responses in macrophages, with tenascin-C leading to a reparative phenotype [30]. Due to alternative splicing, cellular fibronectin contains two extra domains, EDA and EDB. Hardly any fibronectin EDA is found in adult tissue, but it is hugely upregulated upon injury [31]. It was found that the splice variant fibronectin EDA was upregulated in both tissues and serum of SSc patients and was positively regulated by TGF-β. Furthermore, fibronectin EDA signalled through TLR4 and was potently profibrotic. Mice in which the EDA gene was deleted were protected from skin fibrosis [29]. Fibronectin EDA has also been shown to play a key role in cardiac fibrosis [32]. This suggests that EDA is upregulated as a response to injury to maintain homeostasis and a lack of negative regulation of this results in fibrosis (Fig. 2).

TLR2 is another membrane-bound TLR that is protective against bacteria and fungi and whose ligands include lipoproteins, lipoteichoic acid and zymosan [33]. TLR2 forms a heterodimer on the cell surface and can bind with TLR1 or TLR6 dictating its specificity. However, DAMPs have also been described for TLR2, including HMGB1 [18, 34], biglycan [35] and serum amyloid A (SAA) [36].

SAA is an acute phase protein that is synthesized by the liver and can be elevated up to 1000 times in acute settings within 48 h [37]. It is highly elevated in autoinflammatory conditions such as RA [38]. It has been described as a TLR2 ligand and danger signal binding TLR2 [36]. We found that SAA induced the expression of the profibrotic molecule IL-6 and that neutralization of TLR2 retarded this effect [39]. We further showed that this was mediated through the adaptor protein IRAK4 and that TLR2 was regulated by histone acetylation [39]. Elevated levels of SAA are found in the serum of SSc patients [40] and is further elevated in patients with pulmonary disease [41]. This suggests that it could be a possible biomarker of lung disease. Interestingly, a polymorphism in TLR2 is associated with SSc [42]. Other ligands such as S100 have been found to be elevated in SSc tissues [43] and S100A4-mediated fibrosis in pre-clinical models of skin fibrosis [44].

Intracellular TLRs

Intracellular TLRs are employed to sense and respond to nucleic acids such as RNA and DNA [8, 45]. TLR7 (expressed in murine) and 8 are endosomal TLRs that recognize single-stranded RNA and synthetic imidazoquinolines such as imiquimod. These TLRs signal through nuclear factor κB, leading to pro-inflammatory cytokine release.

Using SSc patient monocytes, we found that they had a profibrotic phenotype that led to an upregulation of tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) via single-stranded RNA stimulation [46]. The upregulation of TIMP-1 was mediated by TLR8 and was dependent on intact MyD88 signalling. We found that this increased TIMP1 was functional and inhibited extracellular matrix degradation [46]. Importantly, sera from patients contained factors that led to TLR8-mediated TIMP-1 [46] and this could be diminished with RNase enzyme incubation. Furthermore, we found that TLR8 stimulation led to TIMP-1 upregulation via a MyD88 and Fra2-dependent pathway [47]. Fra2 was found to bind to the TIMP-1 promoter directly and could be enhanced by histone methylation, showing an immune-mediated epigenetic pathway leading to fibrosis [47]. Interestingly, Farina et al. [48] recently demonstrated that Epstein–Barr virus (EBV) infection of SSc monocytes led to activation of TLR8 in these cells and subsequent release of IFN. The authors also showed that skin biopsies contained activated TLR8 gene signatures in coexistence with infectious EBV, suggesting that EBV itself may be driving the initial insult [48]. Indeed, RNA recognition in humanized mouse models of TLR8 transgenics is enough to cause inflammatory disease [49].

Plasmacytoid dendritic cells are a small population of cells in the blood that mediate responses to TLR ligands. It was recently shown that plasmacytoid dendritic cells are chronically activated in SSc and secrete large amounts of IFN-α and that this is driven by TLR8 [50]. Depletion of these cells in vivo retarded skin fibrosis, whereas transgenic TLR8 mice had enhanced skin fibrosis in vivo [50]. These data indicate the importance of TLR8 in mediating fibrosis. Interestingly, it was demonstrated that autoantibody to topoisomerase 1 containing SSc sera led to increased IFN-α in dendritic cells [51]. This immune complex–mediated activation of IFN could be blocked with an antibody to CD32a, which blocks FcγR2, demonstrating a requirement for FcγR2 engagement [51]. It is speculated that immune complexes containing autoantibodies may underlie the phenomenon, at least in the case of anti-topoisomerase 1 antibody. This effect may be amplified, as dendritic cells are more sensitive to stimulation in SSc [52].

TLR3 is another intracellular sensor that has been shown to be elevated in SSc biopsies [53]. However, stimulation of fibroblasts with the TLR ligand polyinosinic:polycytidylic acid led to reduced collagen via increased IFN secretion [54], suggesting an antifibrotic role rather than a profibrotic role.

A recent study demonstrated an interaction between CXCL4, which is highly elevated in SSc, and DNA, leading to a physical complex formation in solution that activated TLR9 to mediate enhanced IFN-α release [55]. CXCL4 has been described as elevated in SSc, but no clear function had been described [56]. Interestingly, TLR9 leads to elevated release of CXCL4 [57], which could potentially fuel a feed-forward loop. Mitochondrial DNA has been described as a TLR9 ligand [58]. TLR9 stimulation was also demonstrated to upregulate SIglec-1 expression on SSc monocytes [59], which could potentially modify their function. Mitochondrial DNA was found to activate SSc fibroblasts and activate TLR9, and significantly elevated levels of mitochondrial DNA were found, particularly in patients with interstitial lung disease, and this is carried in extracellular vesicles [60].

Therapeutic targeting of TLRs

Given the cumulative evidence that TLRs are significantly involved in SSc pathogenesis, targeting of these may potentially provide therapeutic gain. However, one would want a specific inhibitor that does not increase the risk of pathology associated with microbial infection. Given that TLR4 has been shown to play a key fibrotic role in SSc [21, 29], this appears to be a real target in mitigating fibrosis. Rather than specific inhibition of TLR4, it could be that targeting the specific DAMP ligands like fibronectin EDA would have fewer risks. This is because this molecule is barely expressed in the healthy adult. Fibronectin EDA could be targeted using a specific antibody to neutralize the protein.

Alternatively, blocking the interaction through polypeptides may block profibrotic signalling. A recently described polypeptide that blocks interaction with the integrin needed for signalling demonstrated a strong reduction of ECM deposition in vitro [61]. TLR4 signature was associated with the inflammatory molecular subtype of disease, suggesting that this subset would respond most from targeting [62]. A specific MD2 inhibitor called T5342126 significantly attenuated TLR4-mediated fibrosis [62]. This is mediated through inhibition of MD2, which is an important co-receptor for TLR4 to have competent signalling [63]. This suggests that targeting the co-receptor for TLR4 will mitigate fibrosis. An alternative way of inhibiting TLR4 is through epigenetic modification. miRNAs are small RNA species that negatively regulate their cognate mRNAs through post translational repression [22]. miRNA21 is a specific negative regulator of TLR4 signalling and boosts the antifibrotic cytokine IL-10 [64]. Thus, use of an miRNA21 mimic may yield therapeutic benefit. Remlarsen is an miR29a mimic currently in a phase 2 trial for skin fibrosis (NCT03601052), demonstrating mimics are feasible.

While we found no direct effect of TLR2 activation from the DAMP SAA, it could be that stimulation potentiates fibrosis in this system. In light of this, in a pre-clinical model of fibrosis, inhibition of TLR2 via an antibody retarded fibrosis [65], suggesting, at least in pre-clinical models of fibrosis, that inhibition of TLR2 is effective.

Emerging evidence suggests that blocking TLR8 may be useful in SSc [46, 47]. Idera Pharmaceuticals have a TLR7/9 antagonist in clinical trials for plaque psoriasis. Until recently there was no specific inhibitor of TLR8, but Zhang et al. [66] described a molecule called CuCPT8m that is selective for human TLR8. Mechanistically, inhibition of TLR8 is achieved through stabilization of its resting state [66]. The same group has recently described a second generation of this compound that works through the same mechanism of stabilization of TLR8 and has been validated in human peripheral blood cells and spleenocytes from humanized TLR8 transgenic mice [67]. This suggests that targeting TLR8 is viable in this disease. Most recently an inhibitor of TLR9, E6446, has been found to inhibit fibrosis in an animal model of heart disease [68].

Should such inhibitors be given early in disease where a more inflammatory state is present? It is generally accepted in diseases such as RA that there is a ‘therapeutic window of opportunity’ to maximize benefit and change the disease trajectory when biological processes are more reversible [69]. Could such a therapeutic window exist in SSc? Evidence for the concept of a therapeutic window in SSc was recently shown in a clinical trial of the IL-6 receptor antibody tocilizumab, in which patients treated with tocilizumab had an attenuated rate of decline of lung fibrosis when treated early in the disease [70]. It is likely that different patients have different TLRs that underly the disease; e.g. a TLR4-mediated response in individuals, whereas TLR8-mediated fibrosis may underlie another patient, even though both have SSc. A biomarker approach to determine the underlying pathotype could inform which treatment to initiate.

Conclusions

Cumulative evidence has demonstrated direct roles of TLRs in SSc pathogenesis both in vitro and in pre-clinical models of fibrotic disease. Bidirectional communication between fibroblasts and myeloid cells mediate the fibrosis. Therefore drugs that impact on the TLR pathway that is perturbed should be clinically effective. However, while very recent studies have uncovered the role of TLRs, much has yet to be learned regarding pathogenesis and if, for instance, there is a hierarchy to these DAMPs? Are specific molecular subtypes of SSc disease driven by specific DAMPs? For instance, there was a specific TLR4 gene signature associated with the intrinsic inflammatory molecular subset [62], suggesting they would benefit most. Highly enriched SSc patients selected based on molecular classification and significant inflammatory cytokines in sera in clinical trials may yield a positive response to TLR inhibition. Further studies are required with specific molecularly classified patients. A specific inhibitor of TLR signalling ideally would negate the detrimental signalling, say from the DAMP, while preserving innate immune signalling necessary for pathogen clearance.

Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Disclosure statement: The author has declared no conflicts of interest.

Data availability statement

Data are available upon reasonable request by any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). All data relevant to the study are included in the article.

References