SEARCH

SEARCH BY CITATION

Introduction

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

In the early 1990s, Janeway discussed the theory that an immune response could not occur unless antigen-presenting cells (APCs) were first activated, which he called “the immunologist's dirty little secret.” Then, in the coup that caused the immunology community to finally take notice, and following the seminal study by Lemaitre et al (1), Janeway discovered the crucial role of pattern-recognition receptors (PRRs), which recognized evolutionary conserved molecules on infectious nonself organisms (2). Toll-like receptors (TLRs) belong to the PRR family, which was first shown to recognize microbial components, known as pathogen-associated patterns. TLRs are constitutively expressed by numerous immune cells and are designed to detect and eliminate invading pathogens by activating both innate and adaptive immune responses. Accumulating evidence indicates a role of TLRs in the recognition of “host-derived” agonists (so-called endogenous ligands or alarmins), which might be involved in various autoimmune and/or autoinflammatory syndromes. Examples of such endogenous TLR ligands are Hsp60, Hsp70, gp96, small HspB8, hyaluronic acid, and fibronectin, which are all released upon cell stress and are found in various tissues during inflammation (discussed below).

TLRs are of interest to immunologists because of their important role in the initiation of immune responses. Because evidence seems to implicate innate immunity in a wide variety of rheumatic conditions that are seen in the clinical practices of rheumatologists, the question is whether TLRs deserve more of the rheumatologist's attention as well. The role of TLR agonists in animal models of arthritis has been explored and is consistent with Janeway's “dirty little secret.” In this light, the use of Freund's complete adjuvant to boost arthritis has lost its magic with the discovery of TLR binding to constituents of this “miracle potion.” In this review, we discuss how recent advances in TLR-associated research have enhanced our understanding of the role of this receptor family in frequently occurring rheumatic conditions and how continued exploration of their role is likely to change the types of therapy available to battle these chronic diseases.

TLR biology and subsequent immune activation

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Currently, 11 TLR subtypes have been identified in humans, with each having specific ligands, cellular localization, and expression profiles. TLR-2 (as a heterodimer in combination with TLR-1 or TLR-6) and TLR-4 are extracellular receptors that are designed to recognize lipid-based structures from both gram-positive and gram-negative bacteria, from which lipopeptides and lipopolysaccharides (LPS) have been investigated most extensively (3–5) (Table 1). In contrast, TLRs 3, 7, 8, and 9 are located intracellularly, more specifically in the endosomal compartments, and are involved in the recognition of nucleic acids derived from viruses, bacteria, and the host, including single-stranded RNA, double-stranded RNA, and CpG DNA motifs (4–7). TLR-10, which is believed to originate from the TLR-1/TLR-6 precursor, has been identified only in humans, and no specific ligands have been described thus far. TLR-5 is probably the subtype that is studied the least, which perhaps explains why only one ligand, flagellin (8), has been identified thus far. Although profilin has been described as a ligand for mouse TLR-11 (9), ligands for human TLR-11 have not yet been identified. The cytoplasmic region of TLR shares a strong homology with interleukin-1 receptor (IL-1R), and therefore this region is referred to as the Toll/IL-1R (TIR) domain. However, the extracellular TLR regions do not contain Ig-like domains like the IL-1R, but contain several leucine-rich regions instead.

Table 1. Toll-like receptors (TLRs) and their exogenous and endogenous ligands
ReceptorExogenous and endogenous ligands
TLR-1 (+ TLR-2)Bacterial triacyl lipopeptides
TLR-2Necrotic cells; lipoproteins (gram-positive bacteria)
TLR-3Double-stranded viral RNA; endogenous RNA from necrotic cells
TLR-4Lipid-based structures (gram-negative bacteria); heat-shock proteins; hyaluronan; fibronectin
TLR-5Bacterial flagellin
TLR-6 (+ TLR-2)Bacterial diacyl lipopeptides
TLR-7Single-stranded viral RNA
TLR-8Single-stranded viral RNA
TLR-9Bacterial and viral DNA; chromatin IgG complexes; high-mobility group box chromosomal protein 1
TLR-10Not yet identified
TLR-11Not yet identified in humans

Downstream TLR signaling involves a family of 5 adaptor proteins that couple proteins and kinases, which leads to the activation of transcription factors, among which NF-κB and members of the interferon regulatory factor (IRF) family are currently the most thoroughly investigated (for review, see ref.10). In contrast with what was expected in the first years after its discovery, the TLR system is highly specific in that multiple cellular responses are observed, depending upon the ligand used. Much of this specificity is likely due to the use of various comolecules and downstream adaptor pathways by various TLRs (Figure 1). Myeloid differentiation factor 88 (MyD88) was the first adaptor molecule to be discovered (11), and its activation leads to the recruitment of IL-1R–associated kinase (IRAK) and tumor necrosis factor (TNF) receptor–associated factor 6, which subsequently results in the activation of downstream effector pathways (11–14) and finally in the production of proinflammatory cytokines such as TNFα, IL-6, IL-1β, and IL-12. Since mice rendered MyD88-deficient are unresponsive to ligands for TLRs 2, 4, 5, 7, and 9 (15), this indicates that MyD88 acts as a crucial adaptor for the signaling of all TLR pathways except TLR-3, which signals exclusively via TRIF (see below).

thumbnail image

Figure 1. Overview of Toll-like receptor (TLR) signaling and downstream cascades of transcription factor activation. The specificity of several adaptor molecules is shown. Myeloid differentiation factor 88 (MyD88) adaptor–like protein (Mal), for example, is exploited by TLR-2 and TLR-4, whereas TRAM is used by TLR-4 only. TLR-4 downstream signaling occurs along both the MyD88 and TRIF pathways, whereas TLR-2 uses only the MyD88 pathway. In contrast, TLR-3 signals exclusively via TRIF, and TLRs 5, 7, 8, and 9 use only the MyD88 pathway. SARM, the fifth TLR adaptor molecule, is not shown in this scheme since it is actually an inhibitory molecule capable of inhibiting the TRIF pathway. TRAF6 = tumor necrosis factor receptor–associated factor 6; RIP-1 = receptor-interacting protein 1; IRF = interferon regulatory factor.

Download figure to PowerPoint

Another TLR adaptor molecule, MyD88 adaptor–like protein (Mal), has been shown to be solely involved in the TLR-2 and TLR-4 signaling pathway, indicating that there are specific differences between the TIR signaling pathways (16). Mal acts as a bridge that mediates MyD88 to the plasma membrane, thus allowing binding to microdomains that contain TLR-4. Therefore, disturbed function of Mal would lead to diminished signaling of TLR-2 and/or TLR-4 pathways, an assumption that was recently proven by the finding that individuals who carry a variant of Mal are at higher risk for TLR-2/TLR-4–dependent diseases, such as pneumococcal pneumonia, malaria, and tuberculosis, compared with those who do not carry this variant (17). Since Mal was discovered as an adaptor molecule for MyD88 signaling only, this created a gap in our understanding of how TLR-4 could mediate type I interferon (IFN) induction. Later, however, TRIF was identified, which is now known to control TLR-4 MyD88-independent type I IFN production and is the sole adaptor for TLR-3 (18).

The fourth adaptor to be identified was TRAM (also known as TICAM2) (19). TRAM functions only in the TLR-4 pathways and is therefore the most restricted among the TLR adaptor proteins. Similar to TRIF, TRAM is required for the late activation of both NF-κB and IRF. Most recently, Carty et al (20) uncovered the most evolutionarily ancient adaptor protein, named SARM, which acts as a negative regulator of TRIF-dependent TLR signaling.

As mentioned above, TLR activation ultimately leads to the activation of NF-κB and IRF-3, which results in the production of proinflammatory cytokines. In addition, TLR activation initiates a series of events that is often called phenotypic maturation of APCs, which enables these cells to become fully adept at inducing the proper adaptive immune response. Th1-type immune responses are driven by IL-12 and IL-23 and are characterized by the production of IFNγ, whereas IL-10, IL-4, and IL-13 are associated with a Th2-type response. TLR stimulation generally leads to the production of IL-12 and IL-23 and thereby favors a Th1-type response (21). Although there is abundant evidence for the involvement of TLR in Th1-mediated immune responses, data on the role of TLR in the induction of Th2 responses are scarce. Until recently, the development of autoimmune and/or autoinflammatory diseases was thought to be governed by the Th1/Th2 subset model. However, conflicting data regarding this paradigm were introduced, followed by the identification of a newly defined Th cell subset that secretes IL-17 (22). Precisely how TLR signaling is implicated in the production of IL-17 remains to be determined.

TLR regulatory and inhibitory pathways

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

To control the immune responses induced by TLRs, tight negative regulation of these signals is necessary. Many inhibitory mechanisms that are involved in negative TLR regulation have been described recently (for review, see ref.23) (Table 2). First, TLR activation can be controlled by a diminished receptor expression achieved by ubiquitination, which is controlled, for example, by Triad3A (24). Second, the production of specific cytokines, such as IL-4 and transforming growth factor β, can lead to down-regulation of TLR expression and thus to diminished binding of ligands (25). Third, TLR signaling can prohibit the interaction between TLR and its ligand. This phenomenon is achieved by the production of soluble TLR (sTLR), which acts as a decoy receptor. In mice, a naturally occurring form of sTLR-4 has been identified (26). Similarly, sTLR-2 has been found in human plasma, caused by constitutive release from blood monocytes. Soluble TLR-2 is functionally active, since its depletion resulted in an increased cellular response to bacterial lipopeptide (27).

Table 2. TLR inhibitory pathways*
Mechanism, moleculeTLR pathway
  • *

    TLR = Toll-like receptor; MyD88 = myeloid differentiation factor 88; IRAK = interleukin-1 receptor–associated kinase; TRAF = tumor necrosis factor receptor–associated factor; TOLLIP = Toll-interacting protein; RIP-1 = receptor-interacting protein 1; SOCS-1 = suppressor of cytokine signaling 1; Mal = MyD88 adaptor–like protein; SIGIRR = single immunoglobulin interleukin-1 receptor–related molecule; ST2L = ST2 ligand; LPS = lipopolysaccharide; NOD-2 = nucleotide-binding oligomerization domain 2.

MyD88 inhibition 
 MyD88Blocks IRAK-4 to phosphorylate IRAK-1 by MyD88
TRIF regulation 
 SARMInhibits TRIF-dependent transcription factor activation
IRAK regulation 
 IRAK-MInhibits formation of IRAK-1–TRAF6 complex
 TOLLIPInhibits IRAK phosphorylation
Ubiquitination-mediated  degradation 
 A20Terminates TLR signaling by decreasing TRAF6/RIP-1 stability
 SOCS-1Interacts with Mal, leading to instability and degradation
 Triad3ARegulates degradation of TLR-4 and TLR-9
Decoy molecules 
 SIGIRRInhibits recruitment of adaptors of TLR complex
 ST2LInhibits binding with MyD88 and TRIF
 RP105Blocks binding to TLR-4
Soluble factors 
 Soluble TLR-4Inhibits binding between TLR-4 and its ligand
 Soluble TLR-2Blocks association between TLR-2 and its ligand
 Soluble CD14Inhibits association between CD4 and TLR complex
 Soluble ST2Inhibits LPS-induced cytokine secretion
Unknown 
 NOD-2Directly inhibits TLR? Induces Th2 cytokines?
 TRAILRPossibly decreases nuclear translocation of NF-κB

Finally, specific transmembrane and intracellular proteins are involved in the negative regulation of TLR signaling. ST2, for example, inhibits TLR-4–mediated cell activation by sequestering MyD88 and Mal. In addition, TRAILR, which belongs to the TNF superfamily, inhibits TLR activation by IκBα stabilization upon ligation with TRAIL, which reduces the nuclear translocation of NF-κB (28). Similarly, suppressor of cytokine signaling (SOCS) (29), IL-1R–associated kinase M (IRAK-M) (30), and Toll-interacting protein (31) attenuate TLR activation, which leads to suppression of NF-κB activation. Altogether, several mechanisms exist to “control” TLR-mediated immune responses that are designed to prohibit a chronic response leading to tissue damage. Now, evidence supports the concept that disturbed counterregulatory mechanisms, rather than an altered TLR response itself, might explain the higher state of TLR-mediated cell activation seen in several conditions (for review, see ref.32). TLR signaling depends on the selective use of different TLRs, TLR adaptor proteins, and activation of distinct sets of intracellular TLR inhibitors. More insights into these pathways, especially with regard to potential aberrations within rheumatic conditions, might lead to the development of new agents that interfere with these pathways, thus inhibiting or increasing their signaling capacity, depending on the nature of the defect.

TLRs and potential involvement in daily clinical rheumatology

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Systemic lupus erythematosus (SLE).

SLE is a characteristic autoimmune syndrome in which autoantibodies to DNA and antinuclear antibodies (ANAs) develop years before the onset of clinical features (33, 34). The pathogenesis of SLE remains unclear, although the notion of a defective apoptosis “garbage disposal” theory does help explain how the immune system might recognize predominantly intracellular antigens. A remarkably high proportion of the autoantibodies found in SLE bind DNA, RNA, or macromolecular immune complexes (ICs) that contain DNA or RNA. The reason these epitopes play such an important role has been debated for many years, and several explanations have been suggested. The discovery of TLRs and their involvement in the recognition of RNA and DNA has changed our way of thinking. Data continue to accumulate that support the idea that DNA and/or RNA can behave as autoantigens because they have the capacity to stimulate the innate immune system directly via TLRs or indirectly via Fcγ receptors (FcγR) and thereby promote the self-directed immune response, potentially leading to tolerance.

The first evidence that pointed to a link between TLR-mediated immune activation and SLE pathogenesis came from the findings that indicated an important role of IFNα. IFNα constitutes a complex family of cytokines with diverse biologic functions. In SLE, early clinical studies revealed a correlation between IFNα levels and disease (35). Following these studies, the relationship between IFNα and SLE was further supported by the findings that the administration of IFNα as a treatment for malignancies or chronic viral infections led to the production of ANAs and the development of a clinical syndrome closely resembling SLE (36, 37). More recently, studies focusing on the role of IFN have revealed an “IFN signature” in patients with active SLE, an observation that is not restricted to SLE since rheumatoid arthritis synovium also displays such a signature (38, 39).

But what, then, is the link between IFNα production and TLR? Vallin and colleagues (40) were the first to demonstrate that ICs, isolated from SLE patients, are a potent stimulus of IFNα secretion by plasmacytoid dendritic cells (DCs). More intriguingly, Lovgren and colleagues showed that the uptake of such ICs is orchestrated by the concerted action of the activating FcγRIIa that internalizes the IC and transports it to the lysosomal compartment, where it co-segregates with intracellular TLRs such as TLR-7 (41, 42). In addition to the stimulation of plasmacytoid DCs, direct stimulation of antibody production by B cells is also likely to occur in SLE. B cells express both TLR-7 and TLR-9, and access to the appropriate TLR-containing compartment is likely to be mediated by B cell receptor–mediated endocytosis. The activation of B cells by TLR ligand–containing ICs was formally established using TLR-9–deficient cell populations and TLR-9–transfected cell lines (43). The recent discovery of the high-mobility group box 1 facilitating TLR-9–dependent activation by DNA-containing ICs in SLE (44) has only further changed our reasoning about the role of TLRs in this disease.

In addition to these ex vivo experiments, experimental models of SLE have revolutionized our knowledge of TLR activation in SLE. For example, mice homozygous for the lpr mutation, which normally leads to the production of antibodies specific for double-stranded DNA, failed to produce ANAs when rendered deficient for MyD88 (45). Similarly, mice with the Yaa mutation that developed spontaneous SLE, which was later found to be due to a TLR-7 duplication, showed markedly increased mortality rates due to proteinuria and renal disease (46, 47). To date, association studies assessing the role of TLR variants in SLE have yielded conflicting results. Although 2 studies have shown an association of a single-nucleotide polymorphism (SNP) in TLR-5 and TLR-9 with SLE susceptibility (48, 49), several others have failed to confirm these associations (50–53). However, additional studies using larger patient cohorts and other SNPs should provide better insight into the role of TLRs in the susceptibility to and severity of this condition. In this regard, it has been shown that mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 were associated with SLE and familial chilblain lupus (54, 55). Interestingly, TREX1 is involved in nucleic acid processing. A defective TREX1 pathway could thus lead to an overload of nucleic acid binding to TLR. Indeed, the TREX1 mutations were found to be associated with increased levels of IFNα in patients with the monogenetic disorder Aicardi-Goutieres syndrome, a hallmark of SLE reflecting TLR signaling (56).

Rheumatoid arthritis (RA).

The first evidence of TLR involvement in RA came from Leadbetter and colleagues (43), who showed that B cells from MyD88-deficient mice that were rheumatoid factor (RF) positive were completely unresponsive to chromatin-containing ICs. In addition, autoimmune serum that effectively stimulated B cells from RF-positive MyD88-positive mice seemed ineffective in RF-positive MyD88-deficient mice, demonstrating that autoantibody–autoantigen ICs act via an MyD88-dependent receptor, probably TLR-9 (43).

Based on these observations, several research groups have tried to delineate the role of TLR in RA. So far, that role has been shown to not be limited to TLR-9. Human TLRs 2, 3, 4, and 7 were found to be clearly increased in RA synovial tissue compared with that from healthy controls and from patients with osteoarthritis (OA) (57–59). Furthermore, synovial fibroblasts isolated from RA synovial tissue showed clear up-regulation of TLR-2 upon stimulation with the proinflammatory cytokines TNFα and IL-1β (59). Conversely, synovial fibroblasts incubated with TLR-2 agonists showed dramatically increased expression of chemoattractant proteins (60). Similarly, monocyte-derived DCs from RA patients have been shown to produce much higher amounts of IL-6 and TNFα upon TLR-2– and TLR-4–mediated stimulation than cells from healthy controls, whereas cytokine production upon stimulation with TLR-3 and TLR-7 was similar between the groups investigated (58).

More recently, the role of TLR-2 and TLR-4 in synovial inflammation was further substantiated by the observation that genetic deletion of MyD88 and Mal/TIRAP expression led to diminished cytokine production by RA synovial cell membrane cultures (61). One could speculate that the presence of various endogenous and exogenous TLR agonists in the synovial compartment leads to a synergistically enhanced TLR response, which leads to the breakthrough of tolerance. This response might lead to the release of more endogenous TLR ligands, resulting in a vicious circle of inflammation (Figure 2). However, this mechanism is probably not specific to RA, but could underlie joint pathology in general. It is interesting that Chinese hamster ovary reporter cell lines, which were stably transfected with human TLR-4, showed strongly enhanced responses upon incubation with synovial fluid or serum from RA patients with active disease (62).

thumbnail image

Figure 2. Overview of a self-sustaining loop of Toll-like receptor (TLR) activation and TLR-mediated tissue damage as a conceptual framework for the chronic inflammation of rheumatoid arthritis (RA). 1, Both exogenous and endogenous TLR ligands, which are abundant in the synovial joints of RA patients, induce a first immune response mediated by TLRs. 2, Single-TLR stimulation leads to the production of proinflammatory mediators, such as cytokines, chemokines, and metalloproteinases; however, simultaneous stimulation of several TLRs results in a synergistically induced immune response characterized by extremely high levels of proinflammatory mediators. 3, These mediators of inflammation can, in turn, cause tissue damage, which leads to the release of endogenous TLR ligands, such as heat-shock proteins (HSP), fibronectin, and hyaluronan. 4, This results in a self-perpetuating loop of TLR activation and TLR-mediated tissue damage. 5, Subsequent stimulation of TLR-mediated pathways of other immune cells, e.g., fibroblasts and B cells, by endogenous TLR ligands leads to the secretion of metalloproteinases and antibodies, which further contribute to inflammation. 6, In addition, the inflammation mediators, mainly cytokines, can induce TLR up-regulation, which can result in a further lowering of the threshold for TLR-mediated activation, thus also contributing to the vicious circle of inflammation. LPS = lipopolysaccharide; LTA = lipoteichoic acid; APC = antigen-presenting cell.

Download figure to PowerPoint

Recently, 2 novel TLR agonists were identified that are both abundantly expressed during chronic inflammation of the synovial joints. First, Brentano and colleagues (63) found that RNA released from necrotic RA synovial fluid cells could activate synovial fibroblasts via TLR-3. In addition, it was shown that small HspB8, which is present in RA synovial tissue, can induce TLR-mediated cell activation via TLR-4 in vitro (64).

Lately, the effect of TLR polymorphisms, especially genetic variants of TLR-2 and TLR-4, has also been investigated. However, the involvement of these polymorphisms in the susceptibility and severity of RA has not been established thus far (65–67). In contrast, the Asp299Gly TLR-4 polymorphism might influence the treatment response of RA patients, since Kuuliala and colleagues (68) found that RA patients with the genetic TLR-4 variant who were treated with a single disease-modifying antirheumatic drug (DMARD) showed a statistically significantly impaired DMARD response, in terms of the Disease Activity Score (69), compared with patients without the variant. To specify more precisely the possible relationship between genetic variations in TLR subfamilies, disease severity, and treatment response, additional research is required.

Experimental arthritis models have contributed significantly to our understanding of the role of TLR in arthritis. Mice that are MyD88-deficient did not develop streptococcal cell wall (SCW)–induced arthritis, which is indicative of the crucial role of MyD88 in SCW-induced arthritis (70). Furthermore, 2 soluble antagonists of TLR-4 clearly abrogated the onset of and diminished the severity of experimental arthritis (71, 72).

Seronegative spondylarthritides (SpA).

SpA include diseases such as ankylosing spondylitis (AS), psoriatic arthritis, and reactive arthritis, and symptoms consist of spinal inflammation, peripheral arthritis, enthesitis, and organ involvement such as anterior uveitis. Although the discovery 30 years ago of HLA–B27 and its association with SpA contributed to our knowledge of the pathogenesis of this condition (73, 74), the underlying pathways remain largely unknown. However, the notion that the innate and adaptive immune responses have a role is fueled by several observations.

First, the synovitis in SpA displays features of other types of inflammatory arthritis, most closely resembling those seen in RA (75). Relevant differences, though, are a tendency toward greater vascularity (76, 77), more infiltrating CD4+ T cells and CD20+ B cells, and fewer lymphoid aggregates (78). In addition, CD163+ macrophages are more abundant, while CD68+ macrophages are similarly expressed. Interestingly, CD163 is a hemoglobin scavenger receptor and may delineate a population that produces more TNFα and other mediators of inflammation (79). The regulation of CD163 expression is at least partly controlled by TLR. Weaver et al (80) showed that stimulation of peripheral blood mononuclear cells (PBMCs) with ligands of TLRs 2, 4, and 5 led to a clear increase in CD163 surface expression, suggesting that TLR-mediated cell activation might underlie the high expression of CD163 in SpA.

Second, the association between HLA–B27 and seronegative SpA underscores the implication of antigen uptake, processing, and presentation by APCs. The host–pathogen interaction has proved to be a useful model for exploring noncanonical roles for HLA–B27; such exploration is complicated by the sustained difficulties in applying conventional structure and function to a mechanistic hypothesis of how HLA–B27 contributes to disease (72). The conventional role ascribed to HLA class I molecules such as HLA–B27 is presentation of processed peptides to CD8+ cytotoxic T lymphocytes. Thus, HLA–B27–transgenic mice developed a higher incidence of ankylosing enthesopathy, arthritis, and nail changes (81, 82). Following these studies, Hammer and colleagues (83) were the first to demonstrate a direct link between HLA–B27 and disease, since transgenic rats that overexpressed both HLA–B27 and human β2-microglobulin showed spontaneous inflammation of the gastrointestinal tract and joints, along with skin changes that closely resembled psoriasis.

Third, AS patients have repeatedly been shown to have an abnormal mucosal permeability that might lead to the “leaking” of TLR agonists from the gut flora into the circulation (84, 85). Indeed, increased titers of IgA antibodies to LPS from Escherichia coli and Klebsiella pneumoniae were found in patients with AS (86–88), further suggesting the implication of TLR-mediated immune activation. Recently, the involvement of microtrauma and the presence of microorganism-derived TLR ligands in the “synovio-entheseal complex” and the subsequent release of endogenous ligands was further conceptualized and substantiated (89).

Direct evidence of the role of TLRs in AS, though, is scarce. Thus far, 3 groups of researchers have failed to prove an association between the functional variant of TLR-4 and disease susceptibility (90–92). In addition, only 2 ex vivo studies suggest a potential link between TLRs and SpA. De Rycke et al showed that expression of TLR-4 was increased on PBMCs from SpA patients, particularly in the CD163+ subpopulation (93). TLR expression was also found to be higher in inflamed synovium of SpA patients compared with that seen from patients with RA or OA. Although such up-regulation could be a reflection of nonspecific inflammation that is not necessarily dependent on TLR, this might have important consequences for therapeutic interventions, for example, selectively depleting TLR-expressing macrophages. Consistent with this observation, Candia et al (94) observed an increased expression of TLR-2 on immature DCs in SpA patients compared with those in healthy controls and RA patients, whereas expression of TLR-4 was similar among all groups. Collectively, the involvement of TLRs in SpA is unclear and warrants further investigation.

Future perspectives

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Despite the current advances in therapies for rheumatic diseases (TNFα-neutralizing therapies, anti-CD20, CTLA4-Ig), there are still numerous unmet medical needs. The current therapies still show a lack of disease responsiveness, a high socioeconomic burden, failure of long-term efficacy, and unwanted side effects varying from local reactions to life-threatening events. The key challenge, therefore, would be to develop a drug that reduces inflammation to such an extent that it is not harmful to the host, but at the same time allows an efficient host defense. A substantial body of evidence now points toward the role of TLRs in chronic inflammatory conditions. Thus far, TLRs have been implicated only in the activation of immune responses. Whereas the existence of inhibitory TLR subtypes is conceivable, they have yet to be identified.

The inhibition of TLR signaling can be achieved in several ways (Figure 3). First, the TLR–ligand interaction can be blocked by using monoclonal antibodies against TLR. Although this technique might work for extracellular TLR, prohibiting binding to intracellular TLR might be more challenging. Currently, the development of soluble TLR inhibitors and testing in clinical practice are under way. Also, synthetic oligodeoxynucleotides (ODNs), which act as antagonists for TLR-9, and bimodal ODNs, which block both TLR-7 and TLR-9, are currently being tested.

thumbnail image

Figure 3. Methods of modulating the signaling cascade of Toll-like receptors (TLRs). 1, The interaction between TLR ligands and TLR can be blocked by monoclonal antibodies directed against TLR or its specific ligand. 2, One way to interfere with TLR signaling might be to lower TLR expression on antigen-presenting cells (APCs). 3, Many intracellular adaptor proteins and “endogenous inhibitors” involved in TLR signaling or its inhibition have been identified. Interfering with the expression of such molecules might lead to a stronger or weaker TLR response. 4, Several receptors, such as triggering receptor expressed on myeloid cells (TREM) and 4-1BBL, have been implicated in augmenting TLR-mediated immune responses by crosstalk with TLR pathways. Interference with this crosstalk could lower the threshold for TLR signaling; this threshold might even be disease specific. 5, Since every immune response that is ignited must be terminated to prevent tissue damage, counterregulatory pathways that dampen TLR responses must exist. Although they have not been identified thus far, stimulation of such pathways would actively inhibit TLR signaling. 6, Another approach is the active tolerization of dendritic cells (DCs) and subsequently Treg cells by the stimulation of TLR-dependent pathways.

Download figure to PowerPoint

Second, one can attempt to lower TLR expression on immune cells of interest. In our experience, however, even very low levels of TLR expression can lead to full cell activation when triggered with TLR-specific ligands, suggesting that this technique might be successful only when full ablation of TLR expression can be achieved. Furthermore, the complete absence of TLR is likely to lead to serious infectious complications.

Third, interference with downstream TLR signaling cascades might be an attractive alternative. Several molecules that have been implicated in TLR signaling might be candidates. So far, none of these approaches has been tested extensively. In contrast with the former approaches, however, interference with downstream TLR effector pathways is more specific for certain pathways, depending on the targeted molecule. For example, interference with MyD88 would limit signaling of several TLRs, leading to a plethora of unwanted effects. In contrast, blocking of TRAM would specifically inhibit TLR-4.

Fourth, triggering receptor expressed on myeloid cells 1 and 4-1BBL have recently been described as molecules that enhance TLR-mediated responses. The identification of molecules that interfere with TLR signaling, either by augmenting or inhibiting its response, is in its beginning stages. The latter approach seems to be very attractive. Based on the recent finding that RA patients exhibited an augmented TLR-2/TLR-4 response (58), the delineation of molecules responsible for the phenomenon would open novel avenues for a disease-specific treatment, especially when this would lead to the restoration of the “physiologic” TLR response, thus ensuring an adequate response against invading organisms.

Finally, a completely different approach might be the active induction of tolerance, for example, tolerization of professional APCs, such as macrophages and DCs, by an active TLR stimulation protocol. In addition, the regulatory capacity of T cells (Treg cells) might be altered upon TLR-mediated triggering. Further delineation of the exact role of various TLR agonists might facilitate the use of Treg cells. From treatment studies in various autoimmune disorders, there is a small amount of evidence that TLRs could have a role in the treatment of arthritis. Although the underlying mechanisms have yet to be determined, the administration of chaperonin 10 (also known as Hsp10) to RA patients was safe and clinically effective, indicating that such an approach is feasible (95).

In conclusion, substantial evidence indicates that targeting TLR signaling in a variety of rheumatologic diseases might be clinically effective. Further delineation of the precise role of TLRs is therefore warranted, since it will provide novel insights into the pathogenesis of various rheumatic conditions and might create opportunities for discovery of targets for treatment.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Dr. Radstake had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Roelofs, Joosten, van den Berg, Radstake.

Manuscript preparation. Roelofs, Abdollahi-Roodsaz, Joosten, van den Berg, Radstake.

Acknowledgements

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

We thank A. W. T. van Lieshout, M. Wenink, C. Popa, and R. Huijbens for invaluable contributions to the preparation and content of this manuscript. We are grateful for the discussions with many colleagues, particularly I. B. McInnes, A. Marshak-Rothstein, R. Lafyatis, and G. J. Adema, concerning ideas contained in the article. We apologize to those colleagues whose work is cited by review rather than by original work due to space constraints.

REFERENCES

  1. Top of page
  2. Introduction
  3. TLR biology and subsequent immune activation
  4. TLR regulatory and inhibitory pathways
  5. TLRs and potential involvement in daily clinical rheumatology
  6. Future perspectives
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES