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RNA/DNA sensing in SLE—Toll-like receptors and beyond
Introduction
In recent years it has become increasingly clear that aberrant response to nucleic acids (be they endogenous or exogenous) in systemic lupus erythematosus (SLE) patients plays a large role in triggering and sustaining the disruption of immune responses in a genetically predisposed individual. Nucleic acids are recognized by both membrane-bound Toll-like receptors (TLRs) and intracellular RNA and DNA sensors. In predisposed individuals, dysregulation of nucleic acid sensing by these pathways can trigger overproduction of type I interferons (IFN) and the production of antinuclear antibodies (ANAs). Type I IFNs are now well-established for their role in SLE—driving loss of tolerance, etc. (add in sentence here. ANAs have two important roles—first, they form pathogenic immune complexes that are deposited in tissues and drive pathology; second, anti-DNA and anti-RNA containing immune complexes are highly immunostimulatory, acting as danger associated molecular patterns (DAMPs) which can trigger innate immune responses and augment IFN production via recognition by both Toll-like receptors and the intracellular nucleic acid receptors. In this chapter we will review how both TLRs and non-TLRs play a role in driving SLE pathology, how identification of patients with mutations in these pathways has informed our understanding and how we might use this information to inform therapeutic development.
RNA and DNA sensors have evolved principally to detect and respond to viral infection, recognizing microbial RNA and DNA and triggering the production and release of type I IFNs and trigger appropriate innate and adaptive immune responses to clear the virally infected cells and prevent spread. This includes activation of B cells to produce opsonizing antibodies to prevent viral spread and activation of NK and CD8 T cell responses to kill infected cells. In addition, they play a role in detecting cellular damage and cellular stress by being able to detect and respond to the presence of cytosolic DNA or RNA. Two major classes of RNA and DNA sensors exist depending on their ability to detect endogenous or exogenous nucleic acids, respectively—cytosolic sensors that recognize cytosolic RNA (RIG-I or MDA-5) or DNA (cGAS, DDX41, AIM2 and IFI16), and those that respond to RNA/DNA internalized by receptor mediated endocytosis—namely the endosomal TLRs, TLR3, TLR7, TLR8, and TLR9. In general, several specialized immune cells such as dendritic cells, inflammatory monocytes and B cells are the major producers of IFNα by virtue of their ability to express and respond to TLR7/TLR9 engagement. All other cells, both immune and non-immune, tend to respond to cytosolic RNA/DNA via activation of RIG-I/MDA-5 or cGAS and produce IFNβ. Regardless of whether IFNα or IFNβ are produced, they both signal through the IFN receptor complex, and trigger activation of the JAK kinases JAK1 and Tyk2 to drive formation of the transcriptionally active STAT1/STAT2/IRF9 complex and subsequent expression of IFN-stimulated genes (ISGs).
Toll-like receptor family (TLRs)
TLRs are prototype pattern recognition receptors (PRRs). They have evolved to recognize microbial pathogen associated molecular patterns (PAMPs) and drive inflammation and production of anti-viral type I IFNs in or to link innate and adaptive immunity. It has been nearly 30 years since the first Toll receptor was identified in the fruit fly Drosophila. In 1997 the first human ortholog of the Drosophila Toll protein was described, a protein that was later designated Toll-like receptor 4 (TLR4). In humans and mice combined, 13 TLRs have been discovered; however, their expression differs between species. Humans, but not mice, express TLR10, and only mice express TLR11, TLR12, and TLR13. TLRs are an evolutionarily conserved family of type I transmembrane receptors that have an extracellular domain compromising leucine-rich repeats and a cytoplasmic domain that shares significant homology with the mammalian type I IL-1 receptor. The TLR family can be divided into two subgroups, TLRs that are found on the cell surface and TLRs that reside in intracellular vesicles. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are expressed on the cell surface, with TLR2 forming heterodimers with TLR1, TLR6, or TLR10. The intracellular TLRs include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 and they are found localized to intracellular compartments, such as the endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes ( Fig. 20.1 ). TLR11, TLR12, and TLR13 are expressed in mice, but human TLR11 is a nonfunctional pseudogene and human TLR12 and TLR13 are absent from the human genome. TLR activation leads to the initiation of intracellular signaling pathways that elicit the expression of inflammatory genes, such as cytokines essential for host defense and type I IFNs critical for antiviral defenses. How TLRs activate these signaling pathways has been reviewed extensively elsewhere.
In addition to recognizing classical exogenous PAMPs, TLRs also recognize endogenous activators, namely damage associated molecular patterns (DAMPs). While recognition of PAMPs by TLRs is critical for appropriate innate and adaptive immune responses, in contrast it is believed that the inappropriate activation of these receptors by DAMPs can contribute to chronic inflammatory syndromes and autoimmunity. Indeed, members of the TLR family have been implicated in the pathogenesis of several autoimmune diseases, includingSLE. DAMPs are released from the extracellular or intracellular space following tissue injury, stress or cell death. DAMPs originate from four main sources including extracellular matrix, mitochondria, dead and dying cells cells (e.g., nucleic acids), and post-translational modifications of self-proteins. DAMPs mainly activate TLR4 but also TLR3, TLR3, TLR7, and TLR9. In SLE it is thought that intracellular (endosomal) TLRs play a central role by virtue of their ability to recognize and respond to nucleic acids, with antinuclear autoantibodies triggering TLR7 and TLR9 responses. Endosomal TLRs in SLE can also be activated also by non-nuclear autoantibodies such as anti-myeloperoxidase, anti-beta2 glycoprotein, and anti-cardiolipin autoantibodies. The microbial and endogenous ligands for TLRs are shown in Table 20.1 .
Table 20.1
Microbial and endogenous host ligands reported to activate cells through human and murine TLRs.
| Microbial Ligands | Endogenous Ligands | |
|---|---|---|
| TLR1 a | Triacylated lipopeptides, lipoarabinomannan Borrelia burgdorferi (OspA) |
|
| TLR2 | Zymosan, peptidoglycan, M. tuberculosis Leptospiral LPS |
HSP60, HSP70, HMGB1, gp96, MSU |
| TLR3 | dsRNA | mRNA |
| TLR4 | LPS from Gram-negative bacteria GPI from Trypanosoma cruz Proteins from RSV, MMTV, and M. tuberculosis |
HSP60, HSP70, gp96, fibrinogen hyaluron, heparin sulfate, HMGB1 MSU, β-defensin 2 |
| TLR5 | Flagellin | |
| TLR6 a | Diacylated lipopeptides, lipoteichoic acid, zymosan | |
| TLR7 | ssRNA and ssRNA viruses (VSV, NDV, parechovirus) | ssRNA/protein complexes |
| TLR8 | ssRNA, poly(T)/imidazoquinoline complexes G-rich oligonucleotides |
ssRNA/protein complexes |
| TLR9 | CpG DNA | Chromatin, DNA/protein complexes |
| TLR10 | Unknown | |
| TLR11 | Profilin Toxoplasma gondii |
|
| TLR12 | Profilin Toxoplasma gondii |
|
| TLR13 | Bacterial 23S ribosomal RNA (rRNA) |
a TLR1 and TLR6 function only as a heterodimer with TLR2. Murine TLR8 recognizes synthetic RNA analogue complexes (poly(T)/imidazoquinoline), but not other human TLR8 agonists. TLR10 is present only in humans and TLR11, TLR12, and TLR13 are present only in mice.
Toll-like receptor 7 (TLR7)
TLR7 is expressed in intracellular compartments and mediates the recognition of guanosine- and uridine-rich ssRNA and ssRNA viruses, including influenza and vesicular stomatitis virus. TLR7 also recognizes synthetic antiviral nucleoside analogs such as imiquimod (R848), gardiquimod, and loxoribine. TLR7 is highly expressed in human plasmacytoid dendritic cells (pDCs), which secret high amounts of IFN-α on infection with ssRNA viruses.
In SLE, at least during the early phases of the disease, immune complexes are formed between antinuclear antibodies and RNA or DNA, followed by cell internalization (via B cell receptors and Fcγ receptors), thereby leading to the activation of TLR, mainly TLR7 in plasmacytoid dendritic cells. TLR7 has been shown to recognizes ssRNA released from dead and dying cells in an inducible mouse model of SLE and in doing so drive DC activation. Serologically, the hallmark of SLE is a high level of antinuclear antibodies (ANA) present in nearly all affected individuals (95%–99%), with known target antigen specificities of these ANA toward DNA itself and/or nuclear proteins known to form complexes with DNA (i.e., histones) or RNA (i.e., Ro, La, Sm, RNP, others). The Smith antigen is a complex of uridine-rich small nuclear RNA (snRNA) molecules and several proteins. Several studies have demonstrated a role for snRNA-containing autoantibody complexes in the pathogenesis of SLE. In addition, a correlation between the levels of RNA-containing autoantibody complexes in serum and the disease severity has been noted. Furthermore, snRNA-containing immune complexes (ICs) isolated from SLE patient serum are taken up through Fc receptors and delivered to intracellular lysosomes where they stimulate TLR7 ( Fig. 20.2 ). snRNA-ICs can stimulate pDCs to produce inflammatory, immunoregulatory, and chemotatic cytokines. The specificity of TLR7 activation by snRNA-ICs was confirmed in DCs isolated from TLR7-deficient mice and by the demonstration that oligonucleotide-based inhibitors of TLR7 blocked snRNA-IC-induced IFN-α production by pDCs. Together these data suggest that snRNA molecules contained in circulating ICs found in the serum of SLE patients act as endogenous self-ligands for TLR7 and may initiate and/or exacerbate disease by inappropriately and chronically stimulating innate immune cells.
TLR7 expression in SLE
PBMCs isolated from SLE patients have been reported to express significantly higher levels of TLR7 mRNA than those from healthy controls. In addition, the levels of TLR7 expression on SLE patient PBMCs correlated with IFN-α gene expression. TLR7 expression is equivalent in cells isolated from female versus male individuals; however, TLR7 stimulation induced significantly more IFN-α production from female than male cells. While this observation was found in cells isolated from healthy subjects and needs to be repeated in cells isolated from SLE patients, it may provide an explanation for the nearly 10 times higher prevalence of SLE in females than in males. It may also help explain how SLE in remission can undergo flare-up as a result of viral infections such as CMV, EBV, and parvo B19 virus, in a mechanism that activates TLR7 thus leading to the production of type I IFNs.
TLR7 in murine lupus
In vitro experiments demonstrate that autoreactive B cells can bind self-RNA through the B-cell antigen receptor (BCR), which delivers it to endosomes or lysosomes, where it sequentially engages TLR7 ( Fig. 20.2 ). Engagement of TLR7 then leads to B-cell activation, proliferation, and autoantibody production. In this model, self-RNA is an autoantigen that acts as an adjuvant to trigger in this case an unwanted immune response ( Fig. 20.2 ). Moreover, TLR7 expression on B cells can be induced by engagement of the IFN-α/β receptor on B cells ( Fig. 20.2 ). The significance of TLR7 in SLE has been demonstrated in mice overexpressing TLR7. In 1979 the mouse strain BXSB/MP was demonstrated to develop a spontaneous lupus-like syndrome that was restricted to male mice. The susceptibility locus was mapped to the Y chromosome and called the Y-linked autoimmune accelerator (YAA) locus. Nearly 30 years later it has been shown that YAA is not a mutation but rather a duplication of X chromosomal DNA that was transposed to the Y chromosome. This duplicated 4 Mb gene segment contains TLR7 and 16 other genes. The published data support a role for TLR7 as the main gene responsible for the accelerated lupus-like disease, because reduction of TLR7 copy number abrogated the YAA phenotype. Furthermore, experiments in transgenic mice demonstrated that increasing TLR7 gene dosage directly correlated with the production of autoantibodies directed against RNA autoantigens and the severity of the autoimmune disease. These data suggest that strict regulation of TLR7 expression and function is critical (at least in mice) for preventing spontaneous autoimmunity. Studies examining the copy number of the TLR7 gene in human SLE patients and healthy controls have not revealed a significant association with the SLE phenotype. The YAA mice expand B cells at T1 stage, produce class-switched autoantibodies, and severe lupus manifestations. Overexpression of TLR7 in murine lupus also results in functioning plasmacytoid dendritic cells as a result of diminished expression of the transcriptional factor Tcf4, expansion of T1 B cell-intrinsic and autoantibody production. Further, increased expression of TLR7 in B cells leads to both B and T cell activation, the development of antibodies against RNA-protein complexes, and disease exacerbation. Severe manifestations of lupus are displayed when TLR7 is overexpressed in B cells of Sle1-expressing mice. TLR7 expression has also been linked to cardiac neonatal lupus because its capacity to sense immune complexes containing Y RNA. Specifically, immune complexes between Ro60 ribonucleoprotein and anti-Ro60 antibodies result in TLR7 stimulation and lead to the production of TNFα in macrophages.
To test the involvement of TLR7 in the development of SLE, in vivo the lupus-prone MRL lpr/lpr mice were crossed to TLR7-deficient mice. These mice failed to generate autoantibodies to RNA-associated antigens (Sm), but produced levels of anti-DNA autoantibodies typical for the MRL lpr/lpr model. In addition, MRL lpr/lpr mice deficient in TLR7 had less activated B cells and pDCs in circulation and significantly less renal disease. In a separate study, mice engineered to express an autoreactive immunoglobulin that binds to the autoantigens ssDNA, ssRNA, and nucleosomes were created on a nonautoimmune background (C57Bl/6) called 546Igi mice. The B cells in 564Igi mice escape anergy and tolerance and produce pathogenic class-switched autoantibodies that result in kidney disease. Notably, anti-RNA autoantibodies are absent in 564Igi mice deficient in TLR7. Together these observations confirm the model that B-cell activation and regulation of autoantibody production against self-RNA-associated antigens are TLR7 dependent. Expression of TLR7 by B cells mediates aberrant immune cell activation, autoantibody production, and inflammation. B-cell-intrinsic TLR7 signaling promotes expansion and autoantibody production by transitional T1 B cells and the development of spontaneous germinal centers. Thus, TLR7 expression by B cells permits the specific development of autoantibodies to RNA-associated protein complexes that activate TLR7 via a feedback loop, which exacerbates SLE disease. Finally, the expression of TLR7 on B cells requires the expression of the cytosolic RNA sensor mitochondrial antiviral signaling protein (MAVS), also known as VISA, IPS-1, and CARDIF, suggesting that MAVS-mediated signaling may play a role in autoimmunity.
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