Toll Receptors

The toll receptor was originally discovered as a protein that was responsible for dorsoventral polarity in the fly. Subsequent studies demonstrated that the human homolog of the Drosophila toll protein was sufficient to activate NF-κB-dependent genes in mammalian cells. At the time of this writing, 13 mammalian TLR paralogs have been identified, of which 10 functional TLRs have been identified in humans (functional TLRs 11–13 are only expressed in mice). TLRs 1 to 6 are expressed on the cell surface of mammalian cells, whereas TLR 3, 7, and 9 are expressed in intracellular compartments, primarily endosomes and the endoplasmic reticulum, with the ligand-binding domains facing the lumen of the vesicle. TLR10 is the most recent member of the human TLR receptor family discovered; however, its function and direct ligand are still unknown. Humans also encode a TLR11 gene, but it contains several stop codons, and the protein is not expressed.

Messenger RNA (mRNA) for TLRs 1 to 10 have been identified in the human heart. Of note, the relative expression levels of mRNA for TLRs 2, 3, and 4 is approximately 10-fold higher than TLRs 1, and 5 to 10. Although expression levels of TLRs have not been identified in human myocytes, TLR2, 3, 4, and 6 mRNA have been identified in cardiac myocytes from neonatal rats. Although less is known regarding the regulation of TLR expression in heart failure, the experimental literature suggests that sustained activation of TLR signaling following cardiac injury is maladaptive and can lead to heart failure. Two studies have shown that TLR4 expression is increased in the hearts of patients with advanced heart failure. Moreover, the pattern of TLR4 expression in cardiac myocytes differs in heart failure, in that there are focal areas of intense TLR4 staining in failing cardiac myocytes, in contrast to the diffuse pattern of TLR4 staining observed in nonfailing myocytes.

As shown in Fig. 7.2A , the signaling pathway that is used by the TLR family of receptors is highly homologous to that of the interleukin-1 receptor (IL-1R) family (see below). TLRs are type 1 membrane-spanning receptors that have a leucine-rich repeat (LRR) extracellular motif and an intracellular signaling motif that is similar to IL-1. With the exception of TLR3, all TLRs interact with an adaptor protein termed MyD88 (myeloid differentiation factor 88) via their Toll interleukin receptor (TIR) domains ( Fig. 7.2B ). MyD88-dependent signaling through TLR2 and TLR4 requires an adaptor protein termed TIRAP (TIR domain-containing adaptor protein) to initiate signaling. When stimulated, MyD88 sequentially recruits IL-1 receptor associated kinases 4, 1, and 2 (IRAK4, IRAK1, and IRAK2) to the receptor complex. Phosphorylation of IRAK1 on serine/threonine residues by IRAK4 results in recruitment of tumor necrosis receptor-associated factor 6 (TRAF6) to the complex, which is responsible for early responses to TLR signaling. More recent studies have suggested an important role for phosphorylation of IRAK2 by IRAK4 in terms of mediating late responses to TLR signaling. Phosphorylated IRAK1 and TRAF6 dissociate from the receptor and form a complex at the plasma membrane with transforming growth factor-activated kinase 1 (TAK1), a mitogen-activated protein kinase kinase kinase, as well as TAK1-binding protein 1 (TAB1) and TAK1-binding proteins 2 or 3 ( TAB2 or TAB3 ), resulting in the phosphorylation of TAB2 /3 and TAK1. IRAK1 is degraded at the plasma membrane, and the remaining complex (consisting of TRAF6, TAK1, TAB1 and TAB2 or TAB3 ) translocates to the cytosol, where it associates with the ubiquitin ligases ubiquitin conjugating enzyme 13 (UBC13) and ubiquitin-conjugating enzyme E2 variant 1 (UEV1A). This leads to the ubiquitylation of TRAF6, which induces the activation of TAK1. TAK1 subsequently phosphorylates IκB kinase (IKK) α/IKKβ/IKKγ (also known as IKK1, IKK2, and NF-κB essential modulator [NEMO], respectively) and mitogen-activated protein kinase kinase 6 (MP2K6, MKK6, MEK6). The IKK complex then phosphorylates IκB, which leads to its ubiquitylation and subsequent degradation. This allows NF-κB to translocate to the nucleus and to induce the expression of its target genes.

Toll-like receptors ( TLR s) structure and signaling. (A) TLRs and interleukin-1 (IL-1) receptors have a conserved cytoplasmic domain that is known as the Toll/IL-1 R domain. The TIR domain is characterized by the presence of three highly homologous regions (known as boxes 1, 2, and 3). Despite the similarity of the cytoplasmic domains of these molecules, their extracellular regions differ markedly: TLRs have tandem repeats of leucine-rich regions (known as leucine rich repeats, LRR ), whereas IL-1 Rs have three immunoglobulin ( Ig )-like domains. (B) Stimulation of TLRs triggers the association of MyD88, which in turn recruits IRAK4, thereby allowing the association of IRAK1. IRAK4 then induces the phosphorylation of IRAK1. TRAF6 is also recruited to the receptor complex, by associating with phosphorylated IRAK1. Phosphorylated IRAK1 and TRAF6 then dissociate from the receptor and form a complex with TAK1, TAB1, and TAB2 at the plasma membrane (not shown), which induces the phosphorylation of TAB2 and TAK1. IRAK1 is degraded at the plasma membrane, and the remaining complex (consisting of TRAF6, TAK1, TAB1, and TAB2 ) translocates to the cytosol, where it associates with the ubiquitin ligases UBC13 and UEV1A. This leads to the ubiquitination of TRAF6, which induces the activation of TAK1. TAK1, in turn, phosphorylates both MAP kinases and the IKK complex, which consists of IKK-α, IKK-β and IKK-γ (also known as IKK1, IKK2, and NEMO, respectively). The IKK complex then phosphorylates IκB, which leads to its ubiquitination and subsequent degradation. This allows to the nucleus and induce the expression of its target genes. The MyD88 dependent pathway is used by TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9. TIRAP, a second TIR-domain-containing adaptor protein, is involved in the MyD88-dependent signaling pathway through TLR2 and TLR4. In contrast, TLR3- and TLR4-mediated activation of interferon (IFN) -regulatory factor 3 (IRF3) and the induction of IFN-β occur in a MyD88-independent manner. As shown, a third TIR-domain-containing adaptor, TRIF, is essential for the MyD88-independent pathway through TLR3 and TLR4. TRAM, a fourth TIR-domain containing adaptor, is specific to the TLR4-mediated, MyD88-independent/TRIF-dependent pathway. TRIF mediates the activation of the noncanonical IKKs, IKK-ε, and TBK1, and MAP kinase. Note TLR3 is predominately located within endosomes (not illustrated).

Modified from Akira S, Takeda K. Toll like receptor signaling. Nat Rev Immunol. 2004;7:499–511.

TLR4 also can signal through a MyD88 independent pathway by recruiting the adaptor proteins TRIF-related adaptor molecule (TRAM) and TIR-domain-containing adaptor-inducing interferon-β (TRIF) to the receptor complex ( Fig. 7.2B ). TRIF recruits the noncanonical IKKs, the serine-threonine-protein kinase TANK-binding kinase-1 (TBK1) and IKKε, which phosphorylate the transcription factor interferon regulatory factor 3 (IRF3), thereby inducing interferon-β and co-stimulatory interferon-inducible genes. TRIF also recruits TRAF6 and RIP-1, which leads to activation of MAPK and IKKα/IKKβ. These class-specific TLR signaling cascades allow different TLRs to trigger distinct signaling pathways and elicit distinct actions in a cell-specific manner.

TLRs signal by forming homo- or heterodimers, which allows for approximation of the TIR domains, creating “docking” platforms for the recruitment of adaptor proteins and kinases that activate downstream signaling cascades. TLR2 and TLR6 are capable of forming heterodimers or homodimers, whereas TLR3 and 4 signal by forming homodimers. Three general categories of TLR ligands have been identified, including proteins (signal through TLR5), nucleic acids (signal through TLR3, TLR7, TLR9), and lipid-based elements (signal through TLR2, TLR4, TLR6, TLR2/TLR6). Although gram-negative and gram-positive bacteria have been shown to signal through TLR4 and TLR2 in the heart, respectively, the exact ligands that activate TLR signaling in the heart following tissue injury are not known. As noted previously, TLR receptors are activated by proteins released by damage-associated molecular patterns released by injured and/or dying cells, as well as by fragments of the extracellular matrix (see Fig. 7.1 ).

Given the importance TLR signaling, it is not surprising that nature has evolved multiple pathways to negatively regulate TLR signaling. TLR-signaling pathways are negatively regulated by several molecules that are induced following stimulation of TLRs, including IL-1-receptor-associated kinase M IRAK-M, suppressor of cytokine signaling 1 (SOCS1), and Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) a phosphatase that hydrolyzes the 5′phosphate of PI-3,4-P2, which inhibits PI3 kinase-dependent TLR-MyD88 interactions and NF-kβ activation, and thus negatively regulates TLR signaling. TRIM30α destabilizes the TAK1 complex by promoting the degradation of TAB2 and TAB3, whereas myeloid differentiation primary-response protein 88 short (MyD88s), an alternatively spliced variant of MyD88, blocks the association of IRAK4 with MyD88. Sterile-alpha and Armadillo motif containing protein (SARM) is a novel adaptor protein that specifically blocks TRIF-dependent but not MyD88-dependent signaling. TOLLIP (Toll interacting protein) is thought to maintain immune cells in a quiescent state and/or terminate TLR-mediated signaling, by interacting with the cytoplasmic TIR domains of TLR2 and TLR4 and suppressing IRAK1phosphorylation. Finally, the TIR (Toll/IL-1R)-domain-containing receptors single immunoglobulin IL-1 receptor related (SIGIRR) molecule and ST2 have also been shown to negatively regulate TLR signaling. Another highly conserved mechanism for regulating innate immunity is being revealed for microRNAs, so-called immuno-miRs, that regulate innate immune gene expression by preventing mRNA translation by promoting mRNA degradation.

Role of Toll-like Receptors in Myocardial Disease

Deciphering the role that the innate immune system plays in myocardial disease has been challenging, insofar as it has been difficult to reconcile two sets of conflicting observations, one of which suggests that TLR signaling is beneficial, and the other of which suggests that TLR signaling following ischemic injury is deleterious. Recent “reductionist” studies that have been performed ex vivo, or that have employed chimeric TLR-deficient mice that harbor wild-type bone marrow cells have allowed for a clearer understanding of the central (i.e., myocardial) and peripheral (i.e., bone marrow-derived) effects of the innate immune system following ischemic injury. The aggregate data suggest that short-term activation of TLR signaling confers cytoprotective responses within the heart, whereas longer-term TLR signaling is maladaptive and results in the upregulation of proinflammatory cytokines and cell adhesion molecules, which leads to activation and recruitment of the “peripheral” neutrophils, monocytes, and dendritic cells to the myocardium, resulting in increased cell death and adverse cardiac remodeling. The sections that follow will focus on the deleterious long-term effects of the activation of innate immunity in the heart.

Toll-like Receptor Signaling in Ischemia Reperfusion Injury and Myocardial Infarction

TLR-mediated signaling contributes to myocardial damage and adverse cardiac remodeling following ischemia reperfusion injury and/or myocardial infarction. Traditional “loss of function studies” in experimental heart failure models in mice and rats suggest that sustained TLR activation of TLRs is maladaptive and can contribute to LV dysfunction and adverse cardiac remodeling ( Table 7.2 ). Mice with a missense mutation of TLR4 or targeted disruption of TLR4, TLR2, or MyD88 ²⁵ have reduced infarct sizes when compared to wild-type controls. Moreover, mice pretreated with a TLR4 antagonist (Eritoran) had reduced nuclear translocation of NF-κB, decreased the expression of proinflammatory cytokines (e.g., IL-1, IL-6, TNF), and smaller infarct sizes when compared to vehicle treated animals. Mortality and LV remodeling are reduced in mice with targeted disruption of TLR4 or TLR2. Studies performed ex vivo in TLR2-deficient mice suggest that the LV dysfunction that supervenes following I/R is mediated through TLR2-TRAP-mediated upregulation of TNF.

Functional Role of Toll-like Receptor Signaling in Human Heart Failure

The experimental literature reviewed above suggests that sustained activation of TLR signaling following cardiac injury is maladaptive and can lead to a heart failure phenotype. Unfortunately, very little is known with respect to the role of the innate immune system in the failing human heart. To date, two studies have shown that TLR4 expression is increased in the hearts of patients with advanced heart failure. Moreover, the pattern of TLR4 expression in cardiac myocytes differs in heart failure, in that there are focal areas of intense TLR4 staining in failing cardiac myocytes, in contrast to the diffuse pattern of TLR4 staining observed in nonfailing myocytes. To further clarify the role of innate immunity in the failing heart, a recent study examined the expression profiles of 59 innate immune genes using gene arrays that were from explanted hearts from patients with ischemic cardiomyopathy (ICM), idiopathic dilated cardiomyopathy (DCM), and viral cardiomyopathy (VCM); gene arrays from nonfailing hearts were used as the appropriate controls. This bioinformatic study showed that there were distinct gene expression profiles for innate immune genes in failing and nonfailing hearts, and that there were distinct gene expression profiles for innate immune genes in ICM and DCM hearts. Although these provisional studies suggest that the innate immune system is activated in human heart failure, it will be important to more precisely determine the expression levels of the different components of the innate immune system, as well as link activation of the innate immune system to the development and progression of heart failure.

NOD Receptors

NLRs have emerged as important PRR superfamily members in health and disease. NLRs cooperate with TLRs in regulating inflammatory and cellular homeostatic responses. The canonical structure of the NLR consists of three domains, including a central NACHT (NOD nucleotide-binding domain) domain, which is common to all NLRs, and is responsible for ATP mediated oligomerization. As shown in Fig. 7.3 the majority of NLRs contain C-terminal LRRs, which detect the presence of cognate ligands. The N-terminal domain is responsible for homotypic protein–protein interaction and consist of either a caspase recruitment domain (CARD), a pyrin domain (PYD), or an acidic transactivating domain or baculovirus inhibitor repeats (BIRs) domain.

(A) Schematic representation of individual nod-like receptor (NLR) domains. Human NLRs are sub-classified into five categories: NLRA, NLRB, NLRC, NLRP, and NLRX. All 22 human NLRs contain a central NACHT domain and a C-terminal ligand-sensing LRR domain, with the exception of NLRP10. The N-terminal domains ascribe functional properties to the NLRs; however, the function of some of the domains is still unclear. ATD , Acidic transactivation domain; BIR , baculoviral inhibition of apoptosis protein repeat domain; CARD , caspase association and recruitment domain; FIIND , function to find domain; LRR , leucine-rich repeats; MT , targets NLRX1 to the mitochondria; PYD , pyrin domain. (A, from Saxena M, Yeretssian G. NOD-Like Receptors: Master Regulators of Inflammation and Cancer. Front Immunol. 2014;5:327). (B) Pathways for formation of the NLRP3 inflammasome. The formation of the inflammasome in the heart requires two independent steps: priming and triggering. The priming signal is dependent on the activity of nuclear factor (NF) -κB through stimulation of the membrane Toll-like receptors (TLRs) and the downstream signaling mediated by myeloid differentiation primary response protein MyD88 and interleukin-1 (IL-1) receptor-associated kinases (IRAKs) . The TLRs sense extracellular threats through damage-associated molecular patterns (DAMPs) and prime the cells to respond to the potential injurious conditions by increasing the transcription and translation of inflammasome components and the associated cytokines (IL-1β and IL-18). These cytokines function in a paracrine fashion on the membrane cytokine receptor and converge on the same pathways to amplify the signal. A similar priming effect is mediated by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) . The primary signal is necessary but insufficient to form the inflammasome in cardiomyocytes in the absence of the trigger signal. The activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) is mediated by extracellular and/or intracellular pathways. The increase in extracellular ATP (eATP) activates the P2X purinoreceptor 7 (P2X7) and leads to K+ efflux, a step that triggers NLRP3 activation. Lysosomal destabilization by indigestible material is another mechanism that leads to NLRP3 activation by leakage of the lysosomal enzyme cathepsin B and by induction of K+ efflux. The serine/threonine–protein kinase Nek7 senses the K+ efflux and binds NLRP3, allowing its activation. Thioredoxin-interacting protein (TXNIP) links oxidative stress and the unfolded protein response to NLRP3 activation. TXNIP is freed from thioredoxin (TRX) and binds NLRP3. The mitochondria also have an important role in producing reactive oxygen species (ROS) . Furthermore, ineffective clearance of mitochondrial debris by mitophagy contributes to lysosomal destabilization. By contrast, effective mitophagy and autophagy suppress the activation of NLRP3. The tyrosine–protein kinase BTK also binds NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) , leading to inflammasome activation after ischemia. The active NLRP3 oligomerizes, forming a circular structure that functions as a platform for the polymerization of ASC into filaments, which in turn work as a central core from which caspase 1 filaments branch, forming a star-like structure. Active caspase 1 cleaves the inactive pro-IL-1β and pro-IL-18 into the active forms, IL-1β and IL-18. Gasdermin D (GSDMD) is an additional substrate of caspase 1 that oligomerizes upon cleavage and forms pores in the cell membrane with the N-terminal fragments, which allow the extracellular release of active IL-1β and IL-18.

B, modified from Toldo S, Abbate A. The NLRP3 inflammasome in acute myocardial infarction. Nat Rev Cardiol. 2018;15:203–214.

Five subfamilies of NLRs can be distinguished based on their N-terminal effector domains, which impart unique functional characteristics to each NLR. The founding NLR members are NOD1 (NLRC1) and NOD2 (NLRC2), which are part of the larger NLRC subfamily (NLRC 1–5). NLRCs are characterized by the presence of a CARD domain that allows for direct interaction between members of this family and other CARD carrying adaptor proteins. NOD1 (NLRC1) and NOD2 (NLRC2) serve as important sensors of bacterial peptidoglycan (PGN) and are crucial for tissue homeostasis and host defense against bacterial pathogens (48). NLRs with an N-terminal acidic transactivation domain are termed NLRA (CIITA), and serve as transcriptional regulators of MHC class II antigen presentation (44). NLRB (NAIP) proteins have a BIR domain, and play important roles in host defense and cell survival. NLRX1 is the only described member of the NLRX subfamily that contains an N-terminal mitochondria-targeting sequence required for its trafficking to the mitochondrial membrane ( Fig. 7.3A ). Members of the PYD-containing NLRP subfamily (NLRP 1–14) are best known for their role in inducing the formation of the oligomeric inflammatory complex termed the “inflammasome.” The inflammasome ( Fig. 7.3B ) is composed of an NLRP, the proinflammatory caspase-1, and the apoptosis-associated speck-like protein (ASC). The ASC contains an N-terminal PYD and a C-terminal CARD, which enables it to bring caspase-1 and NLRPs into close proximity. Upon activation, NLRP3 recruits ASC and caspase-1, which is required for the cleavage and maturation of the inflammatory cytokines IL-1β and IL-18. More recently, a more complex model for NLRP3-inflammasome activation has been proposed where two adaptors, ASC and mitochondrial antiviral signaling (MAVS) protein, are required for optimal inflammasome triggering.

The NLRP3 inflammasome has been shown to play an important role in the heart following tissue injury ( Fig. 7.3B ). The NLRP3 and other important key components of the inflammasome are not constitutively expressed in cardiac myocytes, but can be upregulated in leukocytes and cardiac myocytes by proinflammatory cytokines and/or DAMPs or PAMPs. After the inflammasome is “primed,” a second “trigger” is required for the activation of the NLRP3, which is largely, but not exclusively, dependent on the intracellular K+ concentration. As shown in Fig. 7.3B , lysosomal destabilization activates a signaling pathway that indirectly leads to increased membrane permeability and K+ efflux. Leakage of cathepsin B from lysosomes also has been shown to activate the inflammasome. Additional triggers include extracellular ATP binding to the P2X purinoreceptor 7 (P2X7), which triggers K+ efflux, leading to a conformational change in NLRP3 that allows for recruitment of ASC ( Fig. 7.3B ). The NLRP3 inflammasome is critically involved in the response to injury during acute myocardial infarction (AMI), and is responsible for the production of IL-1β and the ensuing systemic inflammatory response. Experimental studies in small and large animals show that NLRP3 inflammasome-targeted therapies might be a viable strategy for the reduction of infarct size and the prevention of heart failure following AMI (reviewed in reference ).

Other Pattern Recognition Receptors

The pentraxins, including pentraxin3 (PTX3), serum amyloid P (SAP), and C-reactive protein (CRP), are involved in innate immunity and inflammatory signaling in cardiovascular disease. Pentraxins are useful biomarkers for ischemic heart disease and heart failure. Elevated levels of CRP provide important prognostic information for a variety of clinical settings and have been used to identify patients with coronary artery disease who are more likely to have cardiac events. CLRs are a family of proteins with one or more C-type lectin domains, which require calcium for binding. CLRs display a diverse range of functions, including innate immune response, cellular apoptosis, and cell–cell adhesion. Very little is known about the role of C-type lectins and cardiac function. RIG-I-like receptors (retinoic acid-inducible gene-I-like receptors [RLRs]) are cytosolic innate immune sensors that detect pathogenic RNA and trigger a systemic antiviral response. RLRs cooperate in signaling crosstalk networks with TLRs and activate the innate immunity, as well as modulate adaptive immune response. There are three RLRs: RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). Overexpression of MDA5 in the heart has been shown to protect the heart from viral injury.