Role of Innate Immunity in Heart Failure

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.

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.