TLR receptors, ligands, and signaling pathways

TLRs are a family of transmembrane receptors that play a key role in innate immunity. TLRs prevent pathogenic invasion by recognizing pathogen-associated molecular patterns (PAMPs), which are highly conserved components derived from a range of microbial agents, as well as endogenous macromolecules released by injured tissue.20 To date, ten TLRs have been identified in humans. TLR localization is heterogeneous and varies from the cell surface (TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, and mouse TLR11 and TLR12) to the endosomes (TLR3, TLR7, TLR8, and TLR9),21 according to PAMP localization. The two most eagerly studied TLRs are TLR2 and TLR4, the pattern-recognition receptors for gram-negative and -positive bacterial products, respectively. TLR4 responds to various invading exogenous pathogens through PAMPs and recognizes the endogenous ligands from necrotic cells through damage-associated molecular patterns (DAMPs), including high-motility group box-1 (HMGB1), heat shock protein (HSP), fibrinogen, heparin sulfate, fibronectin, hyaluronic acid, and double- and single-strand RNA.22 While these molecules are released from necrotic cells, they promote tumor cell survival by activating the TLR4 expressed on tumor cells and subsequently upregulating nuclear factor-kappa B (NF-κB) signaling and antiapoptotic proteins. One study found that the levels of these DAMP-derived molecules were high in the tumor microenvironment and that they induced TLR4-related chronic inflammation, leading to carcinogenesis, cancer progression, and metastasis.23 Due to alterations in gene expression and the microenvironment (such as hypoxia and malnutrition) of malignant tumors, HMGB1, as well as hyaluronan expression and secretion in tumors and inflammatory cells, are generally enhanced, which plays a potential role in tumorigenesis, growth, infiltration, and metastasis.24–27 For these reasons, we will further discuss these two endogenous TLR ligands in the context of HCC.

HMGB1 is a DNA-binding protein released from dead cells or secreted by macrophages and hepatocytes at the tumor site that is involved in gene transcription, DNA recombination and repair, and nucleosome remodeling and stabilization.28 Importantly, extracellular HMGB1 released or secreted by damaged cells has been suggested to be a signature DAMP that indicates the presence of necrosis and subsequently triggers inflammation, cell proliferation, and migration.29 Recent data suggest that HMGB1 facilitates and amplifies the inflammatory response to different cytokines and TLR agonists.30,31 Two recent studies found that HMGB1 was highly expressed in HCC tissues and cell lines, induced caspase 1 activation via the TLR4/receptor for advanced glycation endproducts (RAGE) signaling pathway, and mediated the production of various inflammatory cytokines that promote tumor invasion and metastasis.32,33 Hyaluronan is a negatively charged glycosaminoglycan with a high molecular weight that is ubiquitously distributed in the extracellular matrix. When inflammation or cell damage occurs, hyaluronan can be broken down to fragments via reactive oxygen species, which can then be recognized by TLR4 and leads to the activation of immune cells and the inflammatory response. Hyaluronan is able to stimulate chemokine production in peritoneal macrophages in the absence of CD44 through a TLR2- and TLR4-dependent mechanism.34 As a major component of the outer membrane in gram-negative bacteria, lipopolysaccharide (LPS) can bind TLR4 and trigger the downstream signaling pathways.35,36 Ligand-bound TLR4 signals through two pathways. In the myeloid differentiation factor 88 (MyD88)-dependent pathway, the activated TLR4 recruits the TIR domain-containing adaptor protein and MyD88, which subsequently triggers the downstream adapter proteins interleukin-1 receptor-associated kinase 4, tumor necrosis factor (TNF) receptor-associated factor 6, and transforming growth factor (TGF)-β-activated kinase 1, which further activates the I kappa B kinase–NF-κB and mitogen-activated protein kinase (MAPK) pathways, leading to the expression of proinflammatory cytokines. In the MyD88-independent pathway, TLR4 recruits TIR domain-containing adapter-inducing interferon-β (TRIF) and the TRIF-related adapter molecule. TRIF indirectly activates transcription factor interferon regulatory factor (IRF)3 and NF-κB, which eventually leads to the transcription of type I interferon and interferon-related genes to contribute to antibacterial and antiviral immunity (Figure 1).37 TLR stimulation leads to the activation of NF-κB, MAPK, Jun N-terminal kinases, P38, and extracellular signal-regulated kinase, as well as interferon regulatory factor (IRF3, IRF5, and IRF7) signaling pathways, which results in the production of inflammatory cytokines.38,39 TLR activation in antigen-presenting cells also triggers adaptive immunity.

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TLR4 expression in HCC and hepatocarcinogenesis

In recent years, TLR expression in tumor tissue has been reported, which may provide an important mechanism in the recruitment of inappropriate immune enhancement and dysfunctional immunity and lead to antitumor immune tolerance.40–43 TLR4 is associated with cancer in several ways. Diverse cell lines and tissue samples derived from patients with head and neck, esophageal, gastric, colorectal, liver, pancreatic, skin, breast, ovarian, and cervical cancer have been shown to express increased amounts of TLR4.40 In these scenarios of established cancer, TLR4 facilitates an environment that is suitable for continued cancer cell proliferation. Procancer mechanisms could include the evasion of cancer cells from immune surveillance.14,41–43 The liver may be exposed to bacteria from the intestine via the portal vein, leading to an uncontrolled innate immune system that may result in inflammatory liver disorders.

Although intestinal bacteria maintain a dynamic balance and interact with the host under normal circumstances, the significant increase in Enterobacter during chronic liver injury becomes the key pathogenic factor.44 Many liver cells (Kupffer cells, hepatocytes, activated stellate cells) constantly express TLR4.45–48 Kupffer cells are specialized macrophages located in the liver. Kupffer cells are known to promote hepatic stellate cell (HSC) activation and fibrosis. Fibrosis will eventually cause cirrhosis, or loss of function of the liver.49,50 Hepatocytes that express TLR4 are responsive to LPS. Hepatocytes are also believed to play a role in the uptake of endotoxin.51 In response to liver injury, activated HSCs become the major extracellular matrix-producing cell type in the liver. HSCs interact with Kupffer cells and hepatocytes to promote liver fibrosis and inflammatory responses.52 TLR4 expression was significantly elevated in HBV- and HCV-infected livers,53,54 as well as in HCC tissues.55,56 Jing et al57 detected TLR4 expression in 106 cases of HCC patients’ tissue samples; of them, 101 (86%) showed TLR4 positivity. Further studies confirmed that LPS induced the TLR4 signaling that is involved in the invasion and metastasis of HCC and suggested that NF-κB may be directly activated by TLR4 signaling.57