Potential conflict of interest: Nothing to report.
Pattern recognition receptors (PRRs) function as sensors of microbial danger signals enabling the vertebrate host to initiate an immune response. PRRs are present not only in immune cells but also in liver parenchymal cells and the complexity of the cell populations provide unique aspects to pathogen recognition and tissue damage in the liver. This review discusses the role of different PRRs in pathogen recognition in the liver, and focuses on the role of PRRs in hepatic inflammation, cholestasis, ischemia, repair and fibrosis. PRRs as novel therapeutic targets are evaluated. (HEPATOLOGY 2006;44:287–298.)
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The fundamental role of the mammalian innate immune system to sense invading pathogens relies on pattern recognition receptors (PRR).1 PRRs are germline encoded, constitutively expressed molecules that recognize pathogen-associated molecular patterns (PAMPs) that are essential for the survival of the microorganism but not present in eukaryotes.1, 2 Different PRRs recognize specific PAMPs, show distinct expression patterns, activate specific signaling pathways and trigger anti-pathogenic host responses. Toll-like receptors (TLRs) recognize microbes either on the cell surface or on lysosome/endosome membranes, while pathogens that invade the cytosol are detected by cytoplasmic PRRs, including the Nucleotide-binding Oligomerization Domain-Leucine-Rich Repeat (NOD-LRR) proteins, Peptidoglycan Recognition Proteins (PGRP) and the caspase recruitment domain (CARD)-helicase proteins.1–6 Recent studies suggest that PRRs may also recognize damage-associated molecular pattern molecules (DAMPs) from dying host cells2 and may induce inflammatory responses in the absence of microbes. Thus, PRRs play a central role in host homeostasis by recognizing both exogenous pathogen-derived and endogenous danger signals.
All of the ten TLRs discovered in humans contain a conserved intracellular TIR (Toll/ interleukin-1 receptor) domain but the extracellular domain is unique to the individual TLR as it confers specificity for ligand recognition.7 TLRs1, 2,4,5,6 and 10 are expressed on the cellular membrane, while TLRs 3,7, 8 and 9 are found in the endosomal compartment allowing site-specific recognition of pathogens.7, 8
Of the surface expressed TLRs, TLR4 is a major component of the LPS recognition receptor complex, which also involves the co-receptors CD14 and MD-2, and LPS-binding protein (LBP).9, 10 Recent reports revealed that while MD-2 is critical in LPS recognition by TLR4, LBP acts as a LPS-binding unit.9 CD14 facilitates TLR4 induced responses11, 12 and appears to be required for MyD88-independent signaling.13TLR2 is unique due to its diverse ligand recognition profile and its ability to form homodimers or heterodimers with TLR1 and TLR6 and TLR 10.14–16 Among the TLR2 ligands are protozoa, bacteria, fungi and viruses. TLR2 triggers the MyD88-dependent downstream signaling involving the IκB/NFκB pathway.17 Alternatively, TLR2 tyrosine phosphorylation at the intracellular domain can trigger a signaling cascade composed of RacI, PI3K and Akt to target nuclear p65 transactivation independent of IκBα degradation.18 TLR2-mediated cell activation leads to generation of both pro- and anti-inflammatory cytokines but not of type 1 IFNs.11, 12TLR5, a pattern recognition receptor that recognizes flagellin, signals via the MyD88-dependent cell activation pathway.19 The intracellular TLRs, (TLR3, TLR7, TLR8 and TLR9) sense nucleic acids.20TLR3 is expressed in the endosomes of dendritic cells but also found on the cell surface in fibroblasts.3 TLR3 recognizes double-stranded RNA (dsRNA) derived from virus, host mRNA or a synthetic analog polyriboinosinic: polyribocytidylic acid [poly (I: C)].21, 22 Upon ligand engagement, TLR3 is phosphorylated at 2 tyrosine residues and phosphatidyl-inositol 3-kinase (PI3K) is recruited,23 which, together with TRIF, is instrumental for full activation of IRF324 and production of type 1 IFNs. TRIF also associates with receptor-interacting protein 1 (RIP1), leading to NFκB activation, thereby linking TLR3 to the apoptotic cascade via association of the death domain of RIP-1 with caspase 8.25TLR7 and TLR8 recognize unmethylated viral or synthetic ssRNA26 and double-stranded, short-interfering RNA (siRNA).27TLR9 recognizes unmethylated CpG motifs of DNA.28 TLRs7, 8 and 9 are localized to the endosomes and share a common signaling pathway that requires MyD88 and endosomal acidification (maturation) for initiation of signaling.29–31 Interestingly, MyD88 can directly associate with and activate IRF7, leading to type 1 IFN production,32, 33 TLR7-, 8- and 9-induced activation seem to be cell-type specific, leading to high levels of type 1 IFN production in plasmacytoid dendritic cells, or pro-inflammatory cytokine production in myeloid dendritic cells and macrophages, respectively.34–36
Ligand recognition by TLRs triggers signaling from the cytoplasmic TIR domain via recruitment of different intracellular adaptors and culminates in activation of pro-inflammatory cytokines, co-stimulatory molecules, or type I interferons (IFN).11, 12 The specificity of TLR-mediated signaling is determined by the combination of the TLR involved in ligand recognition, its co-receptors, intracellular adaptor and intracellular signaling pathways (Fig. 1). Of the TLR adaptors identified to date, the myeloid differentiation factor 88 (MyD88), is common to all human TLRs except for TLR3 which exclusively utilizes TIR-domain containing adaptor inducing IFN-β (TRIF).12, 37, 38 TLR4 is unique in utilization of both TRIF (MyD88 independent) and MyD88 for downstream signaling.39–41
MyD88-dependent signaling is essential for the induction of pro-inflammatory cytokines.5, 11 MyD88-dependent signaling recruits members of the IL-1 receptor-associated kinase (IRAK) family and hyperphosphorylates IRAK1 leading to dissociation of IRAK1 from MyD88 and interaction with TNF receptor-associated factor 6 (TRAF6).17 IRAK1/TRAF6 form a complex with transforming growth factor beta (TGF-β)-activating kinase (TAK1) and TAK1-binding proteins (TAB) 1 and 2 leading to activation of IκB kinases (IKK) α and β, phosphorylation/degradation of IκB proteins to result in NFκB nuclear translocation and activation of NFκB-dependent genes.11, 17
The MyD88-independent signaling initiated by TLR3 or TLR4 activation leads to activation of IFN-inducible genes.11, 12, 17 The adapter molecule TRIF (and TRAM — TRIF-related adaptor molecule — for TLR4) is recruited and involves TBK1 to activate IRF3 and production of type 1 IFNs.40–43 These interferons then bind to the IFN receptor (IFNR), and initiate STAT-1-dependent activation of IFN-inducible genes, such as IP-10, IRG-1, GARG-16.44
NOD1 and NOD2, members of the NOD-LRR proteins expressed in immune cells recognize bacterial components, such as gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and myramyl dipeptide (MDP), that possesses LRRs and sense a nucleotide binding oligomerization domain (NOD), and a domain for the initiation of signaling such as CARDs (Fig. 2).45 NOD-ligand interaction induces NF-κB activation, leading to production of pro-inflammatory cytokines, and activation of caspase-1, and enzyme which catalyzes the processing of pro-interleukin-1β to produce mature cytokines.46 A missense mutation in the human NOD2 gene is correlated with susceptibility to Crohn's disease and the role of NOD-LRR proteins in the liver diseases is yet to be evaluated.3
Peptidoglycan Recognition Proteins
Peptidoglycan Recognition Proteins (PGRP) represent a novel family of four PRRs in humans (Fig. 2). PGRP-L is expressed in the liver, PGRP-Iα and PGRP-Iβ in the esophagus (and to a lesser extent in tonsils and thymus) and PGRP-S in the bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria and may play a role in recognition of bacteria in the liver.4 PGRPs trigger pro-inflammatory cytokine production via NFκB-dependent pathway. The exact cellular distribution of these PGRPs in the liver is yet to be determined.
Recognition of viruses by RNA helicases represents TLR-independent pathways that culminate in activation of TLR-related signaling molecules and play a role in viral hepatitis. The cytoplasmic protein retinoic acid-inducible gene I (RIG-I) is an RNA helicase with two caspase-recruiting domains (CARD)-like domains that interact with dsRNA and initiate downstream signaling leading to IRF3 and NF-κB activation.47–49 Melanoma differentiation associated gene 5 (Mda5) is a RIG-I-related protein that consists of CARD and a helicase domain, and functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with their helicase domain and transmit a signal downstream by CARD; thus, RIG1 and Mda5 share overlapping functions. The adaptor molecule that connects RIG-I and Mda5 sensing of viral RNA to downstream signaling has recently been identified by four independent research groups and has been named: MAVS, IPS-1, VISA and Cardiff.47–50 It has been shown that this adaptor expressed on mitochondria triggers RIG-1 and Mda5-mediated type I interferon induction.48 IPS-1 activates NF-κB and IPS-1 knockdown blocks interferon responses.48 Recent studies suggest that RNA helicases are targets of viral mechanisms for host invasion.
TLR-independent DNA Sensing
Newly discovered pathways in DNA recognition suggest that TLR-independent pathways are also activated in response to microbial and host nucleic acids.2, 51 Studies from deoxyribonuclease (DNase) II deficient mice demonstrated that accumulation of undigested DNA in liver-derived macrophages resulted in activation of a specific set of host–response genes including the IFNβ, TNFα and CXCL10 genes.51 This response was still maintained after ablation of TLR3 and TLR9 or their adaptor molecule, MyD88, indicating that existence of a TLR-independent sensing mechanism for endogenous DNA that escapes lysosomal degradation to activate innate immunity in the liver. Ishii et al. recently reported that intracellular administration of a double-stranded B-form DNA triggered antiviral responses independently of TLRs or the helicase RIG-I.52 The putative receptor involved in this process is yet to be identified.
PRRs in Liver Diseases
TLR Expression in the Liver.
The extent to which PRRs are expressed in the liver and their roles have yet to be completely delineated. The liver accommodates a network of parenchymal and non-parenchymal cells that express various TLRs53 (Fig. 3). Hepatocytes express mRNA for all TLRs.54, 55 Functional activity of TLR2 and TLR4 was suggested by hepatocyte uptake and clearance of endotoxin from the circulation.56–58 TLR2 or TLR4 ligands activated NFκB in primary hepatocytes,58 while in hepatocyte cell lines TLR3 stimulation with polyI:C resulted in activation of the type 1 IFNs.59 Intracellular TLR trafficking in hepatocytes was found to be similar to that seen in immune cells,60 as LPS challenge in vivo lead to accumulation of cytoplasmic TLR2 in vesicles near the hepatocyte plasma membrane.61 Endothelial cells, lining the hepatic sinusoids, constitutively express TLR4 and upregulate NFκB activity, produce pro-inflammatory cytokine (TNFα) and ROS in response to TLR4-mediated LPS stimulation.62, 63 Biliary epithelial cell lines (MMNK-1, MZChA-1 and H-69) and biliary epithelium in vivo expressed mRNA for all TLRs, the co-receptor, MD-2 and negative regulators of TLR signaling (Tollip, SIGGIR and ST-2).64, 65 TLR2, 3, 4 and 5, but not TLR7, 8, 9, were detected at the protein level and appeared functionally active based on ligand-induced IL-6, IL-8 and MCP-1 production in biliary epithelial cell lines.66, 67In vitro TLR mRNA expression and, to a lesser extent, protein levels were increased by Th1 cytokines (IFNγ and TNFα) in biliary cells.66, 67In vivo, however, LPS failed to elicit an inflammatory response in intrahepatic cholangiocytes.63 This defect in response to the TLR4 ligand, referred to as “TLR tolerance”, has been described in macrophages and represents a natural defense mechanism from gut-derived endotoxin.68, 69 Harada et al.70 identified that human cholangiocytes develop tolerance to TLR2 and TLR4 ligands via induction of IRAK-M, a negative regulator of TLR signaling.71 TLR4 upregulation was shown after infection with Cryptosporidium parvum that led to decreased expression of a micro RNA that is involved in post-transcriptional regulation of TLR4.72 Kupffer cells, the resident macrophages of the liver, express functional TLR2 and TLR4 and respond to their respective ligands resulting in NF-κB activation,61, 73 production of pro- and anti-inflammatory cytokines, and reactive oxygen species (ROS).74–76 In the space of Disse, TLR2 expression was found in the sinusoidal endothelium and in Kupffer cells after LPS stimulation.60 Mulder et al.77 recently reported that inhibition of endotoxin-induced effects in Kupffer cells can be achieved by inhibition of liver-X-receptor (LXR) α and β using targeted pharmacological intervention and may represent a novel strategy to treat inflammation-induced cholestasis. Stellate cells, the main extracellular matrix-producing cells of the liver, express TLR4, TLR2 and CD14 and respond to TLR2 and 4 ligands by activation of JNK, ERK and NFκB and pro-inflammatory cytokines production.78–81
The liver also accommodates a variety of immune cells that widely express TLRs. T lymphocytes and NK cells are rich in TLR1,2,4,5 and 9, while B cells express high levels of TLR1,6,7,9 and 10.82Dendritic cells can be of myeloid (MDC) or lymphoid (plasmacytoid, PDC) origin and represent a major component of innate immunity. While both recognize and present antigens to T cells, plasmacytoid and myeloid DCs are distinct in their TLR expression and cytokine production profile.82 Human PDCs express TLRs 7 and 9 and produce large amounts of IFNα. Myeloid DCs carry TLR2,3,4, and TLR9 and produce pro-inflammatory cytokines and IFNβ but not IFNα.43, 82–84
Acute Liver Failure.
Acute liver failure (ALF) closely resembles the clinical picture of endotoxin shock characterized by inflammatory cytokine induction, hepatocyte damage, hemodynamic compromise, and coma. The development of endotoxin shock is critically dependent on TLRs.85, 86 Activation of pro-inflammatory cytokines, including TNFα, interleukins-1, -6, -12, -18 and IFNγ are pivotal in the pathophysiology and clinical resolution of ALF. Recent reports suggest that TLRs play a major role in an animal model of ALF, elicited by sensitization with heat-killed Propionibacterium acnes (P. acnes) and challenged with the TLR4 ligand LPS.87–91 While P. acnes is recognized by TLR2,92 TLR2-deficient mice were not protected from P. acnes-induced sensitization to LPS.89 In contrast, TLR9 ligand administration induced liver granulomas and sensitization to LPS-induced liver injury.90 A recent report by Kalis et al. also suggested a role for TLR9 in liver sensitization to LPS93 and Romics et al. found that MyD88 expression was indispensable in this process.89 We found that priming with P. acnes or with TLR9 and TLR2 co-stimulation upregulates expression of TLR4 and MD-2 and set the stage for LPS-induced liver injury.89, 90 Consistent with this observation, in isolated Kupffer cells, TLR9 pre-stimulation resulted in sensitization to LPS-induced TNFα production suggesting that TLR9 ligands may sensitize the liver to subsequent stimulation via TLR4.94 In contrast to TLR9, pretreatment with a TLR3 ligand attenuated LPS-induced liver injury by downregulation of TLR4 on macrophages.95
In acetaminophen-induced fulminant hepatic failure there is no evidence for direct involvement of TLRs. However, low dose LPS was protective in acetaminophen-induced liver failure in mice which is similar to the phenomenon of LPS tolerance.96 Deficient LPS recognition by TLR4 and CD14 also protected mice from acetaminophen-induced liver disease suggesting a potential role for LPS and its receptor, TLR4.97
The negative effects of ischemia-reperfusion injury on graft function are limiting factors in liver transplantation. Although a role for ischemic-reperfusion injury in the liver can be independent of LPS recognition and due to oxidative stress,98–100 evidence suggests for a role for TLRs in ischemic-reperfusion injury. Recent studies suggest that LPS levels were elevated early after reperfusion and that multiple components of the LPS signaling pathway, including CD14, LBP, as well as TLR2, are activated during ischemia/reperfusion injury after liver transplantation (98). Interestingly, TLR4 activation mediates inflammatory responses via both IRF3 and MyD88-dependent, pathways after ischemia/reperfusion (99,100). TLR4-deficient, but not wild-type or TLR2-deficient, mice were protected against ischemia/reperfusion induced liver damage, suggesting an important role for TLR4. This was associated neutrophil infiltration, TNFα production, and induction of heme-oxygenase 1.101 Studies by Tsung et al. indicate that the endogenous molecule high mobility group box 1 (HMGB1) in warm I/R may interact with TLR4 on non-parenchymal, phagocytic cells in the liver.102, 103
A recent study by Halangk et al reported no impact of CD14 and TLR4 polymorphism on the incidence and outcome of chronic liver disease.104 In contrast, the authors found significantly increased frequency of homozygotes for CD14-260C>T in patients with persistently elevated aminotransferases in the absence of a readily identifiable etiology, and speculated that hepatic toxicity of microbial components may be mediated by the alterations of the CD14/TLR4 pathway in otherwise healthy individuals.
To date there is no direct evidence that either HBV (dsDNA) or HCV (ssRNA) would directly stimulate TLR9 or, TLR7/8, respectively. Recent reports suggest that in an in vivo model of chronic HBV infection, administration of ligands specific for TLR3, TLR4, TLR5, TLR7, and TLR9, but not TLR2, inhibited HBV replication in the liver within 24 h in a type I interferon-dependent manner.105, 106 Further, there was an activation of the antiviral program in nonparenchymal cells, especially in dendritic cells, but not in hepatocytes during in vivo treatment of HBV infection with TLR ligands. This is in agreement with previous observations that immune cells are the main producers of type 1 IFNs and stimulation via TLR 3, 4,7, 8 and 9, but not TLR2, leads to production of IFNα, suggesting that endogenous IFNs play a critical role in anti-viral defense.107 Indeed, type 1 IFN and IFN-inducing TLR ligands (CpG) could be successfully used as therapeutic agents in viral hepatitis,108–110 or as potent adjuvants for anti-HBV vaccines, as shown both in animal models and in humans.111–112 However, increasing evidence suggest that viruses may use PRRs to escape immune surveillance. A recent report by Cheng showed that HBV surface antigen inhibits LPS-induced COX-2 expression, reduces IL-12 and IL-18 production by blocking the ERK and NFκB pathways, and regulates IFNγ production.113 Recently, Thompson et al.114 hypothesized that TLR2 expression is required for HBV clearance, based on downregulation of TLR2 by HBeAg and significant upregulation of TLR2 expression observed in adefovir+emtricitabine-treated individuals with chronic HBV infection. Hui et al.115 found a correlation between increased levels of TLR7 expression and serological clearance of HbsAg in chronically HBV infected patients.
Unlike HBV, hepatitis C virus exhibits different sensitivity to IFNs depending on the viral genotype.108 High type I IFN response in the liver is seen during acute infections with HCV, however, it does not correlate with viral clearance and it is unclear whether the IFN originates from immune or parenchymal compartment.116 Recombinant type 1 IFN is the main component of anti-HCV therapy, however, the success of viral elimination is limited.117, 118 Recent reports using HCV subgenomic replicon-harboring Huh cells showed that HCV has developed strategies to interfere with IFN pathways through pattern recognition signals. NS3/4A protein interacts directly with TBK1 to decrease TBK1/IRF3 interaction, while NS3 protein alone induces degradation of the TLR adaptor TRIF, both leading to downregulation of IRF3 activity and hamper IRF-3 mediated type 1 IFN induction.119, 120 A recent report suggests that TLR3 plays a role in hepatitis C-associated glomerulonephritis.121 It is unclear to date if HCV proteins may affect TLR7, 8 and 9 signaling, since, at least in dendritic cells, these TLRs use IRF7 but not IRF3 and are TBK-1 independent.41
In hepatocytes, type 1 IFN is induced by activation of PKR or RIG-I, which may occur independently of TLRs, but involve both IRF3 and IRF7.122 Recent studies demonstrated that HCV infection transiently induces RIG-I- and IPS-1- dependent IRF-3 activation.123 This host response initially limits HCV production; however, HCV interferes with this response early in infection via NS3/4A, the major serine protease expressed by HCV. NS3/4A disrupts the RIG-I pathway through proteolysis of essential signaling components of IRF-3 activation and downstream activation of the interferon pathway in hepatocytes.124, 125 These data indicate that HCV subverts RIG-I recognition and TLR signaling pathways to escape from clearance in hepatocytes. In addition, both TLR9- and TLR7/8-induced IFNα production is diminished in peripheral plasmacytoid dendritic cells of HCV-infected patients.126
Prevention of type I IFN induction is not the only strategy that HCV developed to escape immune surveillance. HCV core and NS3 proteins activate TLR2 in monocytes to produce IL-8, IL-6 and TNFα, and inhibit differentiation and antigen-presenting functions of myeloid dendritic cells.122, 127 Marked upregulation of TLR2 and TLR4 was reported in patients with chronic HCV infection irrespective of HCV genotype and viral load128 and was detected in hepatocytes, Kupffer cells, and peripheral blood monocytes.129 TLR2- and TLR4-induced TNFα production and circulating TNFα levels are increased in HCV-infected patients127, 130 while HCV replication, unlike other RNA viruses, is resistant to TNFα.131 Thus, TLR2-mediated activation by HCV proteins may contribute to the increased pro-inflammatory cytokine activation and hepatocyte damage in chronic HCV infection.130, 132
Of the other viruses that cause hepatitis, members of the herpes virus family were shown to activate TLRs.133 Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2,134 while HSV activates via TLR9,135 suggesting that TLR-induced signals may play a role in virus-induced liver damage.
Alcoholic Liver Disease.
Evidence for an essential role of the TLR4 ligand, LPS, in alcoholic liver disease (ALD) was established by studies of Thurman and colleagues.69, 136 Consistent with the key role of LPS in ALD, TLR4 mutant or CD14-deficient mice were protected against alcohol-induced liver disease.137 In the currently accepted model of ALD, LPS promotes hepatic injury via induction of Kupffer cell activation resulting in production of TNFα and other inflammatory mediators.87 In addition, LPS recognition by TLR4 expressed on hepatic stellate cells and sinusoidal epithelial cells may also contribute to the progression of ALD.78, 138 Thus, TLR4-mediated intracellular events are critical in the pro-inflammatory mediator activation and fibrosis. In a mouse model, hepatic expression of TLR2 or TLR4 mRNA was not changed by chronic alcohol feeding or by acute alcohol administration.139
In contrast to chronic alcohol consumption, acute alcohol exposure inhibits TLR4 signaling in monocytes and macrophages after in vitro as well as in vivo alcohol treatment in mice leading to decreased LPS-induced TNFα production.140–142 Acute alcohol administration also suppressed TLR3 downstream signaling.143 Various laboratories have shown distinct roles for TLR-associated molecules such as CD14, TLR4 and LBP in ALD as well as for oxidative stress-related molecules such as NADPH oxydase (p47 phox) and iNOS.144, 145 Recent evidence shows that direct interaction of NADPH oxydase isozyme 4 with TLR4 is involved in LPS-mediated ROS generation and NFκB activation146 and may be responsible for tissue damage in ALD. Thus, TLR-mediated innate immune responses play an important role in liver pathology related to acute and chronic alcohol consumption.
A recently developed animal model of primary biliary cirrhosis is induced by administration of a TLR3 ligand (polyI:C) and reveals a striking similarity between the animal model and human primary biliary cirrhosis (PBC) with respect to increases in alkaline phosphatase, appearance of auto-antibodies and extensive intra- and extra-hepatic inflammation.147 The PBC induced by PolyI: C in mice was also accompanied by low degree of bile duct destruction.147 Biliary cirrhosis induced by common bile duct ligation leads to increased serum and liver levels of LBP and increase in total liver CD14 mRNA, however it does not lead to increase of CD14 or TLR4 at the protein level.148 In human liver tissues from patients with PBC expression of TLR4 was markedly increased in bile-duct epithelial cells and in periportal hepatocytes.149 Recently, it was found that hyper-IgM production in PBC is a result of chronic B cell stimulation likely due to bacterial CpG-induced TLR9 activation.150 This supports the hypothesis that previous contact with pathogens and the consequential mimicry phenomena are potential players in the pathogenesis of autoimmune liver diseases.
PRRs in Liver Regeneration
Increasing evidence suggests that TLR-mediated pathways contribute to liver regeneration. Hepatocyte regeneration was impaired in endotoxin tolerant animals after carbon tetrachloride injection.151 After partial hepatectomy, liver regeneration was delayed in mice deficient in the common TLR adaptor, MyD88, suggesting that MyD88-mediated signals contribute to liver regeneration.152 It remains to be elicited whether this effect of MyD88 is mediated through TLR activation or other mechanisms. In mouse models, liver regeneration is inhibited by viral infection.153 Consistent with this, activation of TLR3 with poly I: C resulted in attenuation of liver regeneration after partial hepatectomy.153
PRRs and Fibrosis
Activation of stellate cells though TLRs has been suggested by recent studies in initiation of fibrosis in the liver.154 Patients with cirrhosis have increased serum endotoxin levels, regardless of the etiology of liver disease.155 Riordan et al.156 reported that in patients with cirrhosis patients the expression of TLR2 is upregulated on peripheral blood mononuclear cells (PBMC) and as a result, recognize Gram-positive bacterial products with a concurrent increase in serum TNFα. Interestingly, TLR4 expression on PBMC did not significantly differ between controls and paitents with cirrhosis.156 In another study, TLR4 mediated inflammation and fibrogenesis after bile duct ligation was reported.157 Many of these observations could be linked to TLR4 expression on stellate cells allowing their direct activation by fibrogenic insults.
PRRs in Liver Cancer
Clinical and epidemiologic studies suggest association between infectious agents, chronic hepatitis, inflammatory disorders and hepatocellular cancer. It was proposed that the infectious agents that are recognized by PRRs lead to activation of NF-κB in myeloid cells resulting in production of growth and survival factors that stimulate tumor progression and development.158 Interestingly, in patients with chronic HCV infection, a population at high risk for hepatocellular carcinoma (HCC), monocytes have increased baseline NF-κB activation and TNFα production. Moreover, HCV core and NS3 proteins can induce both NF-κB activation and TNFα production in monocytes via TLR2.127 In hepatocytes, HCV NS5A inhibits NF-κB activation by TNFα and potentiate TNFα induced JNK activation.159 Importantly, inhibition of NF-κB activation in hepatocytes, the cells that are infected with HCV may lead to increased susceptibility to carcinogen-induced cell death.160 Thus, repeated tissue injury resulting first from acute, then chronic inflammation may serve as major mechanisms in promoting tumor growth in HCC associated with viral hepatitis.158
Therapeutic Approaches Targeting PRRs
Increasing evidence suggests that activation or inhibition of TLR signaling could be used as therapeutic interventions in multiple diseases,161 including liver diseases. Although PRRs are important in liver disease, the extent to which they are expressed in the liver and their roles are yet to be fully delineated. For example, based on the involvement of LPS in various types of liver injury (acute liver failure, alcoholic hepatitis) it is tempting to speculate that pharmacological inhibition of endotoxin responses could benefit some of these conditions. Recently, Visintin et al showed blocking of TLR4 signaling by targeting the TLR4 co-receptor, MD-2.162 Kawata et al. developed a synthetic non-toxic lipid A derivative with TLR4 blocking capacity,163 while Bartfai et al. developed a low molecular weight mimic of Toll-IL-1 receptor/resistance domain that inhibits IL-1R-mediated responses.164 A recent report by Meng et al. indicated that epitope-specific binding of exogenous ligands precedes specific TLR2 signaling and suggested therapeutic application of a neutralizing anti-TLR2 antibody in acute infections.165 Although it seems possible to disrupt TLR signaling at a downstream level (MyD88, TRIF, IRAK or TRAF proteins), by using short interfering RNAs,166 a suitable and safe in vivo application of this approach is yet to be established.
The concept of stimulation of selected TLRs to boost immune responses has advanced for in vivo use in multiple diseases. The therapeutic effect of TLR ligands is mainly based on induction of a specific cytokine milieu that mobilizes the organism's intrinsic immune capacity to reinstate homeostasis. Basic and clinical investigations are ongoing with TLR7 and TLR9 ligands that may prove to be effective both as sole therapeutic agents and as adjuvants for vaccines in diverse liver diseases. In a recent phase 1b trial,167 the treatment of a small number of HCV positive subjects with different doses of a TLR9 agonist, CpG10101, resulted in marked reduction in HCV RNA levels and increased cytokine and chemokines induction, suggesting that TLR9-induced endogenous IFN-induction pathways could be elicited. A recent report by Lee et al. suggests that TLR7 agonists induce anti-HCV immunity not only by direct IFN induction, but also via an IFN-independent mechanism, when used in vitro.168 Further, in vivo administered Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection.169 Drug development targeting non-TLR PRRs revealed that small molecule inhibitors of HCV NS3/4A restored RIG-I signaling of IFNβ induction in vitro.123 This suggests that HCV protease inhibitors effectively prevent IPS-1 proteolysis thereby restoring the HCV-induced defect in PRR functions; thus, represent a virus-specific, PRR targeted therapeutic approach.
The therapeutic application of TLR ligands should be evaluated with cautious optimism because TLR ligands may have multiple detrimental effects. Some TLR ligands, such as the TLR9 ligand, CpG, are toxic and induce destruction of lymphoid follicle after repeated use.170 Excessive production or imbalanced amounts of TLR ligand-induced cytokines may unmask and/or favor development of non-infectious inflammatory or autoimmune diseases.171, 172 An additional concern is that consecutive use of different TLR (TLR2, TLR4, TLR9) ligands may lead to immune paralysis,173, 174 or, as in case of combined TLR2 and TLR9 ligands, can result in sensitization to consecutive TLR4 stimulation and development of acute liver failure.90
In the light of these recent discoveries, the involvement of pattern recognition receptors in therapy provides novel and promising grounds for amelioration of different liver diseases.
We thank Ms. Gail Bird for help with manuscript preparation.