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Keywords:

  • cell and molecular biology;
  • liver immunobiology;
  • viral hepatitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

Hepatitis C virus (HCV) infection is a significant global health problem, affecting over 150 million people worldwide. While the critical role of the adaptive immune system in HCV infection is well-established, the importance of the innate immune system in HCV infection has only been recognized in more recent years. Toll-like receptors form the cornerstone of the innate immune response, and there is considerable evidence for their crucial role in hepatitis C infection. This review outlines recent advances made in our understanding of the role of Toll-like receptor function in HCV infection, exploring how HCV manipulates host immunity to evade immune clearance and establish persistent infection despite leading to inflammatory hepatic damage.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

Hepatitis C virus (HCV) infection is a significant global health problem, affecting over 180 million people worldwide.[1] Despite emerging therapies for HCV infection, the sombre prediction is for the health burden from HCV to steadily increase: by 2020, it is projected that untreated patients with HCV liver cirrhosis will double, the number of patients with HCV cirrhosis developing hepatocellular carcinoma will increase by 80%, and referrals for liver transplantation for HCV-related liver disease are also predicted to double.[1, 2] This makes HCV infection a significant global public health issue, with an expected exponential increase in burden of disease over time.

While the critical role of the adaptive immune system in HCV infection is well-established, the importance of the innate immune system in HCV infection has only been recognized in recent years. Toll-like receptors (TLRs) form the cornerstone of the innate immune response, and there is considerable evidence for their crucial role in HCV infection.

This review outlines recent advances made in our understanding of the role of TLR function in HCV infection, exploring how HCV manipulates host immunity to evade immune clearance and establish persistent infection despite leading to inflammatory hepatic damage. The potential clinical benefits of therapeutic and screening strategies harnessing TLR function will also be addressed.

The innate immune system and TLRs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

The innate immune system forms a stereotyped, highly conserved immune response that is the first line of defense against infection and inflammation in an organism. Even though the innate immune system is evolutionarily conserved, it is now recognized to have a critical role in initial host immune defenses and leads to appropriate activation of the subsequent adaptive immune response.[3] Innate immune responses are specific, triggered by binding of innate immune receptors to their appropriate ligands, thereby initiating a downstream signaling cascade culminating in upregulation of pro-inflammatory cytokine, chemokine, and interferon production. In contrast with adaptive immunity, the innate immune response is rapid in onset and requires no previous exposure to the pathogen.[4, 5]

TLRs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

TLRs are a family of non-clonal, germline-encoded, pattern recognition receptors (PRRs) that give the innate immune system considerable specificity for a large range of pathogen classes.[6] To date, there are 10 functional TLRs identified in humans (TLR 1–10).[7] Each receptor has two domains: an extracellular leucine-rich LRR domain and an intracellular Toll-interleukin (IL-1) receptor (TIR) domain.[8]

TLRs recognize pathogen-associated molecular patterns, or PAMPs, which are highly conserved molecules expressed by classes of invading pathogens. TLR2 and TLR4 also recognize endogenous components derived from dying or damaged host cells (called damage-associated molecular patterns, or DAMPs), allowing inflammatory responses to be initiated by trauma to host cells.[9] Commonly cited PAMPs and DAMPs, and their corresponding TLRs are outlined in Table 1.

Table 1. Human Toll-like receptor (TLR) and their corresponding ligands
TLRLigandLigand type
  1. DAMP, damage-associated molecular pattern (endogenous ligand); HMGB1, high mobility group box 1; PAMP, pathogen-associated molecular pattern (exogenous ligand).

TLR1Complexes with TLR2 
TLR2Lipoproteins (diacylated lipopeptides) and lipoteichoic acidPAMP
HMGB1, hyaluronan, heat shock proteins 60 and 70DAMP
TLR3double-stranded RNA, particularly viralPAMP
TLR4LipopolysaccharidePAMP
hyaluronan, heat shock proteins 60 and 70DAMP
Free fatty acids, heparin sulfate, uric acid 
TLR5FlagellinPAMP
TLR6Complexes with TLR2 
TLR7single stranded RNA, particularly viralPAMP
TLR8single stranded RNA, particularly viralPAMP
TLR9bacterial unmethylated CpG motif containing DNAPAMP
HMGB1DAMP
TLR10Unknown 

Greater breadth of specificity of TLR binding is created by dimerization of TLR2 with TLRs 1 and 6, and accessory proteins such as MD2 that bind to TLRs to alter binding specificity.[10] The localization of TLRs within cells is also important, for example TLRs that bind viral RNA and bacterial DNA are located within endosomes, as these organelles do not contain host RNA or DNA.[11]

There are also other cytosolic pathogen recognition receptors in addition to TLRs that form part of the innate immune system, including the RNA helicases retinoic acid-inducible gene 1, melanoma differentiation-associated gene 5, and laboratory of genetics and physiology 2[12] and nucleotide-binding oligomerization domain-like receptors. However, their involvement in HCV infection is beyond the scope of this review.

Cellular TLR expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

TLRs are expressed ubiquitously; however, levels of expression vary for different cell types. This compartmentalizes TLR function by regulating access to TLR ligands for binding and determining the subsequent signaling pathway and inflammatory response that is activated by TLR ligand interactions.[13] Expression of TLRs by cell type in both peripheral immune cells and liver cells is outlined in Table 2.

Table 2. Toll-like receptor (TLR) expression peripheral blood immune cells and liver cells
 TLR1TLR2TLR3TLR4TLR5TLR6TLR7TLR8TLR9TLR10
  1. HSC, hepatic stellate cell; mDC, myeloid dendritic cell; NK, natural killer; pDC, plasmacytoid dendritic cell.

Monocytes    
mDCs    
pDCs      
Neutrophils   
Eosinophils        
Mast cells      
Myeloid cells      
NK cells  
T cells  
Tregs   
B cells    
Hepatocytes
Kupffer cells     
HSCs      
Biliary cells
Endothelial cells 

The immune system of the liver is highly specialized to prevent constant immune activation in the face of continual bombardment with pathogens, as it receives the entire blood supply of the gastrointestinal tract.[14] TLR messenger RNA (mRNA) expression is therefore low in the liver, favoring TLR ligand tolerance; however, in pathological conditions, TLR expression is induced to allow appropriate TLR activation.[15, 16]

Two key cell types within the liver that express TLRs and have crucial roles in HCV infection and liver fibrosis are Kupffer cells and hepatic stellate cells (HSCs). Kupffer cells are resident macrophages expressing TLR2, TLR3, TLR4, and TLR9, and these signaling pathways mediate phagocytosis, antigen presentation, and secretion of pro-inflammatory mediators.[17, 18] TLR-mediated IL-12 and IL-18 from Kupffer cells induces hepatic natural killer (NK) cells to produce interferon (IFN)-γ, which is critical for viral eradication and inhibition of HSCs and hepatic fibrogenesis.[19] Kupffer cells also play a direct role in fibrogenesis, secreting transforming growth factor beta (TGF-β), matrix metalloproteinases, platelet-derived growth factor, and reactive oxygen species with TLR4 stimulation.[15]

HSCs are the major fibrogenic cell type in the liver.[20] When liver injury occurs, quiescent stellate cells become activated fibrogenic myofibroblasts that produce inflammatory mediators and extracellular matrix and collagen, leading to hepatic fibrogenesis.[21, 22] TLR4 and TLR9 pathways are the most important in HSC activation and fibrogenesis.[23, 24]

TLR signaling pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

When TLRs bind to their appropriate ligand via their leucine-rich LRR domain, they initiate a downstream signaling cascade that leads to upregulation of pro-inflammatory cytokine and chemokine production and interferon signaling.[25] TLRs provide a bridge between innate and adaptive immunity through induction of dendritic cell (DC) maturation, antigen presentation, and T- and B-cell recruitment and activation.[15, 26] These immune responses are critically important in viral infections, including HCV infection.

There are four primary adaptor molecules that bind to intracellular TIR domains of TLRs to transduce signals: myeloid differentiation factor 88 (MyD88), toll-interleukin receptor-associated protein (TIRAP), toll-interleukin-receptor domain containing adaptor protein-inducing interferon beta (TRIF), and TRIF related protein (TRAM). In simple terms, MyD88 is the main adaptor protein for all TLRs except TLR3, which uses TRIF.[27] TIRAP works with MyD88 in TLR2 and TLR4 signaling. TRIF mediates TLR3 and TLR4 antiviral IFN responses and nuclear factor kappa B (NFκB) activation. TRAM mediates TLR4-TRIF signaling.[15] The four key signaling pathways that utilize these four adaptor proteins along with other proteins are outlined in Figure 1. A key paradigm in TLR signaling is overlap of signaling pathways and shared pathways of gene transcription, allowing amplification and built-in redundancy of immune responses.

figure

Figure 1. Schematic overview of Toll-like receptor (TLR) signaling pathways. Internalized viral pathogen-associated molecular patterns (PAMPs) activate TLR3, TLR7/8, and TLR9 in endosomes, whereas bacterial PAMPs activate TLR1, TLR2, TLR4, and TLR6 from outside the cell. TLRs then interact with adaptor proteins (MyD88, TRIF, TRAM, or TIRAP) to induce activation of downstream kinases and transcription factors, leading to upregulation of pro-inflammatory, antiviral, and antibaterial genes in the nucleus, including interferon synthesis. AP-1, activator protein-1; dsRNA, double stranded ribonucleic acid; HMGB1, high mobility group box 1; IκB, I kappa B kinase beta subunit; IKK, I kappa B kinase complex; IRAK, Interleukin 1 receptor associated kinase; IRF, interferon regulating factor; JNK, c-jun-n-terminal kinase; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NFκB, nuclear factor kappa B; RIP, receptor interacting protein; ssRNA, single stranded ribonucleic acid; TAK, transforming growth factor beta activating kinase; TIRAP, toll-interleukin receptor associated protein; TRAF, tumour necrosis factor receptor associated factor 6; TRAM, TRIF-related protein; TRIF, TIR domain containing adaptor protein-inducing interferon β.

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The MyD88-NFκB/AP-1/IRF5/p38 pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

MyD88 induces pro-inflammatory and antibacterial gene transcription by activating the NFκB, activator protein (AP-1), p38, and interferon regulatory factors (IRF) 5 pathways via TLR2, 4, and 5.[28] Upon stimulation with various ligands, I kappa B kinase beta subunits (IKBs) are phosphorylated at serine residues by the I kappa B kinase complex (IκK) complex. This causes degradation of the IκB, allowing NFκB to be released into the nucleus and bind to the KB site. AP-1 activation in TLR signaling mostly mediated by p30, mitogen activated protein kinase (MAPK), and IκK.

Control of TLR signaling: negative feedback and tolerance

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

Because of the potentially deleterious effect of an unchecked pro-inflammatory state, negative feedback exists for TLR signaling and is a critical component of immune activation and modulation.[32] Perturbation of TLR function can occur at multiple levels in the signaling cascade, including synthesis and expression of signaling receptors and proteins, through proteins that negatively interact with signaling and enhanced ubiquination and degradation of signaling proteins.

Another important mechanism of negative feedback is via tolerance or reduced subsequent responses from repeated TLR stimulation after initial stimulation of one TLR type. Cross-tolerance also occurs, whereby activation of one TLR pathway can cross-inhibit another via negative feedback.[33] Potentially, both negative feedback and tolerance can be manipulated by viral infections such as HCV in order to prevent immune clearance.

The HCV

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

Hepatitis C is a positive strand RNA enveloped flavivirus that was first cloned in 1989.[34] HCV virions bind to the cell surface and enter cells via receptor-mediated endocytosis. The structure of HCV is outlined in Figure 2. The core and non-structural proteins shown in the diagram are important sequences recognized by PRRs, including TLRs. They are also important inhibitors of TLR signaling.[35, 36]

figure

Figure 2. Genomic structure of the hepatitis C virus (HCV). Both viral and host cell proteases cleave HCV poly-protein to yield structural and non-structural proteins. These are important pathogen-associated molecular patterns recognized by Toll-like receptors and have several disabling effects on immune function.

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General elements in the immune response against HCV infection

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

In order to understand the context of TLR immune responses in HCV infection, it is necessary to consider general features of the immune response against HCV.

Fundamentally, T-cell responses to HCV are critical for viral eradication and also response to HCV therapy.[37-39] The balance between Th1 antiviral and Th2 viral-permissive T-cell responses determines viral clearance or persistence, and the degree of inflammation and disease progression.[40-43] CD4+ T cells have a protective effect against liver disease progression in chronic HCV infection, and effective CD4+ T-cell responses to HCV are required to mount an active cytotoxic CD8+ T-cell response for viral eradication.[44-47] T-cell responses to HCV proteins are readily detected early during acute HCV infection, but both CD4+ and CD8+ T-cell function is significantly impaired once chronic infection is established, with reduced cytokine production despite ongoing stimulation with circulating HCV antigens.[48-53]

One of the key determinants of T-cell function in HCV infection is the quality of antigen presentation by DCs, as this determines the number of epitopes recognized by T cells that will engender an antiviral response.[38, 54, 55] HCV is associated with a failure of DC function that also leads to impairment in NK cell and natural killer T cell (NKT) function, with reduced IFN-γ secretion leading to reduced inhibition of HCV replication, reduced inhibition of HSCs, and greater hepatic fibrosis.[56-58] Th2-skewed NK cells further downregulate DC function by secreting IL-10 and TGF-β.[56, 59] TLRs play a key role in activation of DCs and NK cells, and initiate inflammatory cytokine responses in other cell types, including liver cells, which contribute to the appropriate cytokine milieu for DC maturation and T-cell activation.[60, 61]

TLRs in HCV infection

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

Arguably, the most important paradigm in the innate immune response against HCV is compartmentalization. HCV has different effects upon TLR pathway stimulation in various cellular compartments and in this way is able to both stimulate pro-inflammatory cytokine production leading to liver damage and evade immune responses to establish viral persistence.[62, 63] A summary of important interactions between HCV viral proteins and TLR signaling pathways are shown in Figure 3 and Table 3.

figure

Figure 3. Positive and negative effects of hepatitis C virus (HCV) viral proteins on Toll-like receptor (TLR) signaling. HCV core and NS3 proteins stimulate TLR2 and TLR4. HCV dsRNA binds to TLR3. The 3′ UTR tail is a pathogen-associated molecular pattern for both TLR7 and TLR9. However, HCV NS3/NS4A degrades TRIF and binds to TBK1, inhibiting IFN production. HCV NS5A also binds to MyD88 to impair TLR2, TLR4, TLR7, and TLR9 signaling. AP-1, activator protein-1; dsRNA, double stranded ribonucleic acid; IκB, I kappa B kinase; IKK, I kappa B kinase complex; IRAK, interleukin 1 receptor associated kinase; IRF, interferon regulating factor; JNK, c-jun-n-terminal kinase; MyD88, myeloid differentiation factor 88; NFκB, nuclear factor kappa B; RIP, receptor interacting protein; TAK, transforming growth factor beta activating kinase; TBK, TANK binding kinase-1; TIRAP, toll-interleukin receptor associated protein; TRAF, tumour necrosis factor receptor associated factor 6; TRAM, TRIF-related protein; TRIF, TIR domain containing adaptor protein-inducing interferon β.

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Table 3. Stimulatory and Inhibitory effects of hepatitis C virus (HCV) on Toll-like receptor (TLR) signaling in different cell types
TLRTLR StimulationTLR Inhibition
 MechanismCell typeEffectMechanismCell typeEffect
  1. DC, dendritic cell; HLA-DR, human leukocyte antigen DR; IFN, interferon; IL, interleukin; ISG, interferon stimulated gene; IRF, interferon regulatory factors; LPS, lipopolysaccharide; mDC, myeloid dendritic cell; NK, natural killer; pDC, plasmacytoid dendritic cell.

TLR2 (TLR1, TLR6)Core, NS3Monocytes, macrophages

[UPWARDS ARROW]TLR2 expression

[UPWARDS ARROW]TLR2 activation/cytokine production

HCV lipoparticlesDCs monocytes

[DOWNWARDS ARROW]Pro-inflammatory cytokines

[UPWARDS ARROW]IL-10 secretion DCs and monocytes

TLR3RNAMonocytes, mDCs, non-parenchymal liver cells

[UPWARDS ARROW]TLR3 expression

[UPWARDS ARROW] IFN-β

RNAMonocytesmDCs[DOWNWARDS ARROW]IRF3 inflammatory cytokines (IL-6)
TLR4NS5AHepatocytes, B cells

[UPWARDS ARROW]TLR4 expression

[UPWARDS ARROW]TLR4 activation/cytokine production

[UPWARDS ARROW]IFN-β/ ISGs

[DOWNWARDS ARROW]Monocyte tolerance to LPS

[UPWARDS ARROW]Liver fibrogenesis

HCV lipoparticlesDCs[DOWNWARDS ARROW]TLR4 expression
TLR7/8

RNA

Poly-U tail

DCs, NK cells, hepatocytes, monocytes

[UPWARDS ARROW]Pro-inflammatory cytokines

[UPWARDS ARROW]TLR7/8 expression monocytes

NS5A

DCs

NK cells

[DOWNWARDS ARROW]TLR7/8 expression

[DOWNWARDS ARROW]TLR7/8 signaling

[DOWNWARDS ARROW]IRF7

[UPWARDS ARROW]Degradation TLR7 liver

[DOWNWARDS ARROW]IFN-α/β

[DOWNWARDS ARROW] NK cell IFN-γ

[DOWNWARDS ARROW] Inhibition of stellate cells/fibrogenesis

TLR9

DNA

Poly-U tail

DCs[UPWARDS ARROW]Pro-inflammatory cytokines mDCs

DNA

Poly-U tail

pDCs

[DOWNWARDS ARROW]IFN-α/β

[DOWNWARDS ARROW]HLA-DR

TLRs in HCV infection: immune activation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

HCV core and non-structural proteins are important PAMPs for TLR2, TLR3, TLR4, TLR7/8, and TLR9. HCV core and non-structural protein 3 (NS3) proteins stimulate TLR2 when associated with TLR1 and TLR6 in peripheral blood mononuclear cells (PBMCs),[64] particularly monocytes and macrophages.

TLR2 stimulation leads to production of TNF-α, IL-6, and IL-8 via the NFκB, c-jun-n-terminal kinase (JNK)/AP-1, p38, and extracellular signal regulator proteins (ERK) pathways, with ERK being the dominant pathway for TNF-α secretion. Some studies have demonstrated that TLR2 expression by PBMCs is increased in HCV infection, and TNF-α production can promote TLR2 expression, thereby providing a potential indirect positive feedback loop for TLR2 activation.[65-68]

TLR4 is also activated by HCV, with NS5A inducing TLR4 expression and thereby increasing IFN-α and IL-6 secretion, especially in B cells and hepatocytes.[65] TLR4 also induces IFN-β production, which leads to paracrine IFN production and upregulation of interferon-sensitive genes within infected cells and surrounding tissues.[69] Monocytes in HCV-infected patients have impaired tolerance for repeated TLR4 challenge and greater TLR4 expression, leading to higher levels of serum and intrahepatic TNF-α, which contributes to inflammation in HCV infection.[64, 70]

TLR3 is important for its antiviral immune effects, and TLR3-stimulated non-parenchymal liver cells are able to regulate HCV replication through production of IFN-β.[71, 72] TLR3 mRNA is significantly increased in monocytes in chronic HCV infection.[73] An IFN-responsive element has been identified in the promotor region of the TLR3 gene, and it therefore seems likely that TLR3 expression is responsive to IFN treatment in HCV infection.[74] Myeloid DCs (mDCs) have normal functioning TLR3 and can produce IL-12, IL-6, IL-10, IFN-γ, and TNF-α with TLR3 stimulation despite HCV infection.[75]

HCV genomic RNA has direct immunostimulatory effects on TLR7 and TLR8, leading to IFN-α production and activation of IRF7 and NFκB.[76] Plasmacytoid DCs (pDCs) can also be activated via TLR7 and TLR9 through the HCV RNA polyuridine tail.[76-81] TLR7 activation of hepatocytes also induces IFN-independent antiviral effects, reducing both HCV RNA levels and NS5A protein expression in cell lines.[82] There is also increased TLR7 and TLR8 expression on monocytes in HCV infection, although the significance of this remains unclear.[64]

TLRs in HCV: immune evasion

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

HCV viral proteins are able to stimulate TLR signaling, which plays an important role in viral immune clearance. However, HCV is able to simultaneously evade immune clearance through specifically targeting and impairing TLR signaling through several mechanisms. First, HCV interferes with signaling via the TRIF-TBK1-IRF3 pathway. The HCV NS3 protein induces degradation of TRIF, while the NS3/4A protein impedes IRF3 and NFκB activation by reducing the amount of TRIF in circulation and by generating cleavage products with dominant-negative activity.[83, 84] NS3/4A also interacts directly with TBK1 to reduce TBK1-IRF3 interaction and therefore inhibit IRF3 activation.[85] HCV also interferes with the TLR-MyD88 pathway through NS5A interaction with MyD88 to prevent IRAK1 recruitment and cytokine production in response to ligands for TLR2, TLR4, TLR7, and TLR9.[86]

The HCV lipoviral particle interferes directly with TLR4 signaling in DCs, while HCV core protein suppresses TLR4 expression.[64, 87] Cellular expression of TLR2 and TLR4 in mDCs is controversial, being reported as both higher and lower in HCV infection patients compared with healthy controls, although signal transduction of TLR2 and TLR4 in mDCs is certainly impaired in HCV infection.[49, 56, 88] Greater anti-inflammatory IL-10 production by macrophages with TLR2 stimulation has been reported and may explain the dichotomous effects of TLR2 activation in different cellular compartments.[89]

HCV is able to reduce TLR7 signaling through a myriad of mechanisms. HCV induces increased instability of TLR7 mRNA transcripts, while the NS5A protein interferes with TLR7 signaling, leading to reduced cytokine responses to stimulation.[64, 86, 90] Interestingly, lower TLR7 expression in HCV-infected livers is restored with successful HCV clearance with treatment.[90]

HCV has been shown to regulate TLR9 expression via Elk-1, which is an important signal integration point between TCR and CD28 in Th1 T-cell activation.[91] HCV also impairs TLR9-mediated IFN-α and IFN-β production, and human leukocyte antigen DR (HLA-DR) expression by pDCs, associated with impaired activation of naïve T cells.[49] TLR9 signaling in mDCs is unaffected.[49, 75]

It is therefore clear that compartmentalization of effects on TLR function is a key strategy by which HCV is able to evade immune clearance yet still lead to chronic inflammatory hepatic damage and liver fibrosis.

Linkage of TLR function and other immune responses in HCV infection

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

We can now start to piece together how HCV-mediated alterations in TLR function may contribute to the immune impairments seen in HCV infection that encourage viral persistence.

Activation of TLR2, TLR3, and TLR4 signaling in monocytes, mDCs, and liver cells leads to upregulation of pro-inflammatory cytokines and chemokines, and recruitment of inflammatory cells to the liver, culminating in cytotoxic and apoptotic death of viral-infected cells and adjacent uninfected cells.[65] Inflammatory hepatocyte damage stimulates fibrogenesis via HSC activation, culminating in hepatic fibrosis. Fibrogenesis is further augmented by impaired TLR7/8 signaling in NK cells, which leads in turn to impaired inhibition of HSCs. Impaired antifibrotic IL-6 production by monocytes with TLR7 and TLR3 stimulation may also contribute.[92-95] Simultaneously, impaired TLR7/8 and TLR9-mediated interferon production by pDCs leads to impaired antigen presentation by DCs and subsequent defective activation of CD4+ T cells, culminating in impaired T-cell responses to HCV antigens, failure of viral clearance, and aborted development of lasting immunity.[49, 82, 83, 96-99]

Clinical applications of TLR function in HCV

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

TLR polymorphisms and clinical outcome prediction

There have been recent considerable advances in our knowledge of TLR function and its role in HCV infection, but a more important question is how this knowledge may be harnessed to improve clinical outcomes.

Pathogen selection pressure has lead to considerably high rates of genetic polymorphism for TLR genes, and many of these polymorphisms affect gene function.[100, 101] There has been great interest in exploring relationships between TLR gene polymorphism carriage and clinical disease, as SNP detection by PCR is a relatively straightforward technique that could be employed for determining response to therapy and risk of adverse clinical outcomes in HCV infection. A summary of these polymorphisms is outlined in Table 4.

Table 4. Toll-like receptor (TLR) gene polymorphisms and association with hepatitis C virus (HCV) infection
TLRPolymorphismEstimated carriageMutation typeDisease association
  1. IFN, interferon.

TLR2Arg753Gln

2–10% Caucasian

Rare Asian

MissenseHCV rapid fibrosis post-liver transplant
TLR4Asp299Gly and Thr399Ile8% Caucasian

Missense

Cosegregate

Reduced fibrosis in HCV
TLR7c.1-120T>G (rs2302267)

> 5% Caucasians

Rare Africans

Intron 1 punitive splicingReduced HCV fibrosis in males
TLR7c.32A>T (rs179008, Gln11Leu)> 5% CaucasiansTransversion amino acid change

Increased chronic HCV in women

Decreased response to IFN-based HCV therapy in women

TLR7c.2403C>A (rs5743781, Ala448Val)> 5% CaucasiansNon-synonymous alteration exon 3Increased chronic HCV men and women

TLR4 gene polymorphism Thr399Gly and cosegregating Asp299Gly have been found to be protective against fibrosis progression in HCV infection,[102] while Li et al. also found TLR4 SNPs rs4986791 and rs960312 were associated with increased fibrosis risk.[103] Carriage of Asp299Gly and Thr399Gly is approximately 8% in Caucasian populations, while SNP rs960312 is important for its high prevalence within Asian populations (up to 25%). It has been shown that protective variants lower the apoptotic threshold of hepatocytes, inhibit TLR4 and NFκB signaling, and are associated with greater spontaneous apoptosis of HSCs.[104]

By contrast, Eid et al.[105] found that in the post-transplant HCV setting, TLR2 polymorphism Arg753Gln homozygosity was strongly associated with rapid HCV fibrosis progression but found no association between TLR4 polymorphisms and adverse outcomes.

The TLR7 gene is located on the X chromosome, and three SNPs in this gene have been identified with > 5% carriage within Caucasian populations: c.1-120T>G (rs2302267), c.32A>T (rs179008, Gln11Leu), and c.2403C>A (rs5743781, Ala448Val).[106] In chronic HCV infection, c.1-120T<G was found to be associated with lower levels of hepatic inflammation and fibrosis in males. PBMCs from patients with this genotype had increased IL-6 production in response to TLR7 ligand, providing a mechanistic clue to explain reduced hepatic fibrosis, as IL-6 has been shown in various studies to be antifibrotic.[92-94] In contrast, c.32A>T was associated with increased susceptibility to HCV in women, with higher levels of viremia, more rapid disease progression, and failure to respond to interferon-based HCV therapy.[107] TLR7-mediated IFN-α secretion is impaired in these women, while TLR7-mediated IL-6 production is preserved.[108]

These data collectively demonstrate that TLR2, TLR4, and TLR7 gene SNP detection may eventually provide potential screening tools for adverse outcomes in HCV-infected patients, guiding timing of therapy. However, further validation studies are warranted.

TLR therapeutics

Given the evidence for impairment of TLR function in HCV infection, restoration of TLR function through TLR agonists is a theoretically attractive approach for potential therapy. In particular, restoration of TLR3-, TLR7-, and TLR9-mediated NK cell and DC interferon secretion so as to improve antigen presentation and T-cell activation is an enticing target for therapy; these effects would not reduce immune responses against other infections, as may be seen if TLR inflammatory pathways were targeted. Importantly, TLR therapies may be less susceptible to viral resistance and broadly active against all HCV genotypes as they do not target HCV proteins directly.

There is evidence that TLR7 agonists are effective at HCV suppression. Isotoribine successfully reduced serum HCV levels in phase I trials but unfortunately has been removed from further studies because of adverse events; other TLR7 agonists are under development.[109] A TLR9 agonist CPG10101 has also been developed; its administration produced promising reductions in HCV viral load in phase I trials.[110] Isotoribine and CPG10101 both increase interferon secretion, engendering robust polyclonal T-cell responses. The side-effect profiles of these agents are therefore similar to interferon-based regimens.

TLR4 antagonists have also been developed to dampen tissue-damaging immune responses. They have shown promise in colitis and sepsis trials,[111, 112] but their use in HCV has not yet been explored. Given the protective effect of TLR4 SNPs that lead to blunted TLR4 responses in HCV hepatic fibrosis, these agents may have therapeutic benefit in HCV infection.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References

The effects of HCV infection on TLR signaling are complex. Compartmentalization of HCV modulation of TLR signaling means that HCV leads to upregulation of non-specific liver inflammation through stimulation of immune cells in an effort to achieve viral clearance. Conversely, suppression of TLR signaling in key antiviral immune effector cells, such as DCs, favors inhibition of inflammation that leads to viral persistence and chronic infection. Preliminary evidence suggests that therapeutic strategies harnessing TLR function will prove to be useful in HCV infection, while TLR polymorphisms offer a potential tool for prediction of adverse HCV-related outcomes.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The innate immune system and TLRs
  5. TLRs
  6. Cellular TLR expression
  7. TLR signaling pathways
  8. The MyD88-NFκB/AP-1/IRF5/p38 pathways
  9. The MyD88-IRF7-IFN pathway
  10. The TRIF-IRF3-IFN pathway
  11. The TRIF-NFκB pathway
  12. Control of TLR signaling: negative feedback and tolerance
  13. The HCV
  14. General elements in the immune response against HCV infection
  15. TLRs in HCV infection
  16. TLRs in HCV infection: immune activation
  17. TLRs in HCV: immune evasion
  18. Linkage of TLR function and other immune responses in HCV infection
  19. Clinical applications of TLR function in HCV
  20. Conclusion
  21. References