The Incidence and Significance of Pattern-Recognition Receptors in Chronic Viral Hepatitis Types B and C in Man

Chronic viral hepatitis B and C are among the most common and devastating liver diseases worldwide. Immune response plays a crucial role in the course of both diseases. In spite of the importance of the adaptive arm of the immune response, there is a growing role of innate immunity, the earliest confronted with viral attack. Pattern-recognition receptors (PRRs) and, in particular, Toll-like receptors (TLRs) are molecules which are able not only to recognize foreign invaders, but also quickly mount an antiviral defense. Activation of PRRs has been demonstrated in both hepatitis types, i.e. in situ in the liver and on while blood cells. Both viruses, HCV and HBV, are able to subvert the PRR-mediated antiviral response by means of various proteins and enzymes. HCV acts via the non-structural proteins NS2 and NS3/4A, while HBV HBeAg is inversely correlated with TLR activity. Viral counterattack is particularly directed toward dendritic cells, those creating the link with the adaptive immune response. Apart from TLRs, other PRRs such as RIG-1 and MDA-5 are also able to recognize viral infection and participate in the activation of type I interferon synthesis. TLRs manifest gene polymorphism, which was shown to affect several consequences associated with chronic viral hepatitis such as liver cirrhosis and the outcome of liver allotransplantation. There have been numerous attempts to take advantage of the existence and activity of PRRs for the patients’ benefit. Several authors examined the role of TLR synthetic agonists as inducers of TLR activation. In hepatitis C the most promising agonists appear to be TLR3, 7, and 9 for potential antiviral therapy. PRRs may also act as potent adjuvants in HBV vaccines. Their baseline mRNA levels may have predictive value in the course of antiviral therapy.     

Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA

Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.     

TLR8: the forgotten relative revindicated

The endosomal Toll-like receptors (TLRs) TLR3, TLR7, TLR8 and TLR9 are important in sensing foreign nucleic acids encountered by phagocytes. Because TLR8 was initially thought to be non-functional in mice, less is known about TLR8 than the genetically and functionally related TLR7. Originally associated with the recognition of single-stranded RNA of viral origin, there is now evidence that human TLR8 is also able to sense bacterial RNA released within phagosomal vacuoles, inducing the production of both nuclear factor (NF)-κB-dependent cytokines and type I interferons (IFNs), such as IFN-β. The functions of TLR8 extend beyond the recognition of foreign pathogens and include cross-talk with other endosomal TLRs, a process that may also have a role in the generation of autoimmunity.     

Activation of dendritic cells via TLR7 reduces Foxp3 expression and suppressive function in induced Tregs

Exogenous and endogenous RNA ligands of Toll‐like receptor (TLR) 7 which are present during viral infection or autoimmune diseases such as systemic lupus erythematosus (SLE) directly activate DCs and B cells and thus support the generation of effector T and B lymphocytes. However, the generation of effective antiviral or autoreactive adaptive immune responses requires blocking of immunosuppression by Tregs. In this study, we show that TLR7 ligands reduce the number of Tregs generated de novo from naïve murine T cells in vitro and in vivo. In the presence of TLR7‐activated splenic DCs, Foxp3 was transiently induced in naïve T cells by TGF‐β but was downregulated at later time points. Neutralization experiments revealed that loss of Foxp3 after initial induction was mostly dependent on IL‐6 produced in the DC–T‐cell cocultures containing TLR7 ligands. Thus, under the influence of TLR7 ligands fewer Tregs were generated and these expressed lower levels of Foxp3 correlating with a reduced capacity to suppress responder T‐cell proliferation. Thus, we provide evidence that TLR7 ligands affect Treg‐dependent immune regulation and may thereby contribute to the development of autoimmune diseases such as systemic lupus erythematosus.     

Human Toll-like receptor-dependent induction of interferons in protective immunity to viruses

Summary: Five of the 10 human Toll-like receptors (TLRs) (TLR3, TLR4, TLR7, TLR8, and TLR9), and four of the 12 mouse TLRs (TLR3, TLR4, TLR7, TLR9) can trigger interferon (IFN)-α, IFN-β, and IFN-λ, which are critical for antiviral immunity. Moreover, TLR3, TLR7, TLR8, and TLR9 differ from TLR4 in two particularly important ways for antiviral immunity: they can be activated by nucleic acid agonists mimicking compounds produced during the viral cycle, and they are typically present within the cell, along the endocytic pathway, where they sense viral products in the intraluminal space. Investigations in mice have demonstrated that the TLR7/9–IFN and TLR3–IFN pathways are different and critical for protective immunity to various experimental viral infections. Investigations in humans with interleukin-1 receptor-associated kinase-4 (IRAK-4) deficiency (unresponsive to TLR7, TLR8, and TLR9), UNC-93B deficiency (unresponsive to TLR3, TLR7, TLR8, and TLR9), and TLR3 deficiency have recently shed light on the role of these two pathways in antiviral immunity in natural conditions. UNC-93B- and TLR3-deficient patients appear to be specifically prone to herpes simplex virus 1 (HSV-1) encephalitis, although clinical penetrance is incomplete, whereas IRAK-4-deficient patients appear to be normally resistant to most viruses, including HSV-1. These experiments of nature suggest that the TLR7-, TLR8-, and TLR9-dependent induction of IFN-α, IFN-β, and IFN-λ is largely redundant in human antiviral immunity, whereas the TLR3-dependent induction of IFN-α, IFN-β, and IFN-λ is critical for primary immunity to HSV-1 in the central nervous system in children but redundant for immunity to most other viral infections.     

Does Toll-like receptor 3 play a biological role in virus infections?

The Toll-like receptor (TLR) family functions to recognize conserved microbial and viral structures with the purpose of activating signal pathways to instigate immune responses against infections by these organisms. For example, in vitro studies reveal that the TLR3 ligand is a double-stranded RNA (dsRNA), a product of viral infections. From this observation, it has been proposed that TLR3 is likely an important first signal for virus infections. We approached this issue by investigating the role of TLR3 in four different infectious viral models (lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), murine cytomegalovirus (MCMV), and reovirus) and in TLR3 genetically deficient ((-/-)) mice. Our results indicate that TLR3 is not universally required for the generation of effective antiviral responses because the absence of TLR3 does not alter either viral pathogenesis or impair host's generation of adaptive antiviral responses to these viruses.     

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily

Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappaB reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pam3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappaB in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappaB. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events.     

Klebsiella pneumoniae Increases the Levels of Toll-Like Receptors 2 and 4 in Human Airway Epithelial Cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IκBα-dependent NF-κΒ pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.     

Expression and function of Toll-like receptors in T lymphocytes

Toll-like receptors (TLRs) are widely expressed in the innate immune system. They recognize conserved microbial ligands such as bacterial lipopolysaccharide, lipopeptides or viral and bacterial RNA and DNA. TLRs play an essential role in innate immune responses and in the initiation of adaptive immune responses. However, certain TLRs are also expressed in T lymphocytes, and the respective ligands can directly modulate T cell function. TLR2, TLR3, TLR5 and TLR9 act as co-stimulatory receptors to enhance proliferation and/or cytokine production of T-cell receptor-stimulated T lymphocytes. In addition, TLR2, TLR5 and TLR8 modulate the suppressive activity of naturally occurring CD25(+)CD4(+) regulatory T cells. The direct responsiveness of T lymphocytes to TLR ligands offers new perspectives for the immunotherapeutic manipulation of T cell responses.     

Cross-talk among Toll-like receptors and their ligands

Toll-like receptors (TLRs) 4, 5, 7 and 9 belong to a familyof proteins that recognize mainly conserved microbial motifs.Though each TLR has a highly specific ability to recognize aparticular microbial pattern, recent papers suggest that someligands are able to affect the expression of different TLRs.In this paper, we have investigated TLR4, 5, 7 and 9 expression,both at mRNA and protein level, following treatment of differentintestinal epithelial cell lines with LPS, flagellin, loxiribine,CpG-oligodeoxynucleotide and peptidoglycan, to assess if thedifferent TLR ligands may modulate the expression of the respectiveTLR and of the unrelated ones. Our results show that a cross-talkexists between TLRs and various ligands, indicating a cross-regulationamong these pattern recognition receptors. In particular, TLR4was generally down-regulated by treatment with ligands otherthan LPS, while flagellin and unrelated microbial-associatedmolecular patterns exerted a general stimulatory activity asregards TLR5 expression. Concerning TLR7 and 9, we have observeda more variable behaviour of the various cell lines with thedifferent ligands. Together, our results demonstrate that theexpression of TLRs in intestinal cells is highly dynamic andtightly regulated in response to encountered microbial stimuli.     

Postulates for validating TLR4 agonists

TLRs play a central role in the innate immune response, recognizing a variety of molecular structures characteristic of pathogens. Although TLR4, together with its co‐receptor myeloid differentiation‐2 (MD‐2), recognize bacterial LPS and therefore Gram‐negative bacterial infections, it also plays a key role in many other pathophysiological processes, including sterile inflammation and viral infection. Specifically, numerous endogenous agonists of TLR4 of notably diverse nature, ranging from proteins to metal ions, have been reported. Direct activation of a single receptor by such a range of molecular signals is very difficult to explain from a structural and mechanistic point of view. It is likely that only a subset of these directly activate the TLR4–MD‐2 complex. We propose three postulates aimed at distinguishing the direct agonists of TLR4 from indirect activators. These postulates are as follows: (i) that the agonist requires the TLR4/MD‐2 receptor complex; (ii) that agonist formed synthetically or in situ must activate the receptor complex in order to eliminate artifacts of contamination by other agonists; and (iii) that a specific molecular interaction between the agonist and TLR4/MD‐2 must be identified. The same type of postulates can be applied to pattern recognition receptors in general.     

Antiviral responses induced by the TLR3 pathway

Antiviral responses are successively induced in virus‐infected animals, and include primary innate immune responses such as type I interferon (IFN) and cytokine production, secondary natural killer (NK) cell responses, and final cytotoxic T lymphocyte (CTL) responses and antibody production. The endosomal Toll‐like receptors (TLRs) and cytoplasmic RIG‐I‐like receptors (RLRs), which recognize viral nucleic acids, are responsible for virus‐induced type I IFN production. RLRs are expressed in most tissues and cells and are primarily implicated in innate immune responses against various viruses through type I IFN production, whereas nucleic acid‐sensing TLRs, TLRs 3, 7, 8 and 9, are expressed on the endosomal membrane of dendritic cells (DCs) and play distinct roles in antiviral immunity. TLR3 recognizes viral double‐stranded RNA taken up into the endosome and serves to protect the host against viral infection by the induction of a range of responses including type I IFN production and DC‐mediated activation of NK cells and CTLs, although the deteriorative role of TLR3 has also been reported in some virus infections. Here, we review the current knowledge on the role of TLR3 during viral infection, and the current understanding of the TLR3‐signalling cascade that operates via the adaptor protein TICAM‐1 (also called TRIF). Copyright © 2011 John Wiley & Sons, Ltd.     

Endogenous ligands of Toll-like receptors

Extensive work has suggested that a number of endogenous molecules such as heat shock proteins (hsp) may be potent activators of the innate immune system capable of inducing proinflammatory cytokine production by the monocyte-macrophage system and the activation and maturation of dendritic cells. The cytokine-like effects of these endogenous molecules are mediated via the Toll-like receptor (TLR) signal-transduction pathways in a manner similar to lipopolysaccharide (LPS; via TLR4) and bacterial lipoproteins (via TLR2). However, recent evidence suggests that the reported cytokine effects of hsp may be a result of the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) being responsible for the putative TLR ligands of hsp include failure to use highly purified hsp free of LPS contamination; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Whether other reported putative endogenous ligands of TLR2 and TLR4 are a result of contamination of pathogen-associated molecular patterns is not clear. It is essential that efforts should be directed to conclusively determine whether the reported putative endogenous ligands of TLRs are a result of the endogenous molecules or of contaminant(s), before exploring further the implication and therapeutic potential of these putative TLR ligands.     

Porcine TLR8 and TLR7 are both activated by a selective TLR7 ligand, imiquimod

Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors (PRR), which are utilized by the innate immune system to recognize microbial components, known as pathogen-associated molecular patterns (PAMP). We cloned and characterized porcine TLR7 and TLR8 genes from pig lymph node tissue. Sequence analysis showed that the aa sequence identities of porcine TLR7 with human, mouse and bovine TLR7 are 85, 78 and 90%, respectively, whereas porcine TLR8 aa sequence identities with human, mouse and bovine TLR8 are 73, 69 and 79%, respectively. Both porcine TLR7 and TLR8 proteins were expressed in cell lines and were N-glycosylated. The stimulatory activity of TLR7 and TLR8 ligands to porcine and human TLR7 and TLR8 in transiently transfected Cos-7 and 293T cells were analyzed using a NF-kappaB reporter assay. Two imidazoquinoline molecules, imiquimod and gardiquimod, markedly activated both porcine TLR7 and TLR8 whereas only human TLR7, but not TLR8, was activated by the ligands. Therefore, receptor specificity for porcine TLR8 is clearly species specific. We further showed that porcine TLR7 and TLR8 are located intracellularly and are mainly within the endoplasmic reticulum. Moreover, activation of transfected cells and porcine PBMC by TLR7 ligands was inhibited by bafilomycin A(1) indicating the requirement of endosomal/lysosomal acidification for activation of the receptors.