Self-Renewal and Toll-like Receptor Signaling Sustain Exhausted Plasmacytoid Dendritic Cells during Chronic Viral Infection

1. Download high-res image (342KB)
2. Download full-size image

     

Natural type I interferon-producing cells as a link between innate and adaptive immunity

Type I interferons (IFNs) are promptly produced upon invasion of pathogens, and activate a broad range of effector cells in the innate and adaptive immune system. Lin⁻CD4⁺CD11c⁻ plasmacytoid dendritic cell precursors (plasmacytoid pre-DCs) produce enormous amounts of type I IFNs in response to viruses and CpG DNA, thus corresponding to the previously described but not fully defined natural type I IFN-producing cells (IPCs). Plasmacytoid pre-DCs strongly express toll-like receptor (TLR) 7 and TLR9, in contrast to monocytes, which mainly express TLR1, 2, 4, 5, and 8, suggesting that these two DC precursors recognize different microbial molecules and that they may have developed through different evolutionary trails. Three different stimuli, CpG DNA plus CD40 ligand, interleukin-3 (IL-3), and herpes simplex virus, stimulate plasmacytoid pre-DCs to differentiate into DCs that induce distinct types of T helper cells, i.e., Th1, Th2, and IFN-γ- and IL-10-producing T cells, respectively. The remarkable versatility of plasmacytoid pre-DCs distinguishes them from other cell types in the immune system that have only limited functions, and suggests that these cells may play a key role in integrating the innate and adaptive aspects of various immune responses.     

Toll-like receptor signaling in endogenous neuroprotection and stroke

Stroke and other cerebral vascular diseases are a leading cause of morbidity and mortality in the United States. Despite intensive research to identify interventions that lessen cerebrovascular injury, no major therapies exist. Development of stroke prophylaxis involves an understanding of the mechanisms of damage following cerebral ischemia, and elucidation of the endogenous mechanisms that combat further brain injury. Toll-like receptors (TLRs) are critical components of the innate immune system that have been shown recently to mediate ischemic injury. Paradoxically, TLR ligands administered systemically induce a state of tolerance to subsequent ischemic injury. Herein we suggest that stimulation of TLRs prior to ischemia reprograms TLR signaling that occurs following ischemic injury. Such reprogramming leads to suppressed expression of pro-inflammatory molecules and enhanced expression of numerous anti-inflammatory mediators that collectively confer robust neuroprotection. Our findings indicate that numerous preconditioning stimuli lead to TLR activation, an event that occurs prior to ischemia and ultimately leads to TLR reprogramming. Thus genomic reprogramming of TLR signaling may be a unifying principle of tolerance to cerebral ischemia.     

Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I

Dendritic cells utilize various sets of Toll-like receptors (TLR) or cytosolic sensors to detect pathogens and evoke immune responses. In patients with hepatitis C virus (HCV) infection, a higher prevalence of various infectious diseases is reported; suggesting that innate immunity against pathogens is impaired. The aim of this study was to clarify whether the TLR and retinoic acid inducible gene-I (RIG-I) system in myeloid dendritic cells is preserved or not in chronic HCV infection. The expression of TLRs, RIG-I and its relatives were compared in myeloid dendritic cells between 39 patients and 52 healthy volunteers. The induction of type-I interferon (IFN) and inflammatory cytokines was examined in response to agonists for TLR2 (palmitoyl-3-cysteine-serine-lysine-4), TLR3/RIG-I (polyinosine-polycytidylic acid) or TLR4 (lipopolysaccharide). The relative expressions of TLR2, TLR4, RIG-I, and LGP2 from the patients were significantly higher than those from the volunteers, whereas TLR3 and MDA-5 expressions did not differ. In search for factors regulating TLR/RIG-I expression, it was shown that IFN-alpha, polyinosine-polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG-I, but TNF-alpha, HCV core or HCV non-structural proteins did not. For the functional analyses, myeloid dendritic cells from the patients induced significantly less amounts of IFN-beta, TNF-alpha and IL-12p70 in response to polyinosine-polycytidylic acid or lipopolysaccharide. It is noteworthy that the expression of TRIF and TRAF6, which are essential adaptor molecules transmitting TLR3 or TLR4-dependent signals, is reduced in the patients. Thus, innate cytokine responses in myeloid dendritic cells are impaired regardless of enhanced expressions of TLR2, TLR4, and RIG-I in HCV infection.     

Pathological role of Toll-like receptor signaling in cerebral malaria

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.     

RIG-I-like receptors: cytoplasmic sensors for non-self RNA

Viral infection results in the generation of non-self RNA species in the cells, which is recognized by retinoic acid inducible gene-I-like receptors (RLRs), and initiates innate antiviral responses, including the production of proinflammatory cytokines and type I interferon. In this review, we summarize reports on virus-specificity of RLRs, structures of non-self RNA patterns, structural biology of RLRs, and the signaling adapter molecules involved in antiviral innate immunity.     

Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious diseases

Toll-like receptors (TLRs) are the best-studied family of pattern-recognition receptors (PRRs), whose task is to rapidly recognize evolutionarily conserved structures on the invading microorganisms. Through binding to these patterns, TLRs trigger a number of proinflammatory and anti-microbial responses, playing a key role in the first line of defence against the pathogens also promoting adaptive immunity responses. Growing amounts of data suggest that single nucleotide polymorphisms (SNPs) on the various human TLR proteins are associated with altered susceptibility to infection. This review summarizes the role of TLRs in innate immunity, their ligands and signalling and focuses on the TLR SNPs which have been linked to infectious disease susceptibility.     

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.     

Toll-like receptor 4 polymorphisms and idiopathic chromosomally normal miscarriage

BACKGROUND: Lipopolysaccharide (LPS or endotoxin) exposure resulting from microbial invasion of the endometrium disturbs the Th1/Th2 balance at the feto-maternal interface and has been linked to the risk of idiopathic miscarriage in a range of human and animal studies. Toll-like receptor 4 (TLR4) mediates LPS signalling, and the human TLR4 gene harbours two single-nucleotide polymorphisms (SNPs) known to reduce LPS responsiveness. We hypothesized that genetic variation altering TLR4 function may influence the risk of idiopathic pregnancy loss. METHODS AND RESULTS: We examined fetal TLR4 genotypes in a case-control cohort of chromosomally normal miscarriages (n=96) and healthy term newborns (n=113). The allele frequencies of the Asp299Gly and Thr399Ile TLR4 SNPs were determined by quantitative PCR using DNA extracted from extraembryonic tissues and umbilical cord blood, respectively. TLR4 genotype frequencies were not significantly different between cases and controls. CONCLUSIONS: There was no association between fetal TLR4 polymorphisms, Asp299Gly and Thr399Ile, known to blunt LPS responsiveness, and the risk of idiopathic, chromosomally normal miscarriage. Nevertheless, TLR4 or perhaps other LPS-binding chaperone molecules are biologically plausible candidate genes that may alter the risk of idiopathic miscarriage.     

Translational mini-review series on Toll-like receptors: networks regulated by Toll-like receptors mediate innate and adaptive immunity

The Toll-like receptor (TLR) family provide key components of mammalian immunity and are part of the earliest surveillance mechanisms responding to infection. Their activation triggers the innate immune response, and is crucial to the successful induction of Th1/Th2-phenotyped adaptive immunity. Innate immunity was long considered to be non-specific and somewhat simple compared to adaptive immunity, mediated via the engulfment and lysis of microbial pathogens by phagocytic cells such as macrophages and neutrophils, and involving no complex protein-protein interactions. The emergence of the TLR field has contributed to a revision of our understanding, and innate immunity is now viewed as a highly complex process, in line with adaptive immunity. This review will give a brief overview of our current knowledge of TLR biology, and will focus on TLRs as key components in complex networks that activate, integrate and select the appropriate innate and adaptive immune responses in the face of immunological danger.     

Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS). MD-2 is associated with TLR4 and imparts LPS responsiveness to it. Little is known, however, as to whether MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of a species-specific pharmacology of lipid IVa, an analogue of lipid A. Lipid IVa acted agonistically on mouse (m) TLR4/MD-2 but not on human (h) TLR4/MD-2. Lipid IVa antagonized the agonistic effect of lipid A on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is conferred by TLR4 or MD-2. hMD-2 was clearly distinct from mMD-2 in the way of influencing LPS recognition by mTLR4. hMD-2 conferred on mTLR4 responsiveness to lipid A but not to lipid IVa. Moreover, lipid IVa acted as a lipid A antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.     

Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity

(TLRs) respond to pathogens to initiate the innate immune response and direct adaptive immunity, and evidence to date suggests that they have a role in the detection of viruses. Many viral macromolecules have been shown to activate anti-viral signalling pathways via TLRs, leading to the induction of cytokines and interferons, while viruses also have means of not only evading detection by TLRs, but also of subverting these receptors for their own purposes. This review discusses the role of TLRs in the context of other known viral detection systems, and examines some of the often surprising results from studies using mice deficient in TLRs and their adaptors, in an attempt to unravel the particular contribution of TLRs to anti-viral immunity.     

Toll-like receptor signaling in neonatal sepsis and inflammation: a matter of orchestration and conditioning

Altered neonatal Toll-like receptor (TLR) function is hypothesized to contribute to the heightened susceptibility to infection and perpetuated inflammation in term and preterm neonates, clinically evident in neonatal sepsis and increased rates of inflammatory disorders. Current data indicate that basal TLR expression in term neonates equals adult expression patterns, while expression in preterm infants seems to increase, depending on gestational age. Regarding TLR signaling, some studies suggest TLR incompetence in neonates associated with impaired pro-inflammatory responses, others describe neonatal TLR function well developed and allude to its hyper-inflammation tendency. We discuss the competing positions and considerable limitations of research approaches and conclude that neonatal innate immunity is not generally less able to respond to TLR stimulation. Moreover, we describe pre-conditioning factors other than immaturity having a comparable impact. In the long term, better understanding of the complex interplay of pre- and postnatal conditions and maturation-dependent neonatal TLR function may provide new therapeutic approaches.     

Toll-like receptor (TLR) 4 polymorphism Asp299Gly is not associated with disease course in Dutch sarcoidosis patients

The aetiology of sarcoidosis, a systemic disorder characterized by the formation of non-caseating granulomas in variable organs, remains enigmatic. Clarification is hampered by heterogeneity in disease phenotypes and course, due partly to the influence of a variety of genetic and environmental factors. Multiple studies have pointed towards bacteria as possible causative agents. Toll-like receptors (TLR) are innate immunity receptors important in the immune response against pathogens. TLR-4, together with CD14 and MD-2, is an essential receptor for the recognition of lipopolysaccharide (LPS), unique to the cell wall of Gram-negative bacteria. Recently, an association between TLR-4 polymorphism Asp299Gly, leading to a change in the extracellular domain of the receptor and possible hyporesponsiveness to LPS, and a chronic course of sarcoidosis was found in German patients. In the present study this polymorphism was genotyped in 156 Dutch sarcoidosis patients and 200 healthy Dutch controls using dual-labelled fluorescent oligonucleotides. No differences were found in allelic distributions between patients and controls (P = 0.79) or within the different clinical entities of the sarcoidosis group (P = 0.44). Importantly, there were no differences between the Dutch and German sarcoidosis patients (P = 0.62). However, the allelic distribution of the Asp299Gly polymorphism differed significantly between both control groups (P = 0.04). This study highlights the importance of testing a reported gene association in a distinct population when performing genetic association studies.     

RNA recognition and signal transduction by RIG-I-like receptors

Summary:  Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid-inducible gene-I-like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.     

Human platelets express Toll-like receptor 3 and respond to poly I:C

Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR’s) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca²⁺]_i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA.     

Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis

The aetiology of sarcoidosis, an inflammatory granulomatous multi-system disorder, is unclear. It is thought to be the product of an unknown exogenous antigenic stimulus and an endogenous genetic susceptibility. Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Lipopolysaccharide (LPS), for example, binds to TLR 4. Two different polymorphisms for the TLR4 gene (Asp299Gly and Thr399Ile) have been described recently. This leads to a change in the extracellular matrix function of TLR4 and to impaired LPS signal transduction. We genotyped a total of 141 Caucasian patients with sarcoidosis and 141 healthy unrelated controls for the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. Among sarcoidosis patients the prevalence for each Asp299Gly and Thr399Ile mutant allele was 15.6% (22/141). In the control group the prevalence was 5.67% (8/141) (P = 0.07). In the subgroup of patients with acute sarcoidosis there was no difference in the control group (P = 0.93), but there was a highly significant association between patients with a chronic course of sarcoidosis and TLR4 gene polymorphisms (P = 0.01).     

The role of Toll-like receptor 10 in modulation of trained immunity

Toll-like receptor 10 (TLR10) is the only member of the human Toll-like receptor family with an inhibitory function on the induction of innate immune responses and inflammation. However, its role in the modulation of trained immunity (innate immune memory) is unknown. In the present study, we assessed whether TLR10 modulates the induction of trained immunity induced by β-glucan or bacillus Calmette–Guérin (BCG). Interleukin 10 receptor antagonist production was increased upon activation of TLR10 ex vivo after BCG vaccination, and TLR10 protein expression on monocytes was increased after BCG vaccination, whereas anti-TLR10 antibodies did not significantly modulate β-glucan or BCG-induced trained immunity in vitro. A known immunomodulatory TLR10 missense single-nucleotide polymorphism (rs11096957) influenced trained immunity responses by β-glucan or BCG in vitro. However, the in vivo induction of trained immunity by BCG vaccination was not influenced by TLR10 polymorphisms. In conclusion, TLR10 has a limited, non-essential impact on the induction of trained immunity in humans.     

Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of Toll-like receptor 2 (TLR2). In this study, we demonstrated that lipoproteins obtained from S. aureus are potent activators of TLR2. A fraction obtained by Triton X-114 phase partitioning activated cells through TLR2. The fraction contained proteins and LTA. The activity was detected in compounds in a mass range of 12-40 kDa. Proteinase K digested the active compounds into lower molecular weight active materials <10 kDa. In contrast, hydrofluoric acid treatment, which decomposes LTA, did not alter the molecular mass of the active compounds. Further, most of the activity was abrogated by lipoprotein lipase digestion. These results suggested that lipoproteins are predominant TLR2 ligands in S. aureus cell wall components.