Innate immunity: structure and function of TLRs

The innate immune system provides the first line of defence against infection. Through a limited number of germline-encoded receptors called pattern recognition receptors (PRRs), innate cells recognize and are activated by highly conserved structures expressed by large group of microorganisms called pathogen-associated molecular patterns (PAMPs). PRRs are involved either in recognition (scavenger receptors, C-type lectins) or in cell activation (Toll-like receptors or TLR, helicases and NOD molecules). TLRs play a pivotal role in cell activation in response to PAMPs. TLR are type I transmembrane proteins characterized by an intracellular Toll/IL 1 receptor homology domain that are expressed by innate immune cells (dendritic cells, macrophages, NK cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). In all the cell types analyzed, TLR agonists, alone or in combination with costimulatory molecules, induce cell activation. The crucial role played by TLR in immune cell activation has been detailed in dendritic cells. A TLR-dependent activation of dendritic cells is required to induce their maturation and migration to regional lymph nodes and to activate na?ve T cells. The ability of different cell types to respond to TLR agonists is related to the pattern of expression of the TLRs and its regulation as well as their intracellular localization. Recent studies suggest that the nature of the endocytic and signaling receptors engaged by PAMPs may determine the nature of the immune response generated against the microbial molecules, highlighting the role of TLRs as molecular interfaces between innate and adaptive immunity. In this review are summarized the main biological properties of the TLR molecules.     

Antiviral signaling through pattern recognition receptors

Viral infection is detected by the host innate immune system. Innate immune cells such as dendritic cells and macrophages detect nucleic acids derived from viruses through pattern recognition receptors (PRRs). Viral recognition by PRRs initiates the activation of signaling pathways that lead to production of type I interferon and inflammatory cytokines, which are important for the elimination of viruses. Two types of PRRs that recognize viral nucleic acids, Toll-like receptors (TLR) and RIG-I-like RNA helicases (RLH), have been identified. Of the TLRs, TLR3 recognizes viral double-stranded (ds) RNA, TLR7 and human TLR8 identify viral single-stranded (ss) RNA and TLR9 detects viral DNA. TLRs are located in endosomal compartments, whereas RLH are present in the cytoplasm where they detect viral dsRNA or ssRNA. Here we review the role of TLRs and RLHs in the antiviral innate immune response.     

Polyinosinic acid is a ligand for toll-like receptor 3

Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.     

Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors

Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. Interestingly, the chimeras between TLR4 and either TLR3, TLR7, or TLR9 were localized completely intracellularly whereas other chimeras were expressed on the cell surface. Lipopolysaccharide (LPS), a ligand for these chimeras, induced the activation of nuclear factor kappa B and mitogen-activated protein kinases and the subsequent production of pro-inflammatory cytokines in macrophages expressing TLR4, TLR4/TLR5, or TLR4/TLR8 chimeras but not in macrophages expressing TLR4/TLR1, TLR4/TLR2, or TLR4/TLR6 chimeras. Co-expression of unresponsive chimeras in some combinations (chimeras with TLR1+TLR2 or TLR2+TLR6 but not TLR1+TLR6) resulted in LPS responsiveness, indicating functional complementarity. Furthermore, the pair of TLR2+TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1+TLR2 pair. Finally, LPS induced effective interferon-beta production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.     

固有免疫应答与动脉粥样硬化关系的研究进展

固有免疫应答在动脉粥样硬化(atherosclerosis,As)的发生和发展中起重要作用．固有免疫应答细胞,包括单核/巨噬细胞、肥大细胞、自然杀伤细胞、中性粒细胞和树突状细胞,是机体抵御微生物和异物入侵的第一道防线．这些细胞广泛参与As中泡沫细胞形成、斑块内基质降解、细胞凋亡、血管新生和斑块破裂等事件．模式识别受体是免疫细胞上识别病原体(或某些内源性成分)相关分子模式的一类受体分子,包括Toll样受体和NOD样受体,介导固有免疫应答反应．Toll样受体在固有免疫应答细胞中具有不同程度的表达,在As中具有不同的作用,如TLR2和TLR4对As起促进作用,而TLR3具有As保护作用．NLRP3炎性体与动脉血管壁的早期损伤有关．对固有免疫应答细胞及模式识别受体在As形成中的作用进行深入研究,不仅有助于理解As的形成过程,而且还能为临床上防治心血管类疾病提供了新的治疗靶点和诊断指标．     

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.     

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.     

Differential recognition of HIV-stimulated IL-1β and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1β and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the “priming” signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1β/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K⁺ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.     

TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity

TLR2 plays a role as a pattern-recognition receptor in the innate immune response involving secreted proteins against microbial pathogens. To examine its possible involvement in the cellular response, we determined the levels of the engulfment and subsequent killing of bacteria by macrophages prepared from TLR2-deficient and wild-type mice. The level of the engulfment of Staphylococcus aureus or Escherichia coli was almost the same between TLR2-lacking and wild-type macrophages. However, the colony-forming ability of engulfed S. aureus, but not of E. coli, decreased to a greater extent in TLR2-lacking macrophages than in the wild-type control. The incubation with S. aureus caused activation of JNK in wild-type macrophages but not in TLR2-lacking macrophages, and the pretreatment of wild-type macrophages with a JNK inhibitor increased the rate of killing of engulfed S. aureus, but again not of E. coli. In addition, the number of colonies formed by engulfed S. aureus increased in the JNK-dependent manner when TLR2-lacking macrophages were pretreated with LPS. Furthermore, JNK seemed to inhibit the generation of superoxide, not of NO, in macrophages. These results collectively suggested that the level of superoxide is reduced in macrophages that have engulfed S. aureus through the actions of TLR2-activated JNK, resulting in the prolonged survival of the bacterium in phagosomes. The same regulation did not influence the survival of E. coli, because this bacterium was more resistant to superoxide than S. aureus. We propose a novel bacterial strategy for survival in macrophages involving the hijacking of an innate immune receptor.     

Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

Toll-like receptor 9, CpG DNA and innate immunity

Innate immunity provides the first line of defense against invading pathogens and is essential for survival in the absence of adaptive immune responses. Innate immune recognition relies on a limited number of germ-line encoded receptors, such as Toll-like receptors (TLRs), that evolved to recognize conserved molecular patterns of microbial origin. To date, ten transmembrane proteins in the TLR family have been described. It is becoming increasingly clear that bacterial CpG DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG are potent inducers of the innate immune system including dendritic cells (DCs), macrophages, and natural killer (NK) and NKT cells. Recent studies indicate that mucosal or systemic delivery of CpG DNA can act as a potent adjuvant in a vaccine combination or act alone as an anti-microbial agent. Recently, it was shown that TLR9 is essential for the recognition of unmethylated CpG DNA since cells from TLR9-deficient mice are unresponsive to CpG stimulation. Although the effects of CpG DNA on bone marrow-derived cells are beginning to unfold, there has been little or no information regarding the mechanisms of CpG DNA function on non-immune cells or tissues. This review focuses on the recent advances in CpG-DNA/TLR9 signaling effects on the activation of innate immunity.     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of pro-inflammatory cytokines by mycobacterial lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.     

Toll样受体识别机制及其生物学意义

Toll样受体介导的信号转导通路在对抗外来病原体的天然免疫应答中起重要作用。Toll样受体是一个天然模板识别受体家族,能识别固有性模板(微生物和哺乳动物所共有的病原相联的分子模板PAMPs)。Toll样受体通过巨噬细胞和其他免疫细胞来识别,其中TLR4识别内毒素、TLR2识别肽聚糖、TLR9识别细菌DNA、TLR5识别鞭毛蛋白、TLR3识别双链RNA等。本探讨了多种Toll受体家族成员在动物体内识别机理及功能,概述了其应用研究进展。     

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV,and MERS-CoV genomes

During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.     

Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense

Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.     

The biology of Toll-like receptors

In 1997, a human homologue of the Drosophila Toll protein was described, a protein later to be designated Toll-like receptor 4 (TLR4). Since that time, additional human and murine TLR proteins have been identified. Mammalian TLR proteins appear to represent a conserved family of innate immune recognition receptors. These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in the activation of genes that mediate innate immune defenses. Numerous studies have now identified a wide variety of chemically-diverse bacterial products that serve as putative ligands for TLR proteins. More recent studies have identified the first endogenous protein ligands for TLR proteins. TLR signaling represents a key feature of innate immune response to pathogen invasion.     

TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells In Vitro

The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC''s) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC''s can alter immune responses of macrophages and dendritic cells (DC''s) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-β dependent. In summary, the results of our study indicate that EEC''s play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.