Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.     

The role of innate immune receptors in the control of Brucella abortus infection: toll-like receptors and beyond

Research into intracellular sensing of microbial products is an up and coming field in innate immunity. Toll-like receptors (TLRs) recognize Brucella spp. and bacterial components and initiate mononuclear phagocyte responses that influence both innate and adaptive immunity. Recent studies have revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella infection. TLR2, TLR4 and TLR9 have been implicated in host interactions with Brucella; however, TLR9 has the most prominent role. Further, the relationship between specific Brucella molecules and various signal transduction pathways needs to be better understood. MyD88-dependent and TRIF-independent signaling pathways are involved in Brucella activation of innate immune cells through TLRs. We have recently reported the critical role of MyD88 molecule in dendritic cell maturation and interleukin-12 production during B. abortus infection. This article discusses recent studies on TLR signaling and also highlights the contribution of NOD and type I IFN receptors during Brucella infection. The better understanding of the role by such innate immune receptors in bacterial infection is critical in host-pathogen interactions.     

Toll样受体4与病毒感染的研究进展

Toll样受体4（TLR4）是固有免疫系统中能够识别病原相关分子模式的受体家族成员,可识别革兰氏阴性菌的脂多糖（LPS）而在细菌感染性疾病的发生中起重要作用。近年来越来越多的研究发现,TLR4还广泛参与病毒感染性疾病的发生和病毒的免疫逃逸,由于其信号转导通路的独特性和细胞定位的可变性,再次引起人们极大的研究兴趣。该文将介绍TLR4的生物学特性、信号转导通路及TLR4与病毒感染的最新研究进展。     

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.     

MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses

Background

Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.

Methodology/Principal Findings

We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.

Conclusions/Significance

These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.     

The role of Toll-like receptor signalling in the pathogenesis of arthritis

Recent evidence highlighted the role of Toll-like receptors (TLRs) as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules of antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. In this review, recent studies on TLR signal transduction pathways activated by corresponding ligands are summarized and evidence for a possible role of TLR signalling in the pathogenesis of rheumatoid arthritis is discussed.     

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.     

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-dependent and toll-independent innate antiviral immunity

Until recently, adaptive immunity and cytotoxic T cells were considered as the only essential components of the antiviral defence arsenal. Additional data that do not rule out the crucial role of these cells in the clearance of viral pathogens have, however, recently emerged. They indicate that innate immune cells such as macrophages, dendritic cells, gammadelta T cells as well as natural killer (NK) cells play a primordial role in this mechanism. It is now well established that innate immune cells can detect various pathogens (bacteria, viruses, fungi or parasites) very rapidly and respond to their presence through the activation of specific receptors. Once activated, these molecules trigger several signalling cascades that culminate in the establishment of very potent defence mechanisms. In addition, cytokines produced during this initial response are essential for the activation of the adaptive immune response which will add specificity and memory to the system. Among the innate immune receptors, attention has focused on the Toll-like receptors (TLR) and many reports indicate that some of the TLRs are clearly involved in defence against viral pathogens. However, new molecules, acting independently from any TLR, have recently been discovered. They define a second antiviral pathway which is presently the subject of intense research. In this article, we will review the role of the different molecules involved in each pathway within the framework of innate antiviral defence.     

Protozoan encounters with Toll-like receptor signalling pathways: implications for host parasitism

Toll-like receptors (TLRs) have emerged as a major receptor family involved in non-self recognition. They have a vital role in triggering innate immunity and orchestrate the acquired immune response during bacterial and viral infection. However, the role of TLRs during infection with protozoan pathogens is less clear. Nevertheless, our understanding of how these parasitic microorganisms engage the host TLR signalling system has now entered a phase of rapid expansion. This Review describes recent insights into how parasitic protozoans are sensed by TLR molecules, and how the TLR system itself can be targeted by these microbial pathogens for their own survival.     

动物TLR4基因多态性及其抗病相关性研究进展

TLR4是天然免疫系统中的一种重要模式识别受体,可选择性识别病原微生物而启动天然免疫,在宿主天然免疫和获得性免疫中具有重要作用。此外,大量研究表明,TLR4基因的多态性与多种动物的细菌、病毒和寄生虫性疾病密切相关,说明通过TLR4基因的研究可为动物分子标记辅助选育研究提供依据。该文简要论述部分动物TLR4基因多态性及其与抗病相关性的研究进展。     

Toll-like receptor pathways in the immune responses to mycobacteria

The control of Mycobacterium tuberculosis infection depends on recognition of the pathogen and the activation of both the innate and adaptive immune responses. Toll-like receptors (TLR) were shown to play a critical role in the recognition of several pathogens. Mycobacterial antigens recognise distinct TLR resulting in rapid activation of cells of the innate immune system. Recent evidence from in vitro and in vivo investigations, summarised in this review demonstrates TLR-dependent activation of innate immune response, while the induction of adaptive immunity to mycobacteria may be TLR independent.     

Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists

Early recognition of invading bacteria by the innate immune system has a crucial function in antibacterial defense by triggering inflammatory responses that prevent the spread of infection and suppress bacterial growth. Toll-like receptor 4 (TLR4), the innate immunity receptor of bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, LBP, CD14 and MD-2 receptors, that bind lipopolysaccharide (LPS) and present it to TLR4 by forming the activated (TLR4-MD-2-LPS)(2) complex. Small molecules active in modulating the TLR4 activation process have great pharmacological interest as vaccine adjuvants, immunotherapeutics or antisepsis and anti-inflammatory agents. In this review we present natural and synthetic molecules active in inhibiting TLR4-mediated LPS signalling in humans and their therapeutic potential. New pharmacological applications of TLR4 antagonists will be also presented related to the recently discovered role of TLR4 in the insurgence and progression of neuropathic pain and sterile inflammations.     

Innate immune recognition of viral infection

Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and triggers innate immune responses. A subset of TLRs recognize viral components and induce antiviral responses by producing type I interferons. Whereas TLR2 and TLR4 recognize viral components at the cell surface, TLR3, TLR7, TLR8 and TLR9 are exclusively expressed in endosomal compartments. After phagocytes internalize viruses or virus-infected apoptotic cells, viral nucleic acids are released in phagolysosomes and are recognized by these TLRs. Recent reports have shown that hosts also have a mechanism to detect replicating viruses in the cytoplasm in a TLR-independent manner. In this review, we focus on the viral recognition by innate immunity and the signaling pathways.     

Insights into innate immune activation via PS-ASO–protein–TLR9 interactions

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary—in part—as a function of 2′modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO–protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

Toll-like receptor 8: the awkward TLR

Toll-like receptors (TLR) sense a variety of microbial products and play an important role in the mounting of innate and adaptive immune responses. TLR1 to TLR9 are common in mice and humans and recognize similar ligands in both species, with the exception of TLR8. Human TLR7 and TLR8 and mouse TLR7 detect viral single-stranded RNA and imidazoquinoline compounds, while mouse TLR8 not. Based on this discrepancy, for long time it was believed that mouse TLR8 is not functional and as a consequence the contribution of TLR8 to innate immunity remained poorly understood. Our recent studies revealed an important role for TLR8 in the regulation of TLR7-mediated autoimmunity in the mouse. This review illustrates our current understanding regarding the function of TLR8 and its potential for future clinical use for the treatment and/or prevention of various pathological conditions.     

Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/-) DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.