Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity

In innate immunity, pattern recognition receptors discriminate between self- and infectious non-self-matter. Mammalian homologs of the Drosophila Toll protein, which are collectively referred to as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS) and lipoproteins, whereas the Drosophila Toll protein does not act as a PAMP receptor, but rather binds to Sp?tzle, an endogenous peptide. In Drosophila, innate immune surveillance is mediated by members of the peptidoglycan recognition protein (PGRP) family, which recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions including the release of antimicrobial peptides and the activation of the prophenoloxidase cascade, the latter effecting localized wound healing, melanization, and microbial phagocytosis. In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte.     

Influence of Toll‐Like Receptor Gene Polymorphisms to Tuberculosis Susceptibility in Humans

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tb), and it remains one of the major bacterial infections worldwide. Innate immunity is an important arm of antimycobacterial host defence mechanism that senses various pathogen‐associated molecular patterns (PAMP) of microbes by a variety of pattern recognition receptors (PRRs). As per the recent discovery, Toll‐like receptors (TLRs) play a crucial role in the recognition of M. tb, this immune activation occurs only in the presence of functional TLRs. Variants of TLRs may influence their expression, function and alters the recognition or signalling mechanism, which leads to the disease susceptibility. Hence, the identification of mutations in these receptors could be used as a marker to screen the individuals who are at risk. In this review, we discuss TLR SNPs and their signalling mechanism to understand the susceptibility to TB for better therapeutic approaches.     

Role of lectins in the innate immunity of horseshoe crab.

We have purified five types of lectins, named tachylectins, from circulating hemocytes and hemolymph plasma of the Japanese horseshoe crab, Tachypleus tridentatus. Tachylectin-1 interacts with Gram-negative bacteria probably through 2-keto-3-deoxyoctonate, one of the constituents of lipopolysaccharides (LPS). Tachylectin-1 also binds to polysaccharides such as agarose and dextran with broad specificity. Tachylectin-2 binds to D-GlcNAc or D-GalNAc and recognizes staphylococcal lipoteichoic acids and LPS from several Gram-negative bacteria. In contrast, tachylectins-3 and -4 specifically bind to S-type LPS from several Gram-negative bacteria through a certain sugar moiety on the O-specific polysaccharides (O-antigens). Tachylectin-5 identified in hemolymph plasma has the strongest bacterial agglutinating activity in the five types of tachylectins, and exhibits broad specificity against acetyl group-containing substances. Thus, the innate immune system of horseshoe crab may recognize invading pathogens through a combinatorial method using lectins with different specificities against carbohydrates exposed on pathogens. An encounter of these lectins derived from hemocytes and hemolymph plasma at injured sites, in response to the stimulation of LPS, suggests that they serve synergistically to accomplish an effective host defense against invading microbes and foreign substances.     

Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models

The vertebrate innate immune system recognizes infectious non-self by employing a set of germline-encoded receptors such as nucleotide-binding oligomerisation domain proteins (NODs) or Toll-like receptors (TLRs). These proteins are involved in the recognition of various microbial-derived molecules, including lipopolysaccharide (LPS), peptidoglycan (PGN) and beta1,3-glucan. Drosophila Toll receptors are not directly dedicated to non-self recognition and insect NOD orthologues have not yet been identified. Studies started more than 20 years ago and conducted on different insect models have identified other receptors on which invertebrate innate systems rely to sense invading microorganisms.     

REVIEW ARTICLE: Toll‐Like Receptors at the Maternal–Fetal Interface in Normal Pregnancy and Pregnancy Disorders

Citation Koga K, Mor G. Toll‐like receptors at the maternal–fetal interface in normal pregnancy and pregnancy disorders. Am J Reprod Immunol 2010 Toll‐like receptors (TLR) form the major family of pattern recognition receptors (PRR) that are involved in innate immunity. Innate immune responses against microorganisms at the maternal–fetal interface may have a significant impact on the success of pregnancy, as intrauterine infections have been shown to be strongly associated with certain disorders of pregnancy. At the maternal–fetal interface, TLRs are expressed not only in the immune cells but also in non‐immune cells such as trophoblasts and decidual cells; moreover, their expression patterns vary according to the stage of pregnancy. Here, we will describe potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR‐mediated innate immune response will be discussed via animal model studies, as well as clinical observations.     

Toll‐like Receptors at the Maternal‐Fetal Interface in Normal Pregnancy and Pregnancy Complications

Toll‐like receptors (TLRs) form the major family of pattern recognition receptors (PRRs) that are involved in innate immunity. Innate immune responses against microorganisms at the maternal‐fetal interface may have a significant impact on the success of pregnancy, as intrauterine infections have been shown to be strongly associated with certain complications of pregnancy. At the maternal‐fetal interface, TLRs are expressed not only in the immune cells but also in non‐immune cells such as trophoblasts and decidual cells; moreover, their expression patterns vary according to the stage of pregnancy. Here, we will update potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR‐mediated innate immune response will be discussed via animal model studies, as well as clinical observations.     

Toll-like receptors: cellular signal transducers for exogenous molecular patterns causing immune responses

Innate immunity initiates protection of the host organism against invasion and subsequent multiplication of microbes by specific recognition. Germ line-encoded receptors have been identified for microbial products such as mannan, lipopeptide, peptidoglycan (PGN), lipoteichoic acid (LTA), lipopolysaccharide (LPS), and CpG-DNA. The Drosophila Toll protein has been shown to be involved in innate immune response of the adult fruitfly. Members of the family of Toll-like receptors (TLRs) in vertebrates have been implicated as pattern recognition receptors (PRRs). Ten TLRs are known and six of these have been demonstrated to mediate cellular activation by distinct microbial products. TLR4 has been implicated as activator of adaptive immunity, and analysis of systemic LPS responses in mice led to the identification of LPS-resistant strains instrumental in its identification as a transmembrane LPS signal transducer. Structural similarities between TLRs and receptor molecules involved in immune responses such as CD14 and the IL-1 receptors (IL-1Rs), as well as functional analysis qualified TLR2 as candidate receptor for LPS and other microbial products. Targeted disruption of the TLR9 gene in mice led to identification of TLR9 as CpG-DNA signal transducer. Involvement of TLR5 in cell activation by bacterial flagellin has been demonstrated. Further understanding of recognition and cellular signaling activated through the ancient host defense system represented by Toll will eventually lead to means for its therapeutic modulation.     

Plasma cell toll‐like receptor (TLR) expression differs from that of B cells,and plasma cell TLR triggering enhances immunoglobulin production

Toll‐like receptors (TLRs) are key receptors of the innate immune system and show cell subset‐specific expression. We investigated the messenger RNA (mRNA) expression of TLR genes in human haematopoietic stem cells (HSC), in naïve B cells, in memory B cells, in plasma cells from palatine tonsils and in plasma cells from peripheral blood. HSC and plasma cells showed unrestricted expression of TLR1–TLR9, in contrast to B cells which lacked TLR3, TLR4 and TLR8 but expressed mRNA of all other TLRs. We demonstrated, for the first time, that TLR triggering of terminally differentiated plasma cells augments immunoglobulin production. Thus, boosting the immediate antibody response by plasma cells upon pathogen recognition may point to a novel role of TLRs.     

Critical roles of Toll-like receptors in host defense

Drosophila Toll is involved not only in dorsoventral patterning of embryos but also in immune responses to microbial infection. Several Toll-like receptors (TLRs) have also been identified in mammals. They are expressed on macrophages or dendritic cells (DCs), which are essential sentinels for innate immunity. These cells utilize TLRs as a recognition and signal transducing receptor for microbial molecular components. The most characterized mammalian TLR, TLR4, is a receptor for lipopolysaccharides (LPS). TLR2 recognizes other components, such as peptideglycans (PGN). This recognition, called pattern recognition, is essential for the establishment of innate immunity, which is the basis for host defense. In this article, we review recent findings about this expanding receptor family.     

Role of Toll-like receptors in pathogen recognition

The innate immune system relies on a vast array of non-clonally expressed pattern recognition receptors for the detection of pathogens. Pattern recognition receptors bind conserved molecular structures shared by large groups of pathogens, termed pathogen-associated molecular patterns. The Toll-like receptors (TLRs) are a recently discovered family of pattern recognition receptors which show homology with the Drosophila Toll protein and the human interleukin-1 receptor family. Engagement of different TLRs can induce overlapping yet distinct patterns of gene expression that contribute to an inflammatory response. The TLR family is characterized by the presence of leucine-rich repeats and a Toll/interleukin-1 receptor-like domain, which mediate ligand binding and interaction with intracellular signaling proteins, respectively. Most TLR ligands identified so far are conserved microbial products which signal the presence of an infection, but evidence for some endogenous ligands that might signal other danger conditions has also been obtained. Molecular mechanisms for pathogen-associated molecular pattern recognition still remain elusive but seem to be more complicated than initially anticipated. In most cases, direct binding of microbial ligands to TLRs still has to be demonstrated. Moreover, Drosophila TLRs bind endogenous ligands, generated through a proteolytic cascade in response to an infection. In the case of endotoxin, recognition involves a complex of TLR4 and a number of other proteins. Moreover, TLR heterodimerization further extends the spectrum of ligands and modulates the response towards specific ligands. The fact that TLR expression is regulated in both a cell type- and stimulus-dependent fashion further contributes to the complexity.     

Toll样受体的信号转导及抗感染免疫研究进展

Toll样受体(Toll-like receptors,TLRs)是进化中比较保守的一个受体家族,至少包括13个成员,Toll样受体能特异识别病原相关分子模式(PAMP),在天然免疫和获得性免疫中都发挥着重要的作用,是连接天然免疫和获得性免疫的桥梁。近年来,对TLRs信号转导的研究,特别是对TLRs负反馈的研究,进展非常迅速,它们在抗感染中起着重要的作用,特别是负反馈机制对信号的平衡调节在抗感染免疫中有重要作用。     

Redundant and regulatory roles for Toll‐like receptors in Leishmania infection

Toll‐like receptors (TLRs) are germline‐encoded, non‐clonal innate immune receptors, which are often the first receptors to recognize the molecular patterns on pathogens. Therefore, the immune response initiated by TLRs has far‐reaching consequences on the outcome of an infection. As soon as the cell surface TLRs and other receptors recognize a pathogen, the pathogen is phagocytosed. Inclusion of TLRs in the phagosome results in quicker phagosomal maturation and stronger adaptive immune response, as TLRs influence co‐stimulatory molecule expression and determinant selection by major histocompatibility complex (MHC) class II and MHC class I for cross‐presentation. The signals delivered by the TCR–peptide–MHC complex and co‐stimulatory molecules are indispensable for optimal T cell activation. In addition, the cytokines induced by TLRs can skew the differentiation of activated T cells to different effector T cell subsets. However, the potential of TLRs to influence adaptive immune response into different patterns is severely restricted by multiple factors: gross specificity for the molecular patterns, lack of receptor rearrangements, sharing of limited number of adaptors that assemble signalling complexes and redundancy in ligand recognition. These features of apparent redundancy and regulation in the functioning of TLRs characterize them as important and probable contributory factors in the resistance or susceptibility to an infection.     

Induction of nuclear factor-κB responses by the S100A9 protein is Toll-like receptor-4-dependent

Interactions between danger‐associated molecular patterns (DAMP) and pathogen‐associated molecular patterns (PAMP) and pattern recognition receptors such as Toll‐like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor‐κB (NF‐κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) ‐free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein‐free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF‐κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP‐1 cells and in mouse bone marrow‐derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF‐κB activation. Both the S100A9‐induced response and the LPS‐induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF‐κB induction were strongly reduced in the presence of specific inhibitors of TLR‐signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488‐labelled S100A9 that was internalized in THP‐1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF‐κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF‐κB in a qualitatively distinct manner.     

Pneumolysin activates neutrophil extracellular trap formation

The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll‐like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co‐ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR‐4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin‐1 receptor‐associated kinase 1 (IRAK‐m) and B cell lymphoma 3 protein (Bcl‐3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator‐activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco‐2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR‐4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD.     

Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6

B‐cell expression of certain Toll‐like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B‐cell localization, deduced from B‐cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three‐colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9‐positive B and non‐B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19⁺ CD27^? CD23⁺ cells and CD19⁺ CD27^high CD80⁺ cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin‐6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B‐cell membrane TLR9.     

Toll‐like receptors and CD40 modulate each other's expression affecting Leishmania major infection

Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite L eishmania major. While polyinosinic‐polycytidylic acid [poly (I:C)], a TLR‐3 ligand, lipopolysaccharide (LPS), a TLR‐4 ligand, imiquimod, a TLR‐7/8 ligand and cytosine–phosphate–guanosine (CpG), a TLR‐9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR‐9 expression. Therefore, we tested the synergism between CD40 and CpG in anti‐leishmanial immune response. In L eishmania‐infected macrophages, CpG was found to reduce CD40‐induced extracellular stress‐regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)‐10, these ligands had differential effects on CD40‐induced IL‐1α, IL‐6 and IL‐12 production. CpG significantly enhanced the anti‐leishmanial function of CD40 with differential effects on IL‐4, IL‐10 and interferon (IFN)‐γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40–TLR cross‐talk that regulated the experimental L . major infection.