Members Of Toll-like Receptor Family Research Articles

Members of the Toll-Like Receptor Family of Innate Immunity Pattern-Recognition Receptors Are Abundant in the Male Rat Reproductive Tract1

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.

Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors

A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.

The effect of systemic iNOS inhibition during human endotoxemia on the development of TLR-tolerance in vitro

The phenomenon of repeated exposure to endotoxin resulting in diminished release of proinflammatory cytokines is called endotoxin tolerance. After receiving a low-dose pretreatment with endotoxin, tolerant animals survive a challenge with a 'lethal dose' of endotoxin. Recent studies suggests a pivotal role for NO arising from iNOS in the development of tolerance. Toll-like receptors (TLRs) are receptors that recognize specific pathogen-associated molecular patterns. Ten members of the so-called TLR family have been identified in humans, and several of them appear to recognize specific microbial products, including LPS (TLR-4), peptidoglycan (TLR-2) and flagelline (TLR-5). Recognition leads to activation of intracellular signaling pathways, which upregulate a wide array of inflammatory modulators that contribute to the early host cell response. The aim of this study was to test whether administration of the selective iNOS inhibitor aminoguanidine during human experimental endotoxemia influences the development of tolerance to several TLR-agonists in vitro.

The Tumor Suppressor Cylindromatosis (CYLD) Acts as a Negative Regulator for Toll-like Receptor 2 Signaling via Negative Cross-talk with TRAF6 and TRAF7

Toll-like receptor 2 (TLR2) plays an important role in host defense against bacterial pathogens. Activation of TLR2 signaling not only induces the activation of innate immunity and instructs the development of the acquired immunity but also leads to the detrimental inflammatory responses in inflammatory and infectious diseases. To avoid detrimental inflammatory responses, TLR2 signaling must be tightly regulated. In contrast to the relative known positive regulation of TLR2 signaling, its negative regulation, however, is largely unknown. In addition the distal signaling components that link TLR2 to its downstream signaling pathways have yet to be further defined. In the present study we have provided direct evidence for the negative regulation of TLR2 signaling by the tumor suppressor cylindromatosis (CYLD). We showed that activation of TLR2 signaling by TLR2 ligands including peptidoglycan (PGN), MALP-2, and Pam3CSK4 induces activation of IKKs-IkappaBalpha and MKK3/6-p38 pathways not only by TRAF6 but also by TRAF7, a recently identified TRAF family member. The activation of both pathways leads to the transcription of TNF-alpha, IL-1beta, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus unveil a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IkappaBalpha/MKK3/6-p38-NF-kappaB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling.

Domain Exchange between Human Toll-like Receptors 1 and 6 Reveals a Region Required for Lipopeptide Discrimination

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)). However, examination of a wide variety of lipopeptide derivatives indicates that recognition by human TLR1 and -2 does not strictly correlate with the number or position of the acyl chains on the modified cysteine residue. Conversely, human TLR2 and -6 exclusively respond to lipopeptides possessing a diacylglycerol group. Most surprisingly, we have found that an R stereoisomer of diacylated macrophage-activating lipopeptide 2 (MALP-2) exclusively activates epithelial cells through TLR6 and -2 but not through TLR1 and -2. These results suggest that the chirality of the central carbon of the diacylglycerol group of these agonists is a structural determinant for human TLR recognition. Examination of chimeric receptors, generated by domain exchange between TLR1 and -6, has revealed that leucine-rich repeats 9-12 of the extracellular domain enable these receptors to discriminate between structurally similar lipopeptides. However, additional chimeric constructs reveal that this region alone is not sufficient to generate receptors that can functionally cooperate with TLR2. Our results support the idea that TLR1 and TLR6 diverged during evolution to differentially recognize natural lipoprotein structures and that this function has been conserved with respect to the human receptors.

Differential expression of CD180 and IgM by B‐cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

Sources of autoantigens in systemic lupus erythematosus

A hallmark of systemic lupus erythematosus is the production of autoantibodies that recognize nuclear antigens. However, the underlying events and mechanisms that lead to the selection of these molecules for the autoimmune response remain poorly understood. In this review, we will examine some of the proposed explanations for sources of systemic lupus erythematosus-specific autoantigens. We will focus on events related to apoptosis, viral infection, cytokine production, innate immune system components, and alternative splicing of pre-mRNA transcripts. Definitive proof of a viral etiology for lupus remains elusive. However, recent observations have added to increasing evidence that viruses contribute to the bypass of tolerance in systemic lupus erythematosus. Also, events associated with apoptosis - most notably proteolytic autoantigen cleavage by caspases and granzyme B - have been implicated in the initiation of autoimmune responses for over a decade. Results obtained from animal models and human systems suggest complex functions for pro-apoptotic pathways in the regulation of immune responses. Inducible antigen expression and alternatively spliced transcripts may represent additional ways of generating autoantigenic material. Finally, toll-like receptor family members may play critical roles in the induction of antibody responses to nucleic acids in systemic lupus erythematosus. Several factors may contribute to the generation of systemic lupus erythematosus-specific autoantigens. Determining the underlying causes of autoantibody production may provide important insight into the etiology and pathogenesis of this disease.

Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-α induction

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-α production was abolished in Irak-1–deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-κB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1–deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-α induction in pDCs.

Current research on the immunostimulatory effects of CpG oligodeoxynucleotides in fish

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within the context of certain flanking bases (CpG motifs) have been shown to induce potent innate and adaptive immune responses. Recognition of CpG motifs by the innate immune system requires engagement of toll-like receptor 9 (TLR-9), a newly identified member of toll-like receptor family, which induces cell signaling and subsequently triggers an immune response. The binding CpG-ODN to its specific membrane receptor and subsequently triggering of CpG-related signaling occurred within acidified endosomes. CpG-ODNs are being harnessed for a variety of therapeutic uses, including as immune adjuvants, for cancer therapy, as anti-allergens, and as immunoprotective agents. However, potential applications for immunostimulatory CpG-ODN in fish are just being explored. This article reviews recent progress in understanding the immunostimulatory effects of CpG DNA in fish.

Pathogen Protection via Toll-like Receptors

Mammalian Toll-like receptor (TLR) proteins have evolved to detect a range of molecular cues displayed by pathogens, such as components of bacterial cell walls and viral nucleic acids (see the Perspective by O'Neill). Zhang et al. present evidence for a new member of the mammalian TLR family, TLR11, which plays a role in protecting the urogenital tract from bacterial pathogens. TLR7 and TLR8 have yet to be assigned specific pathogen-derived ligands. Diebold et al. observed recognition of single-stranded RNA (ssRNA) from influenza virus by TLR7 in mice and found that stimulation of this receptor pathway within the endosome of the dendritic cell induced antiviral cytokine expression. Heil et al. also observed guanosine (G)- and uridine (U)-rich ssRNA sequence detection by TLR7 in mouse dendritic cells. However, in human cells, TLR8, rather than TLR7, detected GU-rich ssRNA sequences of human immunodeficiency virus type-1, highlighting a level of divergence in TLR-mediated viral RNA detection between species. L. A. J. O'Neill, After the Toll rush. Science 303 , 1481-1482 (2004). [Summary] [Full Text] D. Zhang, G. Zhang, M. S. Hayden, M. B. Greenblatt, C. Bussey, R. A. Flavell, S. Ghosh, A Toll-like receptor that prevents infection by uropathogenic bacteria. Science 303 , 1522-1526 (2004). [Abstract] [Full Text] S. S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303 , 1529-1531 (2004). [Abstract] [Full Text] F. Heil, H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, S. Bauer, Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303 , 1526-1529 (2004). [Abstract] [Full Text]

A toll-like receptor that prevents infection by uropathogenic bacteria.

Toll-like receptors (TLRs) recognize molecular patterns displayed by microorganisms, and their subsequent activation leads to the transcription of appropriate host-defense genes. Here we report the cloning and characterization of a member of the mammalian TLR family, TLR11, that displays a distinct pattern of expression in macrophages and liver, kidney, and bladder epithelial cells. Cells expressing TLR11 fail to respond to known TLR ligands but instead respond specifically to uropathogenic bacteria. Mice lacking TLR11 are highly susceptible to infection of the kidneys by uropathogenic bacteria, indicating a potentially important role for TLR11 in preventing infection of internal organs of the urogenital system.

Toll-like receptors as key mediators in innate antifungal immunity

The Toll protein of Drosophila is a transmembrane receptor involved in dorsoventral polarization during embryonic development and recognition of infection. In mammals, Toll-like receptors (TLRs) constitute a novel protein family involved in innate immunity and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Specific agonists for nine of the ten members of the human TLR family have been described to date. TLRs as well as the TLR-associated adaptor molecule MyD88 have been implicated in the recognition of the fungal pathogens Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Pneumocystis carinii. Moreover, several pathogen associated molecular patterns (PAMPs) located in the cell wall or cell surface of fungi have been identified as potential ligands. Yeast zymosan activates TLR2/ TLR6 heterodimers, whereas Saccharomyces cerevisiae- and C. albicans-derived mannan seems to be detected by TLR4. Phospholipomannan, present in the cell surface of C. albicans has been shown to be recognized by TLR2, while TLR4 mainly interacts with glucuronoxylomannan, the major capsular polysaccharide of C. neoformans. MyD88 has been implicated in TLR signalling of linear (1 --> 3)-beta-D-glucan, and of beta-glucan from P. carinii. These data point towards the ability of the innate immune system to utilize TLRs that are specific to different types and components of pathogenic fungi. Recent evidence further suggests that TLRs cooperate with other immune receptors involved in fungal recognition and that the selective induction of adaptor proteins finally leads to distinct signalling events upon fungal challenge.

Innate immunity: sensing the environment and regulating the regulators.

Innate immune cells act as sensors for environmental pathogens and key regulators of pathogen-specific T-cell effector responses. The expression of Toll-like receptors is essential for these functions. Innate immunity is a rapidly evolving field. Great progress has been made in the past year in characterizing the signaling pathways downstream of Toll-like receptors, and the role of Toll-like receptors in the regulation of pathogen responses. The ability to control the activation of regulatory T cells has emerged as a key function of innate immunity. Regulatory T cells are involved in the induction of tolerance and the prevention of harmful immune pathology, particularly T-cell responses directed against self-antigens. Toll-like receptor-mediated interactions between pathogen-stimulated innate immune cells and regulatory T cells result in the release of suppression by regulatory T cells, thus allowing pathogen-specific responses. However, pathogenic microorganisms may exploit this interaction to evade the host's response. In addition, recent studies raise the possibility that regulatory T cells may express some Toll-like receptor family members, thereby responding directly to pathogens. A deeper understanding of the complex role of innate immune cells as sensors of the environment and regulators of pathogen responses will probably influence the current models of immune regulation, particularly those centered on the role of the environment in shaping immune responses.

Innate Adaptations

Viruses, bacteria, and fungi express broadly distinct molecular patterns that activate innate arms of the immune system. Most prominently, the Toll-like receptors (TLRs) use these pathogen-specific cues to elicit intracellular signals that can be either dependent or independent of the Toll/interleukin 1 receptor (TIR) domain-containing adaptor proteins, MyD88 and TIRAP. Yamamoto et al. define a third TIR adaptor, TRIF, as critical for MyD88-independent signaling by particular TLRs. Cells deficient in TRIF failed to initiate either the interferon regulatory factor-3 or nuclear factor κB pathways in response to TLR3 or TLR4 activation, but responded normally to activation of other TLR family members. M. Yamamoto, S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira, Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301 , 640-643 (2003). [Abstract] [Full Text]

Viruses and Toll-like receptors.

Recently a number of viruses, including a poxvirus, herpesvirus, retrovirus and two paramyxoviruses, have been shown to activate cells via Toll-like receptor family members. Here we postulate that although activation via Toll-like receptor molecules can lead to anti-viral innate immune responses, in some cases viruses may use these responses to ameliorate infection.

Role of adapters in Toll-like receptor signalling.

Toll-like receptors (TLRs) play a critical role in the detection of invading pathogens within the body and the subsequent immune response. Individual TLRs recognize distinct microbial components. The TLRs are a type 1 transmembrane receptor that possess an extracellular leucine-rich repeat domain and cytoplasmic domain homologous with that of the interleukin 1 receptor (IL-1R) family. Upon stimulation, TLR recruits the IL-1R-associated kinase (IRAK) via the adapter MyD88, ultimately leading to the activation of nuclear factor-kappaB. Cytokine production in response to all TLR ligands is completely abolished in MyD88-deficient cells, indicating that MyD88 is an essential signalling molecule shared among members of the IL-1R/Toll family. However, several novel adaptor molecules have recently been identified. Evidence is now accumulating showing that differential utilization of these adaptors may activate overlapping as well as distinct signalling pathways, and ultimately give rise to distinct biological effects exerted by individual TLR family members.

Book Review: A Widely Conserved Smell of DangerReview and commentary on Toll-like Receptor Family Members and Their Ligands , edited by Bruce Beutler and Hermann Wagner. Springer-Verlag, Berlin, Heidelberg, 2002, 192 pp. $95.00, ISBN: 3-540-43560-3

With more than 150 original articles and more than a dozen reviews having been published in 2002 on the Toll-like receptor (TLR) family, the book Toll-like Receptor Family Members and Their Ligands provides a timely overview of the role of TLRs in innate immunity. The book provides chapters on the receptors, their ligands, and the signaling cascades activated. Highlighted in this book review are the chapters on the evolution of the TLRs, the receptor-ligand specificity, and the possible cross-talk between TLR signaling pathways and mitogen-activated protein kinase pathways.

Broad expression of Toll-like receptors in the human central nervous system.

The family of Toll-like receptors (TLRs) plays a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules. In this study we investigated expression of TLRs in vitro by purified human microglia, astrocytes, and oligodendrocytes, and in vivo by immunohistochemical examination of brain and spinal cord sections. Cultured primary microglia were found to express mRNA encoding a wide range of different TLR family members while astrocytes and oligodendrocytes primarily express TLR2 and TLR3. Comparisons between microglia derived from a series of control subjects and neurodegenerative cases indicate distinct differences in levels of mRNA encoding the different TLRs indifferent microglia samples. Interestingly, expression of TLR proteins in cultured microglia as revealed by immunocytochemistry was restricted to intracellular vesicles, whereas in astrocytes they were exclusively localized on the cell surface. Finally, in vivo expression of TLR3 and TLR4 was examined by immunohistochemical analysis of brain and spinal cord sections from both control and multiple sclerosis brains, revealing enhanced expression of either TLR in inflamed CNS tissues. Together, our data reveal broad and regulated expression of TLRs both in vitro and in vivo by human glia cells.

Differential involvement of IFN-beta in Toll-like receptor-stimulated dendritic cell activation.

Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.

Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines.

Pattern recognition receptors (PRR) play an important roll in immediate responses to different conserved molecules produced by microbes. In this paper we describe the cloning of the mouse homolog of Toll-like receptor (TLR) 3, and present an analysis of the expression of this gene in innate and adaptive immune cell lines. We also performed a broad expression study on these cells of other TLR, including TLR family members whose expression pattern is not known, i.e. TLR7. The analysis was done in order to understand, and possibly predict, how innate and adaptive immune cells respond to microbial pattern antigens. This first large-scale analysis of immune cell TLR expression in the mouse reveals that cells of the innate immune system express a broader number of TLR than cells of the adaptive immune system, supporting preconceptions concerning the hierarchy of immune cells involved in direct pathogen recognition. Additionally, the expression of TLR transcripts by mast cells, neutrophils and microglial cells observed here suggests that pathogen-associated molecular pattern molecules could induce activation of these cells through TLR. Finally, the mouse homolog of human TLR3 identified here may, like its human counterpart, be an exceptional TLR molecule due to its lack of a conserved proline residue seen to be involved in existing TLR signaling capabilities found in other TLR family members.