Toll-like receptors in systemic autoimmune disease

Toll-like receptors (TLRs) have a crucial role in the early detection of pathogen-associated molecular patterns and the subsequent activation of the adaptive immune response. Whether TLRs also have an important role in the recognition of endogenous ligands has been more controversial. Numerous in vitro studies have documented activation of both autoreactive B cells and plasmacytoid dendritic cells by mammalian TLR ligands. The issue of whether these in vitro observations translate to an in vivo role for TLRs in either the initiation or the progression of systemic autoimmune disease is a subject of intense research; data are beginning to emerge showing that this is the case.     

Functional diversification of the toll-like receptor gene family

Phylogenetic analyses supported the hypothesis that the vertebrate toll-like receptors (TLRs) include two very ancient groups that arose by gene duplication prior to the divergence of protostomes and deuterostomes: (1) the TLR1 family (including mammalian TLR1, TLR2, TLR6, and TLR10); and (2) a clade including the remainder of mammalian TLRs. Correlating data on ligand type, subcellular localization, and gene expression in leukocytes and other tissues with the phylogeny provided evidence that certain major functional specializations within the TLRs occurred after ancient gene duplication events and that these traits have been retained through further events of gene duplication. For example, the recognition of bacterial lipoproteins appears to have arisen in the ancestor of the TLR1 family and continues to characterize members of that family whose ligands are known. Likewise, expression on the endosomal membrane and the recognition of nucleic acids appears to have been arisen in the ancestor of the TLR7 family and some related TLRs. On the other hand, gene expression patterns across tissues appear to have been much more volatile over the evolution of the vertebrate TLRs, since genes may show expression profiles similar to those of distantly related genes but dissimilar to those of closely related genes. Thus, the vertebrate TLRs provide an example of a multi-gene family in which gene duplication has been followed by extensive changes in certain aspects of gene function, while others have been conserved throughout vertebrate history. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Therapeutic targeting of the innate immune system in domestic animals

Since first being described in the fruit fly Drosophila melanogaster, the knowledge regarding Toll-like receptors (TLRs) has transformed our understanding of immunology. TLRs are a family of conserved pattern recognition receptors (PRR) that recognise specific microbial-associated molecular patterns and allow the cell to distinguish between self and non-self materials. The very property of the TLRs, to link innate and adaptive immunity, offers a novel opportunity to develop vaccines that engage TLR signalling. The presence of TLR ligands as adjuvants in conjunction with a vaccine is shown to increase the efficacy and response to the immunisation with a particular antigen. Here, we focus on the findings pertaining to TLR ligands as adjuvants and discuss the importance of these studies in the development of an optimal vaccine in farm and companion animals.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting

Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogen-associated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.     

TLR receptors in laryngeal carcinoma - immunophenotypic, molecular and functional studies

Toll-like receptors (TLRs) have been shown to play crucial role in the recognition of unicellular pathogens. We have shown the expression of three TLRs on tumor cells of human laryngeal carcinoma by means of immunohistochemistry. In the current study we searched presence of TLR1-10 on protein and molecular level in larynx carcinoma cell lines and the impact of respective TLR ligands on TLR expression. Larynx carcinoma cell lines have been used. Cell were subjected to immunocytochemistry. RNA isolated from the cells was tested by RT-PCR. Cells were cultured in the presence of respective TLR ligands. Cells than were harvested and subjected to flow cytometry, using anti TLR1-10 Moabs. The cells were evaluated of membrane and cytoplasmic cell staining. TLR reactivity varied in individual cell lines. RT-PCR allowed to show mRNA for all TLRs tested. After short-term cell culture each cell line exhibited distinct pattern of expression of TLRs following interaction with respective ligand. Cytoplasmic TLR staining had usually higher MFI value than membrane one, but after culture with ligand it became reversed. TLRs 7 and 9 showed highest expression in the majority of tumor cells tested. In conclusion, larynx carcinoma cell lines exhibit rather universal expression of TLRs, both on protein and molecular level. Culture of TLR expressing tumor cells with ligands points out for potential reactivity of tumor cells with TLR agonists, what may have therapeutic implications.     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Beyond Eyeballing: Fitting Models to Experimental Data

Abstract

As we learn more about the biology of the Toll-like receptors (TLRs), a wide range of molecules that can activate this fascinating family of pattern recognition receptors emerges. In addition to conserved pathogenic components, endogenous danger signals created upon tissue damage are also sensed by TLRs. Detection of these types of stimuli results in TLR mediated inflammation that is vital to fight pathogenic invasion and drive tissue repair. Aberrant activation of TLRs by pathogenic and endogenous ligands has also been linked with the pathogenesis of an increasing number of infectious and autoimmune diseases, respectively. Most recently, allergen activation of TLRs has also been described, creating a third broad class of TLR stimulus that has helped to shed light on the pathogenesis of allergic disease. To date, microbial activation of TLRs remains best characterized. Each member of the TLR family senses a specific subset of pathogenic ligands, pathogen associated molecular patterns (PAMPS), and a wealth of structural and biochemical data continues to reveal the molecular mechanisms of TLR activation by PAMPs, and to demonstrate how receptor specificity is achieved. In contrast, the mechanisms by which endogenous molecules and allergens activate TLRs remain much more mysterious. Here, we provide an overview of our current knowledge of how very diverse stimuli activate the same TLRs and the structural basis of these modes of immunity.     

Identification of small heat shock protein B8 (HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant alphaA crystallin, alphaB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both alphaA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP alphaA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.     

Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2

Toll-like receptors (TLRs) are pathogen recognition molecules that activate the immune system as part of the innate immune response. Microbial recognition by TLRs plays a crucial role in the host immune system's decision to respond or not to a particular microbial infection. Lipopolysaccharide (LPS), a membrane glycolipid of Gram-negative bacteria, exhibits strong immunostimulating activity among TLR ligands and has been studied in great detail. Recent studies have shown that cell surface TLR4-MD-2 physically interacts with LPS and triggers the release of an LPS signal, revealing a host-pathogen interaction mediated by TLR.     

Functions of toll-like receptors: lessons from KO mice

The innate immune response is a first-line defense system in which individual Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns (PAMPs) and exert subsequent immune responses against a variety of pathogens. TLRs are composed of an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic domain that is homologous to that of the IL-IR family. Upon stimulation, TLR recruits a cytoplasmic adaptor molecule MyD88, then IL-IR-associated kinase (IRAK), and finally induces activation of NF-kappaB and MAP kinases. However, the responses to TLR ligands differ, indicating the diversity of TLR signaling pathways. Besides MyD88, several novel adaptor molecules have recently been identified. Differential utilization of these adaptor molecules may provide the specificity in the TLR signaling.     

TGF-alpha regulates TLR expression and function on epidermal keratinocytes

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-alpha, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-alpha regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human beta-defensins. These findings provide evidence that TGF-alpha up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.     

Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis

Toll-like receptors (TLRs) play a central role in the innate immune response by recognizing conserved structural patterns in a variety of microbes. TLRs are classified into six families, of which TLR7 family members include TLR7, 8, and 9, which are localized to endolysosomal compartments recognizing viral infection in the form of foreign nucleic acids. In our current study, we focused on TLR8, which has been shown to recognize different types of ligands such as viral or bacterial ssRNA as well as small synthetic molecules. The primary sequences of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the TLR8 pathway is species-specific. Moreover, the factors underlying the receptor's species-specificity remain unknown. To this end, comparative homology modeling, molecular dynamics simulations refinement, automated docking and computational mutagenesis studies were employed to probe the intermolecular interactions between this anti-viral compound and TLR8. Furthermore, comparative analyses of modeled TLR8 (rodent and non-rodent) structures have shown that the variation mainly occurs at LRR14-15 (undefined region); hence, we hypothesized that this variation may be the primary reason for the exhibited species-specificity. Our hypothesis was further bolstered by our docking studies, which clearly showed that this undefined region was in close proximity to the ligand-binding site and thus may play a key role in ligand recognition. In addition, the interface between the ligand and TLR8s varied depending upon the amino acid charges, free energy of binding, and interaction surface. Therefore, our current work provides a hypothesis for previous in vivo studies in the context of TLR signaling.     

Endogenous toll‐like receptor ligands and their biological significance

Toll-like receptors (TLRs), a family of pattern recognition receptors, recognize and respond to conserved components of microbes and play a crucial role in both innate and adaptive immunity. In addition to binding exogenous ligands derived from pathogens, TLRs interact with endogenous molecules released from damaged tissues or dead cells and regulate many sterile inflammation processes. Putative endogenous TLR ligands include proteins and peptides, polysaccharides and proteoglycan, nucleic acids and phospholipids, which are cellular components, particularly extracellular matrix degradation products. Accumulating evidence demonstrates that endogenous ligand-mediated TLR signalling is involved in pathological conditions such as tissue injury, repair and regeneration; autoimmune diseases and tumorigenesis. The ability of TLRs to recognize endogenous stimulators appears to be essential to their function in regulating non-infectious inflammation. In this review, we summarize current knowledge of endogenous TLR ligands and discuss the biological significance of TLR signalling triggered by endogenous ligands in several sterile inflammation conditions.     

Lung cancer and Toll-like receptors

Lung carcinoma is one of the leading causes of death worldwide. It is a non-immunogenic cancer, resistant to immune surveillance. Toll-like receptors (TLRs) connect the innate to the adaptive immune system. Given that cancerous cells evade the immune system, the activation of TLRs could represent a potential target for cancer therapy. The induction of Th1-like and cytotoxic immunity by TLR signalling could lead to tumour cell death, resulting in tumour regression or arrest. However, basic research and clinical trials revealed that the activation of specific TLRs, such as TLR2, TLR4 and TLR9, do not have any anti-tumour activity in lung carcinoma. Increasing evidence suggests that TLRs are important regulators of tumour biology; however, little is known about their function in lung cancer. Thus, in order to develop new therapeutic approaches, further studies are needed to understand the connection between TLRs and lung cancer progression. This review focuses on the potential mechanisms by which TLR ligands can facilitate or not lung cancer and lung metastases establishment/progression.     

Responses with participation of toll-like receptors in protective immunity and in pathologic states

Toll-like receptors (TLRs) are a group of transmembrane receptors which play a key role in both innate and adaptive immune responses. The specific exogenous ligands of TLRs are pathogen-associated molecular patterns such as peptidoglycan, flagellin, teichoic acid, CPG-containing DNA, and others. Stimulation of TLRs induces synthesis and secretion of cytokines, upregulation of co-stimulatory molecules and functional maturation of antigen-presenting cells and leads to the development of both protective and damaging adaptive immune reactions. TLRs are also able to interact with a number of endogenic ligands such as fibronectine, heat shock proteins and extracellular matrix components. Thus, TLRs are involved in the development of many pathological states including sepsis and aseptic inflammation, allergy and autoimmune diseases, cancer. In the recent years several biotechnology and pharmaceutical companies developed new drugs that are either agonists of TLRs to enhance immune responses against tumours and infecious agents or to correct inadequate immune reactions or antagonists designed to reduce the inflammation caused by infection or autoimmune diseases. The paper presents current data concerning TLRs biology, the contribution of TLRs to pathogenesis of human diseases and completed, ongoing and planned clinical trials with immunotherapeutic drugs based on TLR agonists and antagonists.     

The role of Toll-like receptors in chronic inflammation

The role of Toll-like receptors (TLRs) in innate immunity and their ability to recognise microbial products has been well characterised. TLRs are also able to recognise endogenous molecules which are released upon cell damage and necrosis and have been shown to be present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands during inflammation and consequently the activation of TLR signalling pathways may be one mechanism initiating and driving autoimmune diseases. An increasing body of circumstantial evidence implicates a role of TLR signalling in systemic lupus erythematosus (SLE), atherosclerosis, asthma, type 1 diabetes, multiple sclerosis, bowl inflammation and rheumatoid arthritis (RA). Although at present their involvement is not comprehensively defined. However, future therapies targeting individual TLRs or their signalling transducers may provide a more specific way of treating inflammatory diseases without global suppression of the immune system.     

Synthesis of beta-(1-->4)-oligo-D-mannuronic acid neoglycolipids

Mammalian Toll-like receptors (TLRs) play important roles in host immune defense. The activation of TLR and down-stream signaling pathways have great impact on human physiology. Chemically diverse microbial products as well as synthetic ligands serve as agonists for these receptors. Recently, synthetic TLR ligands are being exploited as useful therapeutic agents for a variety of diseases including infections, inflammatory diseases, and cancers. Alginate polymers and oligosaccharides are strong immune stimulants mediated by TLR2/4, but synthesis of alginate oligomers is rarely studied. Reported here are the design and chemical synthesis of two beta-(1-->4)-di- and beta-(1-->4)-tri-d-mannuronic acid neoglycolipids 1 and 2 as potential TLR ligands. By using 4,6-di-O-benzylidene-protected 1-thio mannoside 7 as a glycosyl donor, the diastereoselective beta-d-mannosylation protocol provides the beta-(1-->4)-d-mannobiose and beta-(1-->4)-d-mannotriose derivatives, which upon regioselective oxidation with TEMPO/BAIB oxidation system yield the corresponding beta-(1-->4)-d-mannuronic acid containing neoglycolipids 1 and 2.     

Expression and role of Toll-like receptors on human umbilical cord mesenchymal stromal cells

Background aimsToll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs).MethodsIn the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined.ResultsAt the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon β, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs.ConclusionsTaken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs.