Toll-like Receptors and the Control of Immunity

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1β and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

This paper presents a study of the flow of ice in wedge-shaped converging channels. Such flows are encountered in the relatively constricted waters of the Canadian Arctic Archipelago. Ridging, lead opening patterns, development of a highpressure area, and arch formation are some of the processes which take place during ice flow through converging channels. An idealized geometry and steady wind forcing were used in the testing. The results give ice cover velocity, distribution of stresses, ice thickness, area coverage and ridging. Some of the conditions leading to arch formation at the constricted exit of the channel are explored.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKe) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKe and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was…

CATALYTIC RECEPTORS

A Functional Polymorphism of Toll-Like Receptor 4 Gene Increases Risk of Gastric Carcinoma and Its Precursors

Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disorder marked by autoantibody-mediated dysfunction at the neuromuscular junction, resulting in fluctuating muscle weakness. The pathogenesis of MG involves a complex interplay between genetic predisposition, environmental factors, and immune system dysregulation. Among these, the innate immune system, particularly Toll-like receptors (TLRs), has emerged as a critical player in disease progression by influencing both innate and adaptive immunity. TLRs are a family of pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering immune responses. Dysregulation of TLRs expression and signaling in MG has been implicated in chronic inflammation, breakdown of immune tolerance, and activation of autoreactive T and B cells. Overexpression of specific TLRs, such as TLR4 and TLR9, has been reported in MG patients, particularly in thymic tissues and peripheral immune cells, correlating with increased pro-inflammatory cytokine production and autoantibody generation. These aberrant responses contribute to the autoimmune cascade that underlies MG. Emerging evidence highlights the therapeutic potential of targeting TLRs pathways in MG. Strategies include using TLRs antagonists, modulating downstream signaling pathways, and leveraging epigenetic regulators to normalize TLRs activity. This review examines the role of TLRs in MG by exploring their expression profiles, their involvement in inflammatory signaling pathways, their impact on the adaptive immune system, and their potential as therapeutic targets. A better understanding of the role of TLRs in MG pathogenesis could open new avenues for modulating immune responses and precision therapies targeting the innate immune system.

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.

Objective: The immune system regulates inflammation associated with chronic cardiovascular disease. The primary aim of this study was to compare gene expression profiles of three innate and adaptive immune signaling pathways --the toll-like receptor (TLR), T-cell receptor (TCR), and B-cell receptor (BCR) signaling pathways in patients with subclinical atherosclerosis, acute ischemic stroke (IS), and myocardial infarction (MI). Methods: Peripheral blood gene expression profiles from human atherosclerotic diseases were downloaded from Gene Expression Omnibus (GEO): Atherosclerosis (GSE20129: 48 patients, 71 controls), IS (GSE16561: 39 patients - samples within 24 hours from symptom onset, 24 controls), and MI (GSE29111, 18 MI patients (7 and 30 days) samples after experiencing MI). Genes in the TLR (106 genes), TCR (104 genes), and BCR (72 genes) pathways were retrieved from the NCBI BioSystems database. Gene set enrichment analysis was used to evaluate the statistical significance of gene enrichment in each signaling pathway for each study. Weighted Gene Co-expression Network Analysis was used to identify gene modules and evaluate network connectivity across the disease states. Results: All three immune pathways significantly enriched differentially expressed genes in each disease state (P<0.01) - TLR (38% in atherosclerosis, 47% in IS, 30% in MI); TCR (51% in atherosclerosis, 54% in IS, and 53% in MI); BCR (57% in atherosclerosis; 60% in IS; 43% in MI). Transcriptional changes in the pathways are highly concordant across the diseases. The correlation of expression changes between any two diseases ranges from 0.53 to 0.76 (mean±SD: 0.66±0.09, P<0.00001). Although TLR genes were all up-regulated in each condition, network analysis revealed a striking down-regulation of gene modules in the TCR and BCR pathways. Of significance, the TCR-CD3 complex and CD79A/B were significantly down-regulated in patients of all three disease states. Conclusion: We found a strong overlap in expression between innate and adaptive immune pathways in three atherosclerotic related diseases. The down regulation of the TCR-CD3 complex suggests immune suppression is a common occurrence in either chronic or acute inflammation associated atherosclerotic disease.

Toll-like receptors (TLRs), a recently discovered family of pattern recognition receptors, play an important role in mediating the activation of innate and adaptive immunity in vertebrate animals. TLRs express on a diverse variety of cells and tissues especially on dendritic cells, macrophages and granulocytes, recognizing pathogens like Gram-positive and -negative bacteria, viral RNA, unmethylated CpG DNA and fungi. Recent studies show that TLRs signaling is involved in either the initiation or the progression of systemic autoimmune disease and chronic inflammatory diseases. Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease and its aetiology remains unclear. In this study, we investigate the expression of TLR2 and TLR4 on polymorphonuclear neutrophil (PMN) and mononuclear cell (MNC) in SLE patients and healthy subjects. We also try to identify the synergistic molecule related to TLR2 or TLR4 signaling and the mechanism involved in the pathogenesis of SLE. Our results show that the expression of TLR2 or TLR4 on PMN and MNC are not significantly different between healthy subjects and SLE patients. Nevertheless, we detect that the expression level of soluble CD14 (sCD14), the mediator of LPS-TLR4 signaling, in serum are significantly elevated in SLE patients. Moreover, we show that the expression level of soluble TLR2 (sTLR2), the modulator of TLR2 signaling, in serum are reduced in SLE patients. In addition, we utilize anti-TLR2 and anti-TLR4 antibodies to mimic immune stimulations to examine the immune response on PMN and MNC. These findings demonstrate that the production of sTLR2 seems unaffected by those stimulations. Only MNC is down- regulated by those stimulations in sCD14 expression. Likely sTLR2 is an existed molecule in normal serum and sCD14 is related to the immunity in SLE. Although further studies elucidating the detailed mechanisms of sTLR2 and sCD14 are required, this study provides a new direction for SLE treatment and prevention.

BackgroundAtopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease that can be triggered by various precipitating factors, including colonization by Staphylococcus aureus (S. aureus). The toll-like receptor (TLR), which belongs to the family of pattern recognition receptors (PRR), can recognize components of S. aureus, such as staphylococcal enterotoxin B (SEB). This receptor is known to be expressed on monocytes. However, the understanding of the role of SEB in the pathogenesis of AD through the TLR pathway, especially TLR2 and TLR6, is not widely known.PurposeTo investigate the expression of TLR2 and TLR6 on peripheral blood monocytes induced by SEB during AD exacerbations. Patients and MethodsTwenty AD patients and 20 healthy subjects as a control group were selected. A 5 mL blood sample from each subject was taken for monocyte culture, which was induced by SEB for three days, and the outcomes were assessed by flow cytometry to evaluate TLR2 and TLR6 expression.ResultsThe expression of TLR2 on peripheral blood monocytes in AD patients was increased compared to healthy controls (p = 0.000), but not for the expression of TLR6 (p = 0.304). In the AD group, TLR2 and TLR6 expression on peripheral blood monocytes after being induced by SEB was significantly increased compared to before induction (p = 0.025 and p = 0.023, respectively), but not in the control group (p = 0.737 and p = 0.100, respectively).ConclusionThere is significantly increased expression of TLR2 and TLR6 on peripheral blood monocytes induced by SEB during exacerbation in AD patients.

Protein kinase C (PKC) is a family of kinases that are implicated in a plethora of diseases, including cancer and cardiovascular disease. PKC isoforms can have different, and sometimes opposing, effects in these disease states. Toll-like receptors (TLRs) are a family of pattern recognition receptors that bind pathogens and stimulate the secretion of cytokines. It has long been known that PKC inhibitors reduce LPS-stimulated cytokine secretion by macrophages, linking PKC activation to TLR signaling. Recent studies have shown that PKC-α, -δ, -ε, and -ζ are directly involved in multiple steps in TLR pathways. They associate with the TLR or proximal components of the receptor complex. These isoforms are also involved in the downstream activation of MAPK, RhoA, TAK1, and NF-κB. Thus, PKC activation is intimately involved in TLR signaling and the innate immune response.

The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development. The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development.

The immune system's first line of defense is innate immunity, largely based on a large family of pattern recognition receptors (PRRs) that recognize evolutionary conserved molecular motifs on pathogens called pathogen-associated molecular patterns (PAMPs). The most extensively studied family of PRRs is Toll-like receptors (TLRs), which can trigger various cellular pathways after ligand stimulation. Their role in cancer is still unresolved as there are many different studies showing contradictory results. TLRs have been associated with both tumor progression and immunosuppression as well as with apoptosis and immune system activation. With their ability to induce apoptotic response and activation of innate and adaptive immunity, TLRs are an interesting pharmacological target for the development of anticancer therapy. There are numerous studies including the clinical trials reviewed in this paper, indicating that TLR agonists, especially combined with other more conventional therapies such as chemotherapy and radiotherapy, are promising adjuvants or components of newly developed treatment regimens. Still, the increasing number of studies indicating protumorigenic consequences of TLR activation in various cancer types and recent reports of the existence of endogenous TLR ligands, forewarn that more studies on this topic are required before their inclusion into regular clinical practice.

Toll-like receptors (TLRs) belong to the family of pattern recognition receptors (PRRs), playing critical roles in linking innate with adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). TLRs and TLR signaling pathways serve as not only the first line of pulmonary defense against pathogens infection but crucial factors in maintaining pulmonary immune homeostasis. However, aberrant activation of TLR signaling leads to inflammation and immune dysregulations, contributing to various pulmonary diseases, including inflammation, infection, fibrosis, and malignancy. This review summarizes the updated roles of TLRs and TLR signaling in lung development and the establishment and regulation of pulmonary region-specific immunity. We further elucidate the involvement of TLRs and TLR signaling in the onset and progression of lung diseases, such as infections, fibrosis, malignancies, and immune disorders. It would provide updated insights into the exploration of novel diagnostic and therapeutic strategies targeting TLRs and TLR signaling in pulmonary diseases.