靶向Toll样受体4的小分子调节剂

Toll样受体(Toll-like receptors,TLRs)是进化保守的天然免疫模式识别受体,能够识别外源的病原菌相关分子模式(Pathogen-associated molecular patterns,PAMPs)、内源的损害相关分子模式(Damage-associated molecular patterns,DAMPs)和异源物相关分子模式(Xenobiotic-associated molecular patterns),诱导炎症免疫反应。 其中,TLR4(Toll-like receptor 4)是目前研究最为广泛的Toll样受体之一,TLR4是脂多糖(lipopoiysaccharide,LPS)的主要受体,LPS激活的TLR4信号通路在炎症信号的传递中发挥着重要作用,而此信号转导需要通过LPS与TLR4及其附属蛋白髓样分化因子2(myeloid differentiation factor 2,MD-2)的相互作用来实现。 因此,TLR4/MD-2成为炎症反应和免疫调控最重要的研究热点。 本文综述靶向TLR4/MD-2的小分子激动剂和抑制剂的研究进展,以进一步理解TLR4小分子调节剂与其相互作用的复杂性,帮助靶向TLR4/MD-2的免疫调节剂药物发现。     

Regulation of antiviral innate immune signaling and viral evasion following viral genome sensing

A harmonized balance between positive and negative regulation of pattern recognition receptor (PRR)-initiated immune responses is required to achieve the most favorable outcome for the host. This balance is crucial because it must not only ensure activation of the first line of defense against viral infection but also prevent inappropriate immune activation, which results in autoimmune diseases. Recent studies have shown how signal transduction pathways initiated by PRRs are positively and negatively regulated by diverse modulators to maintain host immune homeostasis. However, viruses have developed strategies to subvert the host antiviral response and establish infection. Viruses have evolved numerous genes encoding immunomodulatory proteins that antagonize the host immune system. This review focuses on the current state of knowledge regarding key host factors that regulate innate immune signaling molecules upon viral infection and discusses evidence showing how specific viral proteins counteract antiviral responses via immunomodulatory strategies.Subject terms: Innate immunity, Post-translational modifications     

Toll-like receptors for pathogen detection in water: challenges and benefits

The need for effective and efficient methods for pathogen detection in water is as serious as ever due to the health risk posed to human population by the consumption of pathogen-contaminated water. One of the important research streams which have been focused on by researchers for development of novel techniques for this purpose is biosensor technologies. Using different bio-recognition elements and transduction methodologies, biosensors have the potential to detect their analyte of interest in a fast and highly specific manner. Different pathogenic agents can be recognised by toll-like receptors (TLRs). The innate immune system of higher organisms employs TLRs for triggering intracellular signalling and induction of the expression of immune response genes. In this report, we explore the challenges associated with employing TLRs for pathogen detection in water samples. Although methods using TLR expressing cells also have been discussed, the focus of this review is on using TLR proteins as the bio-recognition elements in biosensors.     

Interactions between tumor-derived proteins and Toll-like receptors

Damage-associated molecular patterns (DAMPs) are danger signals (or alarmins) alerting immune cells through pattern recognition receptors (PRRs) to begin defense activity. Moreover, DAMPs are host biomolecules that can initiate a noninflammatory response to infection, and pathogen-associated molecular pattern (PAMPs) perpetuate the inflammatory response to infection. Many DAMPs are proteins that have defined intracellular functions and are released from dying cells after tissue injury or chemo-/radiotherapy. In the tumor microenvironment, DAMPs can be ligands for Toll-like receptors (TLRs) expressed on immune cells and induce cytokine production and T-cell activation. Moreover, DAMPs released from tumor cells can directly activate tumor-expressed TLRs that induce chemoresistance, migration, invasion, and metastasis. Furthermore, DAMP-induced chronic inflammation in the tumor microenvironment causes an increase in immunosuppressive populations, such as M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Therefore, regulation of DAMP proteins can reduce excessive inflammation to create an immunogenic tumor microenvironment. Here, we review tumor-derived DAMP proteins as ligands of TLRs and discuss their association with immune cells, tumors, and the composition of the tumor microenvironment.Subject terms: Immune cell death, Cancer microenvironment     

Recognition of lipopolysaccharide pattern by TLR4 complexes

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins.     

Involvement of tumor necrosis factor receptor superfamily(TNFRSF) members in the pathogenesis of inflammatory diseases

Current therapies for autoimmune diseases are not cures but merely palliatives, aimed at reducing symptoms. For the most part, these treatments provide nonspecific suppression of the immune system and thus do not distinguish between a pathogenic autoimmune response and a protective immune response. Recently emerging evidence not only has indicated the involvement of members of the TNF receptor/ligand superfamilies but also has revealed exciting innovative strategies for the treatment of autoimmune diseases and other chronic inflammatory diseases without depressing the immune response in general. In this review, we will discuss the regulatory mechanisms of TNF receptor/ligand family members, such as HVEM/ LIGHT, 4-1BB/4-1BBL, and GITR/GITRL that regulate T and B cell functions and participate in the process of inflammatory diseases. We will also discuss how intervening in the costimulatory pathways mediated by these molecules might have some potential as a therapeutic approach to immune disorders.     

The Development of Maillard Reaction,and Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling Inhibitors as Novel Therapeutic Strategies for Patients with AGE-Related Diseases

Advanced glycation end products (AGEs) are generated by nonenzymatic modifications of macromolecules (proteins, lipids, and nucleic acids) by saccharides (glucose, fructose, and pentose) via Maillard reaction. The formed AGE molecules can be catabolized and cleared by glyoxalase I and II in renal proximal tubular cells. AGE-related diseases include physiological aging, neurodegenerative/neuroinflammatory diseases, diabetes mellitus (DM) and its complications, autoimmune/rheumatic inflammatory diseases, bone-degenerative diseases, and chronic renal diseases. AGEs, by binding to receptors for AGE (RAGEs), alter innate and adaptive immune responses to induce inflammation and immunosuppression via the generation of proinflammatory cytokines, reactive oxygen species (ROS), and reactive nitrogen intermediates (RNI). These pathological molecules cause vascular endothelial/smooth muscular/connective tissue-cell and renal mesangial/endothelial/podocytic-cell damage in AGE-related diseases. In the present review, we first focus on the cellular and molecular bases of AGE–RAGE axis signaling pathways in AGE-related diseases. Then, we discuss in detail the modes of action of newly discovered novel biomolecules and phytochemical compounds, such as Maillard reaction and AGE–RAGE signaling inhibitors. These molecules are expected to become the new therapeutic strategies for patients with AGE-related diseases in addition to the traditional hypoglycemic and anti-hypertensive agents. We particularly emphasize the importance of “metabolic memory”, the “French paradox”, and the pharmacokinetics and therapeutic dosing of the effective natural compounds associated with pharmacogenetics in the treatment of AGE-related diseases. Lastly, we propose prospective investigations for solving the enigmas in AGE-mediated pathological effects.     

Toll-like Receptors Gene Expression in the Gastrointestinal Tract of Salmonella Serovar Pullorum-Infected Broiler Chicken

Salmonella enterica serovar Pullorum causes substantial mortality in chicks as well as results in persistent infection and vertical transmission in layer birds. An effective innate immune response in the early stages of infection could reduce bacterial colonization and mortality in chicks and persistency of infection in later stages. Toll-like receptors (TLRs), important components of innate immune response, plays a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, we quantified the expression levels of chicken TLRs (1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15, and 21) mRNA by quantitative real-time PCR in the gastrointestinal (GI) tissues (duodenum, jejunum, ileum, and cecum) of 3-day-old broiler chicks after 24 h of oral infection with S. enterica serovar Pullorum. We found significant upregulation of TLRs (TLR2, TLR4, TLR21) mRNA expressions in GI tract tissues after S. Pullorum infection. The exceptions were for TLR3 and TLR15 with decrease in the expression levels in the jejunum after infection. TLR4 gene expression was significantly (P?<?0.05) upregulated in the duodenum and ileum of infected chicks. Gene expression for some of the TLRs (TLR1LA, ILB, 2B, and TLR5) remained unchanged after infection with S. Pullorum in all the GI tissues studied. Most substantial change in gene expression was found for TLR21, being significantly (P?<?0.05) upregulated in all the tissues investigated. The differential expression levels of TLRs shed light on tailored innate immune response induced by S. Pullorum during the early stages of infection in chicks.     

Discovery and characterization of 2-aminobenzimidazole derivatives as selective NOD1 inhibitors

NLR family proteins play important roles in innate immune response. NOD1 (NLRC1) activates various signaling pathways including NF-κB in response to bacterial ligands. Hereditary polymorphisms in the NOD1 gene are associated with asthma, inflammatory bowel disease, and other disorders. Using a high throughput screening (HTS) assay measuring NOD1-induced NF-κB reporter gene activity, followed by multiple downstream counter screens that eliminated compounds impacting other NF-κB effectors, 2-aminobenzimidazole compounds were identified that selectively inhibit NOD1. Mechanistic studies of a prototypical compound, Nodinitib-1 (ML130; CID-1088438), suggest that these small molecules cause conformational changes of NOD1 in?vitro and alter NOD1 subcellular targeting in cells. Altogether, this inaugural class of inhibitors provides chemical probes for interrogating mechanisms regulating NOD1 activity and tools for exploring the roles of NOD1 in various infectious and inflammatory diseases.     

Techniques to Study Inflammasome Activation and Inhibition by Small Molecules

Inflammasomes are immune cytosolic oligomers involved in the initiation and progression of multiple pathologies and diseases. The tight regulation of these immune sensors is necessary to control an optimal inflammatory response and recover organism homeostasis. Prolonged activation of inflammasomes result in the development of chronic inflammatory diseases, and the use of small drug-like inhibitory molecules are emerging as promising anti-inflammatory therapies. Different aspects have to be taken in consideration when designing inflammasome inhibitors. This review summarizes the different techniques that can be used to study the mechanism of action of potential inflammasome inhibitory molecules.     

Pellino1 promotes chronic inflammatory skin disease via keratinocyte hyperproliferation and induction of the T helper 17 response

Psoriasis is one of the most common immune-mediated chronic inflammatory skin diseases. However, little is known about the molecular mechanism underlying the immunological circuits that maintain innate and adaptive immune responses in established psoriasis. In this study, we found that the Pellino1 (Peli1) ubiquitin E3 ligase is activated by innate pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs), and is highly upregulated in human psoriatic skin lesions and murine psoriasis-like models. Increased Peli1 expression is strongly correlated with the immunopathogenesis of psoriasis by activating hyperproliferation of keratinocytes in the S and G2/M phases of the cell cycle and promoting chronic skin inflammation. Furthermore, Peli1-induced psoriasis-like lesions showed significant changes in the expression levels of several T helper 17 (Th17)-related cytokines, such as IL-17a, IL-21, IL-22, IL-23, and IL-24, indicating that overexpression of Peli1 resulted in the sequential engagement of the Th17 cell response. However, the overexpression of Peli1 in T cells was insufficient to trigger psoriasis, while T cells were indispensable for disease manifestation. In summary, our findings demonstrate that Peli1 is a critical cell cycle activator of innate immunity, which subsequently links Th17 cell immune responses to the psoriatic microenvironment.Subject terms: Chronic inflammation, Immunoproliferative disorders     

Radiofrequency Irradiation Attenuates High-Mobility Group Box 1 and Toll-like Receptor Activation in Ultraviolet B–Induced Skin Inflammation

Ultraviolet B (UVB) exposure activates various inflammatory molecules of keratinocytes in the epidermis layer. Such UVB-mediated skin inflammation leaves post-inflammatory hyperpigmentation (PIH). Reports show a close relationship between PIH and high-mobility group box 1 (HMGB1) and its receptors. General clinical treatments of PIH, such as oral medication and laser treatment, have reported side effects. Recent studies reported the effects of radiofrequency (RF) irradiation on restoring dermal collagen, modulating the dermal vasculature, and thickening the basement membrane. To validate how RF regulates the inflammatory molecules from UVB-irradiated keratinocytes, we used UVB-radiated keratinocytes and macrophages, as well as animal skin. In addition, we examined two cases of RF-irradiated skin inflammatory diseases. We validated the effects of RF irradiation on keratinocytes by measuring expression levels of HMGB1, Toll-like receptors (TLRs), and other inflammatory factors. The results show that the RF modulates UVB-radiated keratinocytes to secrete fewer inflammatory factors and also modulates the expression of macrophages from HMGB1, TLRs, and inflammatory factors. RF irradiation could alleviate inflammatory skin diseases in patients. RF irradiation can regulate the macrophage indirectly through modulating the keratinocyte and inflammatory molecules of macrophages reduced in vitro and in vivo. Although the study is limited by the low number of cases, it demonstrates that RF irradiation can regulate skin inflammation in patients.     

Toll-Like Receptor 22 in Labeo rohita: Molecular Cloning,Characterization, 3D Modeling,and Expression Analysis Following Ligands Stimulation and Bacterial Infection

Toll-like receptors (TLRs) are a class of innate immune receptors that sense pathogens or their molecular signatures and activate signaling cascades to induce a quick and non-specific immune response in the host. Among various types of TLRs, TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. This report describes molecular cloning, three-dimensional (3D) modeling, and expression analysis of TLR22 in rohu (Labeo rohita), the most commercially important freshwater fish species in the Indian subcontinent. The open reading frame (ORF) of rohu TLR22 (LrTLR22) comprised of 2,838 nucleotides (nt), encoding 946 amino acid (aa) residues with the molecular mass of ～107.6 kDa. The secondary structure of deduced LrTLR22 exhibited the presence of signal peptide (1–22 aa), 18 leucine-rich repeat (LRR) regions (79–736 aa), and TIR domain (792–935 aa). The 3D model of LrTLR22-LRR regions together elucidated the horse-shoe-shaped structure having parallel β-strands at the concave surface and few α-helices at the convex surface. The TIR domain structure revealed alternate presence of five α-helices and β-sheets. Phylogenetically, LrTLR22 was closely related to common carp and exhibited significant similarity (92.2 %) and identity (86.1 %) in their amino acids. In rohu, TLR22 was constitutively expressed in all embryonic developmental stages, and tissue-specific analysis illustrated its expression in all examined tissues, highest was in liver and lowest in brain. In vivo modulation of TLR22 gene expression was analyzed by quantitative real-time PCR (qRT-PCR) assay following stimulation with lipopolysaccharide (LPS), synthetic double stranded RNA (polyinosinic-polycytidylic acid), and bacterial (Aeromonas hydrophila) RNA. Among these ligands, bacterial RNA most significantly (p?A. hydrophila infection, induction of TLR22 gene expression was also observed in majority of the tested tissues. Together, these data suggested that in addition to sensing other microbial signatures, TLR22 can recognize bacterial RNA and may play the important role in augmenting innate immunity in fish.     

Botanically-Derived Δ9-Tetrahydrocannabinol and Cannabidiol,and Their 1:1 Combination,Modulate Toll-like Receptor 3 and 4 Signalling in Immune Cells from People with Multiple Sclerosis

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 μM), CBD (10 μM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 μM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-β in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 μM) attenuated TLR3-induced CXCL10 and IFN-β protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 μM, and in combination with THC (in 1:1 combination at 10 μM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB₁ (encoded by CNR1 gene) and CB₂ (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.     

Recent Advancements on Immunomodulatory Mechanisms of Resveratrol in Tumor Microenvironment

Immunomodulation of the tumor microenvironment is emerging as an important area of research for the treatment of cancer patients. Several synthetic and natural agents are being investigated for their ability to enhance the immunogenic responses of immune cells present in the tumor microenvironment to impede tumor cell growth and dissemination. Among them, resveratrol, a stilbenoid found in red grapes and many other natural sources, has been studied extensively. Importantly, resveratrol has been shown to possess activity against various human diseases, including cancer. Mechanistically, resveratrol has been shown to regulate an array of signaling pathways and processes involving oxidative stress, inflammation, apoptosis, and several anticancer effects. Furthermore, recent research suggests that resveratrol can regulate various cellular signaling events including immune cell regulation, cytokines/chemokines secretion, and the expression of several other immune-related genes. In this review, we have summarized recent findings on resveratrol’s effects on immune regulatory cells and associated signaling in various cancer types. Numerous immunomodulatory effects of resveratrol suggest it may be useful in combination with other cancer therapies including immunotherapy for effective cancer management.     

Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators

Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein–protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis.Subject terms: Apoptosis, Necroptosis, Glycosylation, Phosphorylation, Ubiquitylation     

Rationally designed macrocyclic peptides as synergistic agonists of LPS-induced inflammatory response

Toll-like receptor 4 (TLR4) plays an important role in the regulation of the innate and adaptive immune response. Both agonists and antagonists of TLR4 are of considerable interest as drug leads for various disease indications. We herein report the rational design of two myeloid differentiation factor 2 (MD2)-derived macrocyclic peptides as TLR4 modulators, using the Rosetta Macromolecular Modeling software. The designed cyclic peptides, but not their linear counterparts, displayed synergistic activation of TLR signaling when co-administered with lipopolysaccharide (LPS). Although the understanding of the mechanism of action of these peptides remains elusive, these results underscore the utility of peptide cyclization for the discovery of biologically active agents, and also provide valuable tools for the investigation of TLR4 signaling.     

Baicalin Protects Keratinocytes from Toll‐like Receptor‐4 Mediated DNA Damage and Inflammation Following Ultraviolet Irradiation

UVB radiation contributes to both direct and indirect damage to the skin including the generation of free radicals and reactive oxygen species (ROS), inflammatory responses, immunosuppression and gene mutations, which can ultimately lead to photocarcinogenesis. A plant‐derived flavonoid, baicalin, has been shown to have antioxidant, anti‐inflammatory and free radical scavenging activities. Previous studies from our laboratory have shown that in murine skin, Toll‐like receptor‐4 (TLR4) enhanced both UVB‐induced DNA damage and inflammation. The aim of this study was to investigate the efficacy of baicalin against TLR4‐mediated processes in the murine keratinocyte PAM 212 cell line. Our results demonstrate that treating keratinocytes with baicalin both before and after UV radiation (100 mJ cm^?2) significantly inhibited the level of intracellular ROS and decreased cyclobutane pyrimidine dimers and 8‐Oxo‐2′‐deoxyguanosine (8‐oxo‐dG)—markers of DNA damage. Furthermore, cells treated with baicalin demonstrated an inhibition of TLR4 and its downstream signaling molecules, MyD88, TRIF, TRAF6 and IRAK4. TLR4 pathway inhibition resulted in NF‐κB inactivation and down‐regulation of iNOS and COX‐2 protein expression. Taken together, baicalin treatment effectively protected keratinocytes from UVB‐induced inflammatory damage through TLR pathway modulation.