Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.     

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.     

The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophages

Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.     

Comparison of Cellular Uptake and Inflammatory Response via Toll-Like Receptor 4 to Lipopolysaccharide and Titanium Dioxide Nanoparticles

The innate immune response is the earliest cellular response to infectious agents and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) play an important role in these interactions. We have already shown that TLRs are involved with the uptake of titanium dioxide nanoparticles (TiO₂ NPs) and promote inflammatory responses. In this paper, we compared role of cellular uptake and inflammatory response via TLR 4 to lipopolysaccharide (LPS) and TiO₂ NPs. In the case of LPS, LPS binds to LPS binding protein (LBP) and CD 14, and then this complex binds to TLR 4. In the case of TiO₂ NPs, the necessity of LBP and CD 14 to induce the inflammatory response and for uptake by cells was investigated using over-expression, antibody blocking, and siRNA knockdown experiments. Our results suggested that for cellular uptake of TiO₂ NPs, TLR 4 did not form a complex with LBP and CD 14. In the TiO₂ NP-mediated inflammatory response, TLR 4 acted as the signaling receptor without protein complex of LPS, LBP and CD 14. The results suggested that character of TiO₂ NPs might be similar to the complex of LPS, LBP and CD 14. These results are important for development of safer nanomaterials.     

TLR/WNT: A Novel Relationship in Immunomodulation of Lung Cancer

The most frequent cause of death by cancer worldwide is lung cancer, and the 5-year survival rate is still very poor for patients with advanced stage. Understanding the crosstalk between the signaling pathways that are involved in disease, especially in metastasis, is crucial to developing new targeted therapies. Toll-like receptors (TLRs) are master regulators of the immune responses, and their dysregulation in lung cancer is linked to immune escape and promotes tumor malignancy by facilitating angiogenesis and proliferation. On the other hand, over-activation of the WNT signaling pathway has been reported in lung cancer and is also associated with tumor metastasis via induction of Epithelial-to-mesenchymal-transition (EMT)-like processes. An interaction between both TLRs and the WNT pathway was discovered recently as it was found that the TLR pathway can be activated by WNT ligands in the tumor microenvironment; however, the implications of such interactions in the context of lung cancer have not been discussed yet. Here, we offer an overview of the interaction of TLR-WNT in the lung and its potential implications and role in the oncogenic process.     

Toll-Like Receptors (TLRs), NOD-Like Receptors (NLRs), and RIG-I-Like Receptors (RLRs) in Innate Immunity. TLRs,NLRs, and RLRs Ligands as Immunotherapeutic Agents for Hematopoietic Diseases

The innate immune system plays a pivotal role in the first line of host defense against infections and is equipped with patterns recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Several classes of PRRS, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) recognize distinct microbial components and directly activate immune cells. TLRs are transmembrane receptors, while NLRs and RLRs are intracellular molecules. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of overlapping and unique genes involved in the inflammatory and immune responses. The innate immune system also influences pathways involved in cancer immunosurveillance. Natural and synthetic agonists of TLRs, NLRs, or RLRs can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment, and are being explored as promising adjuvants in cancer immunotherapies. In this review, we provide a concise overview of TLRs, NLRs, and RLRs: their structure, functions, signaling pathways, and regulation. We also describe various ligands for these receptors and their possible application in treatment of hematopoietic diseases.     

Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview

Hepatitis C virus (HCV) infection remains a major global health burden, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that detect pathogen-associated molecular patterns and activate downstream signaling to induce proinflammatory cytokine and chemokine production. An increasing number of studies have suggested the importance of TLR responses in the outcome of HCV infection. However, the exact role of innate immune responses, including TLR response, in controlling chronic HCV infection remains to be established. A proper understanding of the TLR response in HCV infection is essential for devising new therapeutic approaches against HCV infection. In this review, we discuss the progress made in our understanding of the host innate immune response to HCV infection, with a particular focus on the TLR response. In addition, we discuss the mechanisms adopted by HCV to avoid immune surveillance mediated by TLRs.     

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

House dust mites (HDMs) are a common source of respiratory allergens responsible for allergic asthma and innate immune responses in human diseases. Since HDMs are critical factors in the triggering of allergen-induced airway mucosa from allergic asthma, we aimed to investigate the mechanisms of Toll-like receptors (TLR) in the signaling of the HDM extract that is involved in mucus hypersecretion and airway inflammation through the engagement of innate immunity. Previously, we reported that the somatic nuclear autoantigenic sperm protein (sNASP)/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis controls the initiation of TLRs to maintain the homeostasis of the innate immune response. The present study showed that the HDM extract stimulated the biogenesis of Mucin 5AC (MUC5AC) in bronchial epithelial cells via the TLR2/4 signaling pathway involving MyD88 and TRAF6. Specifically, sNASP binds to TRAF6 in unstimulated bronchial epithelial cells to prevent the activation of TRAF6-depenedent kinases. Upon on HDMs’ stimulation, sNASP is phosphorylated, leading to the activation of TRAF6 downstream of the p38 MAPK and NF-κB signaling pathways. Further, NASP-knockdown enhanced TRAF6 signaling and MUC5AC biogenesis. In the HDM-induced mouse asthma model, we found that the HDM extract promoted airway hyperresponsiveness (AHR), MUC5AC, and allergen-specific IgE production as well as IL-5 and IL-13 for recruiting inflammatory cells. Treatment with the PEP-NASP peptide, a selective TRAF6-blocking peptide, ameliorated HDM-induced asthma in mice. In conclusion, this study indicated that the sNASP/TRAF6 axis plays a regulatory role in asthma by modulating mucus overproduction, and the PEP-NASP peptide might be a potential target for asthma treatment.     

细菌感染对斑马鱼TLR5表达的影响研究

Toll样受体家族（TLRs）在天然免疫中发挥着重要作用。用杀鲑气单胞菌（革兰氏阴性菌）和金黄色葡萄球菌（革兰氏阳性菌）分别感染宽马鱼后,检测了斑马鱼TLR5表达的变化。结果表明,具有鞭毛的杀鲑气单胞菌和不具有鞭毛的金黄色葡萄球菌均可以诱导TLR5表达的上调。认为具有鞭毛的杀鲑气单胞菌可以直接激活TLR5通路,而不具有鞭毛的金黄色葡萄球菌可能是先激活了TLRs的其它通路后再诱导TLR5表达的上调。从而推断TLR5是斑马鱼天然免疫的核心。     

Toll-like Receptor Signaling Inhibitory Peptide Improves Inflammation in Animal Model and Human Systemic Lupus Erythematosus

Toll-like receptors (TLRs) play a major role in the innate immune system. Several studies have shown the regulatory effects of TLR-mediated pathways on immune and inflammatory diseases. Dysregulated functions of TLRs within the endosomal compartment, including TLR7/9 trafficking, may cause systemic lupus erythematosus (SLE). TLR signaling pathways are fine-tuned by Toll/interleukin-1 receptor (TIR) domain-containing adapters, leading to interferon (IFN)-α production. This study describes a TLR inhibitor peptide 1 (TIP1) that primarily suppresses the downstream signaling mediated by TIR domain-containing adapters in an animal model of lupus and patients with SLE. The expression of most downstream proteins of the TLR7/9/myeloid differentiation factor 88 (MyD88)/IFN regulatory factor 7 signaling was downregulated in major tissues such as the kidney, spleen, and lymph nodes of treated mice. Furthermore, the pathological analysis of the kidney tissue confirmed that TIP1 could improve inflammation in MRL/lpr mice. TIP1 treatment downregulated many downstream proteins associated with TLR signaling, such as MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor 6, and IFN-α, in the peripheral blood mononuclear cells of patients with SLE. In conclusion, our data suggest that TIP1 can serve as a potential candidate for the treatment of SLE.     

A Novel 1,8-Naphthyridine-2-Carboxamide Derivative Attenuates Inflammatory Responses and Cell Migration in LPS-Treated BV2 Cells via the Suppression of ROS Generation and TLR4/Myd88/NF-κB Signaling Pathway

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.     

Pseudomonas aeruginosa DnaK Stimulates the Production of Pentraxin 3 via TLR4-Dependent NF-κB and ERK Signaling Pathways

Microbe-derived factors trigger innate immune responses through the production of inflammatory mediators, including pentraxin 3 (PTX3). PTX3 is a soluble pattern recognition molecule that stimulates the clearance of clinically important bacterial pathogens such as Pseudomonas aeruginosa. However, the P. aeruginosa factors responsible for the production of PTX3 have not been elucidated. In this study, we found that P. aeruginosa DnaK, a homolog of heat shock protein 70, induced PTX3 production. Induction was mediated by intracellular signals transmitted through the Toll-like receptor 4 (TLR4) signaling pathway. Following receptor engagement, the stimulatory signals were relayed initially through the nuclear factor kappa B (NF-κB) signaling pathway and subsequently by extracellular signal-regulated kinases (ERK), which are mitogen-activated protein kinases. However, ERK activation was negatively controlled by NF-κB, implying the existence of negative crosstalk between the NF-κB and the ERK pathways. These data suggest that P. aeruginosa DnaK acts as a pathogen-associated molecular pattern to trigger modulation of host defense responses via production of PTX3.     

Regulatory Mechanisms of Mitogen-Activated Protein Kinase Cascades in Plants: More than Sequential Phosphorylation

Mitogen-activated protein kinase (MAPK) cascades play crucial roles in almost all biological processes in plants. They transduce extracellular cues into cells, typically through linear and sequential phosphorylation and activation of members of the signaling cascades. However, accumulating data suggest various regulatory mechanisms of plant MAPK cascades in addition to the traditional phosphorylation pathway, in concert with their large numbers and coordinated roles in plant responses to complex ectocytic signals. Here, we highlight recent studies that describe the uncanonical mechanism of regulation of MAPK cascades, regarding the activation of each tier of the signaling cascades. More particularly, we discuss the unusual role for MAPK kinase kinases (MAPKKKs) in the regulation of MAPK cascades, as accumulating data suggest the non-MAPKKK function of many MAPKKKs. In addition, future work on the biochemical activation of MAPK members that needs attention will be discussed.     

Toll-Like Receptors: Expression and Roles in Otitis Media

Otitis media is mainly caused by upper respiratory tract infection and eustachian tube dysfunction. If external upper respiratory tract infection is not detected early in the middle ear, or an appropriate immune response does not occur, otitis media can become a chronic state or complications may occur. Therefore, given the important role of Toll-like receptors (TLRs) in the early response to external antigens, we surveyed the role of TLRs in otitis media. To summarize the role of TLR in otitis media, we reviewed articles on the expression of TLRs in acute otitis media (AOM), otitis media with effusion (OME), chronic otitis media (COM) with cholesteatoma, and COM without cholesteatoma. Many studies showed that TLRs 1–10 are expressed in AOM, OME, COM with cholesteatoma, and COM without cholesteatoma. TLR expression in the normal middle ear mucosa is absent or weak, but is increased in inflammatory fluid of AOM, effusion of OME, and granulation tissue and cholesteatoma of COM. In addition, TLRs show increased or decreased expression depending on the presence or absence of bacteria, recurrence of disease, tissue type, and repeated surgery. In conclusion, expression of TLRs is associated with otitis media. Inappropriate TLR expression, or delayed or absent induction, are associated with the occurrence, recurrence, chronicization, and complications of otitis media. Therefore, TLRs are very important in otitis media and closely related to its etiology.     

C-Src Is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gβγ and β-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.     

A Light‐Controlled TLR4 Agonist and Selectable Activation of Cell Subpopulations

The spatial and temporal aspects of immune cell signaling are key parameters in defining the magnitude of an immune response. Toll‐like receptors (TLRs) on innate immune cells are important in the early detection of pathogens and initiation of an immune response. Controlling the spatial and temporal signaling of TLRs would enable further study of immune synergies and assist in the development of new vaccines. Here, we show a light‐based method for the spatial control of TLR4 signaling. A TLR4 agonist, pyrimido[5,4‐b]indole, was protected with a cage at a position critical for receptor binding. This afforded a photocontrollable agonist that was inactive while caged, yet effected NF‐κB activity in cells following UV photocontrolled deprotection. We demonstrated spatial control of NF‐κB activation within a population of cells by treating all cells with the caged TLR4 agonist and constraining light exposure and consequent activation to a region of interest.     

TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production

Emerging evidence has demonstrated that Toll-like receptors (TLRs) are associated with autoimmune diseases. In this study, we investigated the role of TLR2 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although TLR2 signaling is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, TLR2 deficiency unexpectedly exacerbated psoriasiform skin inflammation. Importantly, messenger RNA (mRNA) levels of Foxp-3 and IL-10 in the lesional skin were significantly decreased in TLR2 KO mice compared with wild-type mice. Furthermore, flow cytometric analysis of the lymph nodes revealed that the frequency of regulatory T cells (Tregs) among CD4-positive cells was decreased. Notably, stimulation with Pam3CSK4 (TLR2/1 ligand) or Pam2CSK4 (TLR2/6 ligand) increased IL-10 production from Tregs and DCs and the proliferation of Tregs. Finally, adoptive transfer of Tregs from wild-type mice reduced imiquimod-induced skin inflammation in TLR2 KO mice. Taken together, our results suggest that TLR2 signaling directly enhances Treg proliferation and IL-10 production by Tregs and DCs, suppressing imiquimod-induced psoriasis-like skin inflammation. Enhancement of TLR2 signaling may be a new therapeutic strategy for psoriasis.     

Epigenetic Regulation of Autophagy in Cardiovascular Pathobiology

Cardiovascular diseases (CVDs) are the number one cause of debilitation and mortality worldwide, with a need for cost-effective therapeutics. Autophagy is a highly conserved catabolic recycling pathway triggered by various intra- or extracellular stimuli to play an essential role in development and pathologies, including CVDs. Accordingly, there is great interest in identifying mechanisms that govern autophagic regulation. Autophagic regulation is very complex and multifactorial that includes epigenetic pathways, such as histone modifications to regulate autophagy-related gene expression, decapping-associated mRNA degradation, microRNAs, and long non-coding RNAs; pathways are also known to play roles in CVDs. Molecular understanding of epigenetic-based pathways involved in autophagy and CVDs not only will enhance the understanding of CVDs, but may also provide novel therapeutic targets and biomarkers for CVDs.