Targeting Toll-like Receptors Research Articles

MicroRNA-146a Protects Against Ischemia/Reperfusion Liver Injury Through Inhibition of Toll-like Receptor 4 Signaling Pathway in Rats

Hepatic ischemia/reperfusion injury (IRI) is a severe and common clinical challenge involved in liver surgery and transplantation. MicroRNA-146a (miR-146a) has recently been reported to be abnormally expressed in hepatic IRI, but the underlying mechanism is not fully elucidated. Accumulating evidences showed miR-146a targets Toll-like receptor 4 (TLR4) signaling pathway. Here, we found that miR-146a inhibited TLR4 signaling pathway accompanied by attenuated liver dysfunction, histologic injury and inflammation. Conversely, miR-146a inhibition increased TLR4 and interleukin-1 receptor-associated kinase, accompanied by exacerbated hepatic IRI and inflammation. Taken together, these data indicated that miR-146a protect against hepatic IRI via inhibiting TLR4 signaling pathway. In addition, we verified ultrasound microbubble-mediated gene transfection improved miR-146a transfection efficacy.

MiR-370-3p Alleviates Ulcerative Colitis-Related Colorectal Cancer in Mice Through Inhibiting the Inflammatory Response and Epithelial-Mesenchymal Transition.

IntroductionUlcerative colitis (UC) is a chronic and inflammatory bowel disease. UC-associated colorectal cancer (UC-CRC) is one of the most severe complications of long-standing UC. In the present study, we explored the effects of miR-370-3p on UC-CRC in vivo and investigated its underlying mechanisms in vivo and in vitro.MethodsAzoxymethane (AOM) and dextran sodium sulfate (DSS) were used to induce UC-CRC in C57BL/6 mice. AOM/DSS-induced mice were treated with 5×108 pfu miR-370-3p overexpressing-adenovirus via tail-vein injection every two weeks.ResultsWe found that miR-370-3p significantly improved the body weights and survival rates and inhibited the tumorigenesis of UC-CRC in AOM/DSS mice. Mechanically, miR-370-3p inhibited AOM/DSS-induced inflammatory response by decreasing tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) through targeting toll-like receptor 4 (TLR4), as demonstrated by down-regulation of TLR4, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and phosphorylated epidermal growth factor receptor (pEGFR). miR-370-3p decreased the expression of tumor-associated proteins, including p53, β-catenin, and ki67 in AOM/DSS-treated mice. Additionally, miR-370-3p remarkably inhibited epithelial-mesenchymal transition (EMT) via increasing E-cadherin expression and reducing N-cadherin and Vimentin expression in vivo. Further studies showed that miR-370-3p repressed proliferation and EMT of colon cancer cells in vitro. Moreover, we proved that miR-370-3p decreased the expression of tumor-associated proteins and reversed EMT by regulating β-catenin in colon cancer cells.ConclusionTaken together, miR-370-3p alleviated UC-CRC by inhibiting the inflammatory response and EMT in mice, which suggested miR-370-3p as a novel potential target for UC-CRC therapy.

Development of RNA/DNA Hydrogel Targeting Toll-Like Receptor 7/8 for Sustained RNA Release and Potent Immune Activation.

Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity.

Exploring Methamphetamine Nonenantioselectively Targeting Toll-like Receptor 4/Myeloid Differentiation Protein 2 by in Silico Simulations and Wet-Lab Techniques.

Methamphetamine (METH) is one of the highly addictive nonopioid psychostimulants, acting as a xenobiotic-associated molecular pattern (XAMP) to target TLR4 and activate microglia. However, the molecule recognition of METH by innate immune receptor TLR4/MD-2 is not well-understood. METH exists in two enantiomeric forms, and it is unclear whether the TLR4 innate immune recognition with METH is stereoselective. Herein, molecular dynamics (MDs) simulations were performed to dissect the recognition of (+)-METH and (-)-METH by TLR4/MD-2 at the atomic level. Amphetamine (AMPH), which is an analogue of METH, was also investigated for comparison. Computational simulations indicate that METH binds into the interaction interface between MD-2 as well as TLR4* that is from the adjacent copy of TLR4-MD-2, therefore stabilizing the active heterotetramer (TLR4/MD-2)2 complex. The calculated binding free energies and potential of mean force (PMF) values show that (-)-METH and (+)-METH have similar TLR4/MD-2 binding affinity. Further dynamics analyses of bindings with TLR4/MD-2 indicate that (-)-METH and (+)-METH behave similarly. Unlike the stereoselective neuron-stimulating activities of METH, no enantioselectivity was observed for METH interacting with TLR4/MD-2 complex as well as activating TLR4 signaling. Compared to METH, AMPH showed much weaker interactions with TLR4/MD-2, indicating that the substituted methyl group is critical in the molecular recognition of METH by TLR4/MD-2. In all, this study provides molecular insight into the innate immune recognition of METH, which demonstrates that METH could be nonenantioselectively sensed by TLR4/MD-2.

Expression profile of Toll‑like receptors in human breast cancer.

Toll-like receptors (TLRs) are the most widely studied pattern recognition receptors. Mounting evidence suggests an important association between TLRs and the occurrence and development of breast cancer. Thus, targeting these receptors may be a potential strategy for breast cancer treatment. The current study analyzed the data of 1,215 patients with breast cancer obtained from The Cancer Genome Atlas (TCGA) database. It was observed that, in addition to TLR6, TLR7 and TLR8, the expression of the remaining TLRs in breast cancer tissues was lower than that in normal tissues. In addition, TLR3 and TLR9 displayed significantly different expression levels in ER-/PR-negative breast cancer compared with the control tissues, while TLR5 expression was significantly reduced in HER2-enriched breast cancer. Furthermore, TLR10 exhibited lower expression levels in advanced stages of the disease as compared with that observed in earlier stages. Survival analysis revealed that the expression of TLR4 and TLR7 had a significant impact on survival, and higher expression levels suggested worse prognosis. Finally, the expression levels of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 were correlated with those of the inflammatory cytokines interleukin-1β and tumor necrosis factor-α, while the expression levels of TLR3, TLR7, TLR8 and TLR9 were correlated with those of interferon-β and C-X-C motif chemokine ligand 10. Taken together, the current study results suggest that TLR expression may serve as a biomarker of cancer pathogenesis and progression, and may provide new insights for the treatment of breast cancer through the regulation and targeting of TLRs.

Treatment with toll-like receptor 2 inhibitor ortho-vanillin alleviates lipopolysaccharide-induced acute kidney injury in mice.

Reducing inflammation is a promising approach for the prevention and treatment of septic acute kidney injury (AKI), since AKI is characterized by excessive inflammation in the kidney. Previous studies have demonstrated that toll-like receptor 2 (TLR2) is overstimulated, which promotes inflammation by activating the NF-κB signaling pathway, in a lipopolysaccharide (LPS)-induced model of AKI mice. For the present study, it was hypothesized that TLR2 inhibition could reduce inflammation and consequently prevent septic AKI. Therefore, the potential renal protective effects of ortho-vanillin (OV), an inhibitor of TLR2, were investigated in the present study in vitro and in vivo. In vitro treatment with OV on LPS-stimulated mouse podocyte cell line MPC5 did not affect TLR2 expression but interrupted the interaction between TLR2 and its downstream adaptor MyD88, resulting in the reduction of inflammatory cytokines IL-6 and TNF-α expression. In vivo OV treatment in an LPS-challenged mouse model effectively alleviated LPS-induced kidney injury as indicated by histology analysis and the significantly reduced blood urea nitrogen and serum creatinine levels. Additionally, inflammatory cytokines TNF-α, IL-6 and IL-1β expression were also significantly reduced in mice with OV treatment. Signaling pathway analysis further demonstrated that OV treatment did not affect the expression of TLR2 and p65 but suppressed p65 phosphorylation. Taken together, data from the present study demonstrated that OV was effective in protecting renal function against LPS-induced AKI through the inhibition of TLR2/NF-κB signaling and subsequent inflammatory cytokine production. These findings indicated that OV or targeting TLR2 signaling in general, represents a novel therapeutic approach for use in the prevention and treatment of AKI.

Tannic acid prevents macrophage-induced pro-fibrotic response in lung epithelial cells via suppressing TLR4-mediated macrophage polarization.

Polarized macrophages induce fibrosis through multiple mechanisms, including a process termed epithelial-to-mesenchymal transition (EMT). Mesenchymal cells contribute to the excessive accumulation of fibrous connective tissues, leading to organ failure. This study was aimed to investigate the effect of tannic acid (TA), a natural dietary polyphenol on M1 macrophage-induced EMT and its underlying mechanisms. First, we induced M1 polarization in macrophage cell lines (RAW 264.7 and THP-1). Then, the conditioned-medium (CM) from these polarized macrophages was used to induce EMT in the human adenocarcinomic alveolar epithelial (A549) cells. We also analysed the role of TA on macrophage polarization. We found that TA pre-treated CM did not induce EMT in epithelial cells. Further, TA pre-treated CM showed diminished activation of MAPK in epithelial cells. Subsequently, TA was shown to inhibit LPS-induced M1 polarization in macrophages by directly targeting toll-like receptor 4 (TLR4), thereby repressing LPS binding to TLR4/MD2 complex and subsequent signal transduction. It was concluded that TA prevented M1 macrophage-induced EMT by suppressing the macrophage polarization possibly through inhibiting the formation of LPS-TLR4/MD2 complex and blockage of subsequent downstream signal activation. Further, our findings may provide beneficial information to develop new therapeutic strategies against chronic inflammatory diseases.

Galactan isolated from Cantharellus cibarius modulates antitumor immune response by converting tumor-associated macrophages toward M1-like phenotype

Tumor-associated macrophages (TAMs) with an M2-like phenotype have been linked to the proliferation, invasion and metastasis of tumor cells. Resetting tumor-associated macrophages represents an attractive target for an effective cancer immunotherapy. WCCP-N-b, a novel linear 3-O-methylated galactan, isolated from Cantharellus cibarius, can convert tumor-promoting M2-like macrophages to tumor-inhibiting M1-like phenotype. On a cellular mechanistic level, WCCP-N-b inhibited M2-like macrophages polarization through suppression of STAT6 activation. Furthermore, WCCP-N-b increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and degradation of IκB-α through targeting Toll-like receptor 2 (TLR2). The activation of MAPKs and degradation of IκB-α were responsible for converting M2-like macrophages to M1-like macrophages. Importantly, cell culture supernatants of WCCP-N-b-treated M2-like macrophages could inhibit the cell viability of B16F1 and B16F10. Our findings provide a potential natural and harmless polysaccharide for macrophage-based tumor immunotherapy.

Tanshinone IIA ameliorates non-alcoholic fatty liver disease through targeting peroxisome proliferator-activated receptor gamma and toll-like receptor 4.

ObjectiveTo investigate the cellular mechanisms of action of tanshinone IIA on the fatty liver disease induced by a high-fat diet in an animal model of non-alcoholic fatty liver disease (NAFLD).MethodsAdult male Sprague Dawley rats were randomized into one of three groups: regular rat diet (CON group) for 4 months; high-fat diet (HFD group) for 4 months; HFD for 2 months followed by tanshinone IIA treatment plus HFD (TAN group) for a further 2 months. A range of physical and biochemical markers of lipid accumulation and fatty liver disease were measured and compared between the groups.ResultsTanshinone IIA treatment significantly reduced fat accumulation in the liver and plasma lipid levels that had been increased by HFD. The toll-like receptor (TLR4)/nuclear factor kappa B (NF-κB) signalling pathway was silenced by tanshinone IIA treatment. Tumour necrosis factor-α and interleukin-6 were reduced by tanshinone IIA. Hepatocyte apoptosis was inhibited by tanshinone IIA. Tanshinone IIA upregulated peroxisome proliferator-activated receptor gamma (PPAR-γ), which resulted in an improvement in the oxidative status.ConclusionTanshinone IIA ameliorates NAFLD by targeting PPAR-γ and TLR4, resulting in decreased plasma lipids and oxidative stress, suggesting this strategy may form the basis of novel NAFLD therapies.

MiR-140-5p Attenuates Neuroinflammation and Brain Injury in Rats Following Intracerebral Hemorrhage by Targeting TLR4.

The Toll-like receptor 4 (TLR4)-mediated neuroinflammation plays a key role in inducing secondary brain injury after intracerebral hemorrhage (ICH). However, how TLR4 is regulated during this pathological process is not well understood. In the present study, by taking advantage of a rat ICH model, we show that miR-140-5p is reversely correlated with TLR4 expression in the peri-hematomal striatum following ICH. In vitro, miR-140-5p directly targets TLR4 and suppresses its expression in a rat neuronal PC12 cell line. Moreover, an intracerebral ventricular injection of miR-140-5p mimics improves neurological function and reduces apoptotic cell death and limits the production of inflammatory cytokines following ICH, indicating that miR-140-5p attenuates brain injury and neuroinflammation in vivo. Furthermore, miR-140-5p suppresses TLR4 expression and inhibits the downstream MyD88/TRIF inflammatory pathway and NF-κB activity following ICH, suggesting that the inhibition of TLR4-mediated neuroinflammation at least in part accounts for the neuroprotective role of miR-140-5 against ICH-induced brain injury in rats. Collectively, these results identify miR-140-5 as a negative regulator of TLR4 and downstream inflammatory pathway following ICH, implicating that miR-140-5 might represent as a potential therapeutic target for alleviating ICH-induced brain injury.

Toll-like receptor 4: A promising therapeutic target for pneumonia caused by Gram-negative bacteria.

Gram‐negative bacteria (GNB) emerge as important pathogens causing pulmonary infection, which can develop into sepsis due to bacterial resistance to antibiotics. GNB pneumonia poses a huge social and economic burden all over the world. During GNB infection in the lung, Toll‐like receptor 4 (TLR4) can form a complex with MD2 and CD14 after recognizing lipopolysaccharide of GNB, initiate the MyD88‐ and TRIF‐dependent signalling pathways and stimulate host non‐specific immune response. In this review, we summarize recent progress in our understanding of the role of TLR4 in GNB pneumonia. The latest experimental results, especially in TLR4 knockout animals, suggest a promising potential of targeting TLR4 signalling pathway for the treatment of GNB pneumonia. Furthermore, we highlight the benefits of Traditional Chinese Medicine as novel candidates for the therapy of GNB pneumonia due to the modulation of TLR4 signalling pathway. Finally, we discuss the promise and challenge in the development of TLR4‐based drugs for GNB pneumonia.

Development of a Nanostructured RNA/DNA Assembly as an Adjuvant Targeting Toll-Like Receptor 7/8.

Adjuvants are essential for efficiently inducing an antigen-specific immune response in vaccine therapy. Single-stranded RNA (ssRNA) containing guanosine- and uridine-rich sequences is recognized by Toll-like receptor (TLR)7 and/or TLR8 and induces strong immune responses; thus, the application of ssRNA as an adjuvant is desirable. The development of a ssRNA-based adjuvant, however, requires the efficient delivery of ssRNA into the endosomes of antigen-presenting cells, where the TLRs exist. To achieve this, we developed a nanostructured RNA/DNA assembly using DNA nanotechnology, which can be efficiently recognized by antigen-presenting cells. The nanostructured RNA/DNA assembly, named tetrapodRD3, was designed using a 40-mer phosphorothioate-stabilized RNA and three 40-mer phosphodiester DNAs. TetrapodRD3 was more stable than ssRNA under serum conditions. The secreted alkaline phosphatase assay using HEK-Blue hTLR cells showed that tetrapodRD3 triggered human TLR8-specific responses. Fluorescently labeled tetrapodRD3 was efficiently taken up by murine dendritic DC2.4 cells and induced a high level of tumor necrosis factor-α release from the cells. Antigen presentation by the major histocompatibility complex class I on bone marrow-derived dendritic cells was significantly increased by the addition of an antigen along with tetrapodRD3. These results indicate that tetrapodRD3 constructed using DNA nanotechnology can be a useful adjuvant targeting human TLR8.

Cell Type Specific Expression of Toll-Like Receptors in Human Brains and Implications in Alzheimer's Disease.

Toll-like receptors mediate important cellular immune responses upon activation via various pathogenic stimuli such as bacterial or viral components. The activation and subsequent secretion of cytokines and proinflammatory factors occurs in the whole body including the brain. The subsequent inflammatory response is crucial for the immune system to clear the pathogen(s) from the body via the innate and adaptive immune response. Within the brain, astrocytes, neurons, microglia, and oligodendrocytes all bear unique compositions of Toll-like receptors. Besides pathogens, cellular damage and abnormally folded protein aggregates, such as tau and Amyloid beta peptides, have been shown to activate Toll-like receptors in neurodegenerative diseases such as Alzheimer's disease. This review provides an overview of the different cell type-specific Toll-like receptors of the human brain, their activation mode, and subsequent cellular response, as well as their activation in Alzheimer's disease. Finally, we critically evaluate the therapeutic potential of targeting Toll-like receptors for treatment of Alzheimer's disease as well as discussing the limitation of mouse models in understanding Toll-like receptor function in general and in Alzheimer's disease.

The Defensive Action of LYRM03 on LPS-Induced Acute Lung Injury by NF-κB/TLR4/NLRP3 Signals

The aim of the current investigation was to study the role of 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine (LYRM03) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and investigate its potential pathogenesis. An LPS-induced ALI model was produced with LPS (5 mg/kg) followed by 24 h of injury. Rats were randomly assigned to 6 groups for in vivo experiments: (1) Sham, (2) LYRM03 (20 mg/kg), (3) LPS, (4) LPS plus LYRM03 (5 mg/kg), (5) LPS plus LYRM03 (10 mg/kg), and (6) LPS plus LYRM03 (20 mg/kg). The rat alveolar macrophage cell line (NR8383) cells were divided into 6 groups for in vitro experiments: (1) Sham, (2) LYRM03 (200 μmol/L), (3) LPS (100 ng/mL), (4) LPS plus LYRM03 (50 μmol/L), (5) LPS plus LYRM03 (100 μmol/L), and (6) LPS plus LYRM03 (200 μmol/L). Further study about siRNA targeting NF-κB p65, TLR4, and NLRP3 to explore the potential mechanism of LYRM03 in the LPS-induced ALI models have been done. Therefore, LYRM03 decreased LPS-induced ALI and NR8383 activation as demonstrated through hematoxylin-eosin staining and western blot analysis in vivo and in vitro. LYRM03 ameliorated the content of protein in bronchoalveolar lavage fluid, myeloperoxidase in the lung and malondialdehyde (MDA) in serum. In addition, LYRM03 ameliorated the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in the serum of rats and the supernatant of NR8383 cells. Moreover, LYRM03 significantly inhibited the activities of nuclear factor kappa B (NF-κB), myeloid differentiation factor 88 (MyD88), and toll-like receptor 4 (TLR4). LYRM03 also reduced the increase in the inflammasome, including apoptosis-related speck-like protein containing CARD (ASC), and NOD-like receptor 3 (NLRP3), in LPS-stimulated rats and NR8383 cells. The extent of injury and lung injury scores in the LYRM03 (20 mg/kg) + siRNA targeting NF-κB p65, TLR4, or NLRP3 + LPS-treated rats were higher than that in the LYRM03 (20 mg/kg) + LPS-treated rats. In summary, LYRM03 conferred an intensely lung defensive action on LPS-induced ALI in vivo and in vitro, which could be associated with the abatement of TLR4-induced NLRP3/NF-κB.

Regulation of Toll-Like Receptor (TLR) Signaling Pathway by Polyphenols in the Treatment of Age-Linked Neurodegenerative Diseases: Focus on TLR4 Signaling.

Neuronal dysfunction initiates several intracellular signaling cascades to release different proinflammatory cytokines and chemokines, as well as various reactive oxygen species. In addition to neurons, microglia, and astrocytes are also affected by this signaling cascade. This release can either be helpful, neutral or detrimental for cell survival. Toll-like receptors (TLRs) activate and signal their downstream pathway to activate NF-κB and pro-IL-1β, both of which are responsible for neuroinflammation and linked to the pathogenesis of different age-related neurological conditions. However, herein, recent aspects of polyphenols in the treatment of neurodegenerative diseases are assessed, with a focus on TLR regulation by polyphenols. Different polyphenol classes, including flavonoids, phenolic acids, phenolic alcohols, stilbenes, and lignans can potentially target TLR signaling in a distinct pathway. Further, some polyphenols can suppress overexpression of inflammatory mediators through TLR4/NF-κB/STAT signaling intervention, while others can reduce neuronal apoptosis via modulating the TLR4/MyD88/NF-κB-pathway in microglia/macrophages. Indeed, neurodegeneration etiology is complex and yet to be completely understood, it may be that targeting TLRs could reveal a number of molecular and pharmacological aspects related to neurodegenerative diseases. Thus, activating TLR signaling modulation via natural resources could provide new therapeutic potentiality in the treatment of neurodegeneration.

Long noncoding RNA UBE2R2-AS1 promotes glioma cell apoptosis via targeting the miR-877-3p/TLR4 axis.

Introduction: Brain glioma is the most common type of primary malignancy in the central nervous system (CNS), with high recurrence and mortality rate, especially glioblastoma (GBM). Recent evidence suggests a role for many long noncoding RNAs (lncRNAs) in the pathogenesis, proliferation, apoptosis, metastasis, and chemotherapeutic resistance of cancer cells. Although the functions of some lncRNAs in the occurrence and development of gliomas have been confirmed, detailed mechanisms of action are lacking. Furthermore, the biological roles of many other lncRNAs in glioma have not been reported at all.Methods: In this study, we identified a novel lncRNA, UBE2R2-AS1, which was dramatically downregulated in glioma compared with normal tissue, by performing microarray detection of six pairs of glioma samples and adjacent normal tissues. In vitro experiments demonstrated that UBE2R2-AS1 regulated glioma cell proliferation, apoptosis, and migration.Results: UBE2R2-AS1 acted as a competing endogenous RNA (ceRNA) to target Toll-like receptor 4 (TLR4) mRNA by binding to miR-877-3p. Furthermore, lncRNA UBE2R2-AS1 suppressed glioblastoma cell growth, migration, and invasion, as well as promoting cell apoptosis by targeting miR-877-3p/TLR4 directly.Conclusion: This information regarding UBE2R2-AS1 and its glioma-related molecular mechanisms will aid the future identification of new lncRNA-directed diagnostics and drug-targeting therapies.

Cell-traversal protein for ookinetes and sporozoites (CelTOS) formulated with potent TLR adjuvants induces high-affinity antibodies that inhibit Plasmodium falciparum infection in Anopheles stephensi

BackgroundPlasmodium falciparum parasite is the most deadly species of human malaria, and the development of an effective vaccine that prevents P. falciparum infection and transmission is a key target for malarial elimination and eradication programmes. P. falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate. A comparative study was performed to characterize the immune responses in BALB/c mouse immunized with Escherichia coli-expressed recombinant PfCelTOS (rPfCelTOS) in toll-like receptor (TLR)-based adjuvants, CpG and Poly I:C alone or in combination (CpG + Poly I:C), followed by the assessment of transmission-reducing activity (TRA) of anti-rPfCelTOS antibodies obtained from different vaccine groups in Anopheles stephensi.MethodsThe aim of the current work was achieved by head-to-head comparison of the vaccine groups using conventional and avidity enzyme-linked immunosorbent assay (ELISA), immunofluorescence test (IFAT), and standard membrane feeding assay (SMFA).ResultsComparing to rPfCelTOS alone, administration of rPfCelTOS with two distinct TLR-based adjuvants in vaccine mouse groups showed a significant increase in responses (antibody level, IgG subclass analysis, avidity, and Th1 cytokines) and was able to induce reasonable transmission-reducing activity. Also, comparable functional activity of anti-rPfCelTOS antibodies was found in group that received antigen in either CpG or Poly I:C (69.9%/20% and 73.5%/24.4%, respectively, reductions in intensity/prevalence). However, the vaccine group receiving rPfCelTOS in combination with CpG + Poly I:C showed a significant induction in antibody titers and inhibitory antibodies in oocysts development (78.3%/19.6% reductions in intensity/prevalence) in An. stephensi.ConclusionsA key finding in this investigation is that rPfCelTOS administered alone in BALB/c mouse is poorly immunogenic, with relatively low IgG level, avidity, inhibitory antibodies, and mixed Th1/Th2 responses. However, immunological characteristic (IgG level, cytophilic IgG2a and IgG2b, avidity, and Th1 cytokines) and TRA of anti-rPfCelTOS significantly enhanced in the presence of co-administration of TLR-based adjuvants, confirming that targeting TLRs would be an effective means for the enhancement of inducing TRA against rPfCelTOS.

TLR4 blockade using TAK-242 suppresses ovarian and breast cancer cells invasion through the inhibition of extracellular matrix degradation and epithelial-mesenchymal transition

Numerous links exist between inflammation and tumor development. Toll-like receptor 4 (TLR4) expression by tumor cells can be a contributing factor that promotes tumor cell proliferation, survival, migration, and metastasis. In this study, we explored the impact of TLR4 inhibition using TAK-242, a specific inhibitor of TLR4, on the invasion properties of ovarian (A2780CP, 2008C13, SKOV3, and A2780S) and breast (MCF7, SKBR3, MDA-MB-231, and BT-474) cancer cell lines. Six out of eight cell lines expressed TLR4 and its downstream mediators (MyD88, NF-ĸB1, and RELB), indicating that these cell lines could be proper candidates for the TLR4 inhibition. TAK-242 induced a cytotoxic effect on all tested cell lines; however, a different cell sensitivity pattern was noticeable. Interestingly, in the TLR4-expressing cell lines, there was a significant correlation between the TLR4/MyD88 expressions and the cancer cell response to TAK-242: the higher the expression, the higher the IC50. To the best of our knowledge, no study has addressed the effects of TAK-242 on invasive abilities of cancer cells and our study suggests for the first time that TAK-242 could considerably decrease invasion properties of ovarian and breast cancer cell lines. We found that not only did TAK-242 reduce the enzymatic activity of MMP2 and MMP9, but also down-regulated gene expressions of epithelial-mesenchymal transition (EMT)-related genes. In sum, it seems that targeting TLR4 using TAK-242 possesses novel promising potential in cancer treatment strategies and may prevent invasion in patients suffering from ovarian and breast cancers, especially in those with over-expression of TLR4.

MicroRNA-217 relieved neuropathic pain through targeting toll-like receptor 5 expression.

Neuropathic pain is the most common chronic pain that is caused by nerve injury or disease that influences the nervous system. Increasing evidence suggested that microRNAs (miRNAs) play a crucial role in neuropathic pain and neuroinflammation development. However, the functional role of miR-217 in the development of neuropathic pain remains unknown. In this study, we used rats to establish a neuropathic pain model and showed that the miR-217 expression level was upregulated in the spinal dorsal horn of bilateral sciatic nerve chronic constriction injury (bCCI). However, the expression of miR-217 was not changed in the anterior cingulated cortex (ACC), hippocampus, and dorsal root ganglion (DRG) of bCCI rats. Ectopic expression of miR-217 attenuated neuropathic pain and suppressed neuroinflammation expression in vivo. We identified toll-like receptor 5 (TLR5) as a direct target gene of miR-217 in the PC12 cell. In addition, we demonstrated that the expression level of TLR5 was upregulated in bCCI rats. Moreover, restoration of TLR5 rescued the inhibitory roles induced by miR-217 overexpression on neuropathic pain and neuroinflammation development. These data suggested that miR-217 played a pivotal role in the development of neuropathic pain partly through regulating TLR5 expression.

18β-glycyrrhetinic acid inhibited mitochondrial energy metabolism and gastric carcinogenesis through methylation-regulated TLR2 signaling pathway.

The natural phenolic substance, 18β-glycyrrhetinic acid (GRA), has shown enormous potential in the chemoprevention of cancers with rich resources and biological safety, but the GRA-regulated genetic and epigenetic profiles are unclear. Deregulated mitochondrial cellular energetics supporting higher adenosine triphosphate provisions relative to the uncontrolled proliferation of cancer cells is a cancer hallmark. The Toll-like receptor 2 (TLR2) signaling pathway has emerged as a key molecular component in gastric cancer (GC) cell proliferation and epithelial homeostasis. However, whether TLR2 influenced GC cell energy metabolism and whether the inhibition effects of GRA on GC relied on TLR2 signaling were not illustrated. In the present study, TLR2 mRNA and protein expression levels were elevated in gastric tumors in the K19-Wnt1/C2mE (Gan) mice model, GC cell lines and human GCs, and the overexpression of TLR2 was correlated with the high histological grade and was a poor prognostic factor in GC patients. Further gain and loss of function showed that TLR2 activation induced GC cell proliferation and promoted reactive oxygen species (ROS) generation, Ca2+ accumulation, oxidative phosphorylation and the electron transport chain, while blocking TLR2 inhibited mitochondrial function and energy metabolism. Furthermore, GRA pretreatment inhibited TLR2-activated GC cell proliferation, energy metabolism and carcinogenesis. In addition, expression of TLR2 was found to be downregulated by GRA through methylation regulation. Collectively, the results demonstrated that GRA inhibited gastric tumorigenesis through TLR2-accelerated energy metabolism, suggesting GRA as a promising therapeutic agency targeting TLR2 signaling in GC.