Activation Of Toll-like Receptor 4 Signaling Research Articles

2-Pentadecyl-2-oxazoline inhibits lipopolysaccharide-induced microglia activation interfering with TLR4 signaling

Aim2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of N-palmitoylethanolamine, exerts anti-inflammatory activity; however, very little is known about the molecular mechanisms underlying this effect. Here, we tested the anti-neuroinflammatory effect of PEA-OXA in primary microglia and we also investigated the possible interaction of the molecule with the Toll-like receptor 4 (TLR4)–myeloid differentiation protein-2 (MD-2) complex. Main methodsThe anti-inflammatory effect of PEA-OXA was analyzed by measuring the expression and release of pro-inflammatory mediators in primary microglia by real-time PCR and ELISA, respectively. The effect of PEA-OXA on the activation of TLR4 signaling was assessed using two stably TLR4-transfected cell lines (i.e., HEK-293 and Ba/F3 cells). Finally, the putative binding mode of PEA-OXA to TLR4–MD-2 was investigated by molecular docking simulations. Key findingsTreatment with PEA-OXA resulted in the following effects: (i) it down-regulated gene expression of several pro-inflammatory molecules and the secretion of pro-inflammatory cytokines in LPS stimulated microglia cells; (ii) it did not prevent microglia activation after stimulation with TLR2 ligands; (iii) it prevented TLR4/NF-κB activation triggered by LPS in HEK-Blue™ hTLR4 cells; and (iv) it interfered with the binding of LPS to TLR4–MD-2 complex. Furthermore, molecular docking studies suggested that PEA-OXA could bind MD-2 with a 1:3 (MD-2/PEA-OXA) stoichiometry. ConclusionWe show for the first time that the anti-neuroinflammatory effect of PEA-OXA involves its activity against TLR4 signaling, making this molecule a valuable tool for the development of new compounds directed to control neuroinflammation via inhibiting TLR4 signaling.

Increased Gal-3 Mediates Microglia Activation and Neuroinflammation via the TREM2 Signaling Pathway in Prion Infection.

Galectin 3 (Gal-3) is one of the major elements for activating microglia and mediating neuroinflammation in some types of neurodegenerative diseases. However, its role in the pathogenesis of prion disease is seldom addressed. In this study, markedly increased brain Gal-3 was identified in three scrapie-infected rodent models at the terminal stage. The increased Gal-3 was mainly colocalized with the activated microglia. Coincidental with the increased brain Gal-3 in prion-infected animals, the expression of brain trigger receptor expressed in myeloid cell 2 (TREM2), one of the Gal-3 receptors, and some components in the downstream pathway also significantly increased, whereas Toll-like receptor 4 (TLR4), another Gal-3 receptor, and the main components in its downstream signaling were less changed. The increased Gal-3 signals were distributed at the areas with PrPSc deposit but looked not to colocalize directly with PrPSc/PrP signals. Similar changing profiles of Gal-3, the receptors TREM2 and TLR4, as well as the proteins in the downstream pathways were also observed in prion-infected cell line SMB-S15. Removal of PrPSc replication in SMB-S15 cells reversed the upregulation of cellular Gal-3, TREM2, and the relevant proteins. Moreover, we presented data for interactions of Gal-3 with TREM2 and with TLR4 morphologically and molecularly in the cultured cells. Stimulation of prion-infected cells or their normal partner cells with recombinant mouse Gal-3 in vitro induced obvious responses for activation of TREM2 signaling and TLR4 signaling. Our data here strongly indicate that prion infection or PrPSc deposit induces remarkably upregulated brain Gal-3, which is actively involved in the microglia activation and neuroinflammation mainly via TREM2 signaling.

METTL3-mediated m6A mRNA methylation regulates neutrophil activation through targeting TLR4 signaling

N6-methyladenosine (m6A) modification accounts for the most prevalent mRNA internal modification and has emerged as a widespread regulatory mechanism in multiple physiological processes. We address a role of methyltransferase-like protein 3 (METTL3) in neutrophil activation. METTL3 controls neutrophil release from bone marrow to circulation through surface expression of CXC chemokine receptor 2 (CXCR2) in a Toll-like receptor 4 (TLR4) signaling-dependent manner in lipopolysaccharide (LPS)-induced endotoxemia. We show that the mRNA of TLR4 is modified by m6A, exhibiting increased translation and slowed degradation simultaneously, leading to elevated protein levels of TLR4, which eventually promotes the TLR4 signaling activation of neutrophil. The reduced expression of TLR4 lowers cytokine secretion in METTL3-deleted neutrophils upon LPS stimulation through TLR4/Myd88/nuclear factor κB (NF-κB) signaling. Collectively, these data demonstrate that METTL3 modulation of TLR4 expression is a critical determinant of neutrophil activation in endotoxemia.

MiRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling.

Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme responsible for the production of inositol polyphosphates and phosphoinositide. IPMK in macrophages was identified as a key factor for the full activation of the Toll-like receptor 4 (TLR4) signaling pathway and inflammation by directly interacting with tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, dynamic changes of IPMK levels in lipopolysaccharide (LPS)-stimulated macrophages and their functional significance were investigated. Both the mRNA and protein levels of IPMK were acutely decreased in mouse and human macrophages when cells were stimulated with LPS for between 1 and 6 h. Analysis of the 3' untranslated region (UTR) of mouse IPMK mRNA revealed a highly conserved binding site for miR-181c. Transfection of miR-181c mimics into RAW 264.7 macrophages led to decreased IPMK 3'UTR-luciferase reporter activity and lowered endogenous IPMK levels. When the genomic deletion of a 33-bp fragment containing a putative miR-181c-binding site was introduced within the IPMK 3'UTR of RAW 264.7 macrophages (264.7Δ3'UTR), LPS-triggered downregulation of IPMK levels was prevented. LPS treatment in 264.7Δ3'UTR macrophages decreased TLR4-induced signaling and the expression of proinflammatory cytokines. In response to LPS stimulation, K63-linked ubiquitination of TRAF6 was impaired in 264.7Δ3'UTR macrophages, suggesting an action of IPMK in the suppression of TRAF6 activation. Therefore, our findings reveal that LPS-mediated suppression of IPMK regulates the full activation of TLR4 signaling and inflammation in macrophages.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4

Ethnopharmacological relevanceRadix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. Aim of the studyThe purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). Materials and methodsA model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. ResultsWe observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1β. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10−7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. ConclusionOur findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

Exposure to hydroxyapatite nanoparticles enhances Toll-like receptor 4 signal transduction and overcomes endotoxin tolerance in vitro and in vivo

Emerging studies indicate hydroxyapatite nanoparticles (HANPs) exhibit modest immunogenicity to elicit innate immune response which might involve Toll-like receptor 4 (TLR4) activation. This study was proposed to elucidate how HANPs direct over TLR4 signal activity in macrophage in response to TLR4 ligand, lipopolysaccharide (LPS). The present study for the first time reveals that HANPs themselves can induce TLR4 endocytosis and activate pathways both of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which potentially trigger the production of inflammatory cytokine by macrophage. Further, HANPs dose-dependently reprogram over LPS driven TLR4 signaling transduction in macrophage, leading to synergistically augmented innate immune response. In particular, HANPs synergize with LPS to promote macrophage polarization toward M1 phenotype. Moreover, HANPs abrogate the endotoxin tolerance in macrophages by restoring the production of inflammatory cytokines from macrophage in response to secondary LPS stimulation, and enhance the responsiveness of the body to LPS re-challenge in the endotoxin tolerance mice model. Therefore, this study sheds a new light on the mechanism by which HANPs drive the innate immune response, and offers a powerful strategy to potentiate LPS mediated TLR4 signaling activation in macrophage. Statement of significanceIn recent years, increasing attention has been given to hydroxyapatite nanoparticles (HANPs) on how they interact with immune cells for achieving appropriate biological effect such as bone tissue repair, soft tissue filler, tumor treatment, vaccine delivery, et al. This study indicated HANPs can induce TLR4 signaling activation. In the further, HANPs dose-dependently synergize with LPS to program over LPS induced TLR4 signaling transduction in macrophage, to favor macrophage polarizing toward M1 phenotype, as well as to abrogate immune tolerance in macrophage in response to repeated LPS stimulation. This work opens a window for the intrinsic mechanism of HANPs to drive immune response and facilitate to direct the rational use or design of HANPs for their better biomedical application.

Dorsal root ganglion toll-like receptor 4 signaling contributes to oxaliplatin-induced peripheral neuropathy.

Activation of toll-like receptor 4 (TLR4) in the dorsal root ganglion (DRG) and spinal cord contributes to the generation of paclitaxel-related chemotherapy-induced peripheral neuropathy (CIPN). Generalizability of TLR4 signaling in oxaliplatin-induced CIPN was tested here. Mechanical hypersensitivity developed in male SD rats by day 1 after oxaliplatin treatment, reached maximum intensity by day 14, and persisted through day 35. Western blot revealed an increase in TLR4 expression in the DRG of oxaliplatin at days 1 and 7 after oxaliplatin treatment. Cotreatment of rats with the TLR4 antagonist lipopolysaccharide derived from Rhodobacter sphaeroides ultrapure or with the nonspecific immunosuppressive minocycline with oxaliplatin resulted in significantly attenuated hyperalgesia on day 7 and 14 compared with rats that received oxaliplatin plus saline vehicle. Immunostaining of DRGs revealed an increase in the number of neurons expressing TLR4, its canonical downstream signal molecules myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β, at both day 7 and day 14 after oxaliplatin treatment. These increases were blocked by cotreatment with either lipopolysaccharide derived from Rhodobacter sphaeroides or minocycline. Double staining showed the localization of TLR4, MyD88, and TIR-domain-containing adapter-inducing interferon-β in subsets of DRG neurons. Finally, there was no significant difference in oxaliplatin-induced mechanical hypersensitivity between male and female rats when observed for 2 weeks. Furthermore, upregulation of TLR4 was detected in both sexes when tested 14 days after treatment with oxaliplatin. These findings suggest that the activation of TLR4 signaling in DRG neurons is a common mechanism in CIPN induced by multiple cancer chemotherapy agents.

Olanzapine-Induced Activation of Hypothalamic Astrocytes and Toll-Like Receptor-4 Signaling via Endoplasmic Reticulum Stress Were Related to Olanzapine-Induced Weight Gain.

The antipsychotic drug olanzapine is associated with serious obesity side effects. Hypothalamic astrocytes and associated toll-like receptor-4 (TLR4) signaling play an essential role in obesity pathogenesis. This study investigated the effect of olanzapine on astrocytes and TLR4 signaling both in vitro and in the rat hypothalamus and their potential role in olanzapine-induced weight gain. We found that olanzapine treatment for 24 h dose-dependently increased cell viability, increased the protein expression of astrocyte markers including glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), and activated TLR4 signaling in vitro. In rats, 8- and 36-day olanzapine treatment caused weight gain accompanied by increased GFAP and S100B protein expression and activated TLR4 signaling in the hypothalamus. These effects still existed in pair-fed rats, suggesting that these effects were not secondary effects of olanzapine-induced hyperphagia. Moreover, treatment with an endoplasmic reticulum (ER) stress inhibitor, 4-phenylbutyrate, inhibited olanzapine-induced weight gain and ameliorated olanzapine-induced changes in hypothalamic GFAP, S100B, and TLR4 signaling. The expression of GFAP, S100B, and TLR4 correlated with food intake and weight gain. These findings suggested that olanzapine-induced increase in hypothalamic astrocytes and activation of TLR4 signaling were related to ER stress, and these effects may be related to olanzapine-induced obesity.

Antiviral efficacy of orally delivered neoagarohexaose, a nonconventional TLR4 agonist, against norovirus infection in mice

The neoagarohexaose (NA6) is an oligosaccharide that is derived from agarose, the major component of red algae cell walls, by enzymatic hydrolysis. Here we show that NA6 is a noncanonical Toll-like receptor 4 (TLR4) agonist with antiviral activity against norovirus. Its TLR4 activation was dependent on myeloid differentiation factor 2 (MD2) and cluster of differentiation 14 (CD14), leading to interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) production. This effect was abolished by TLR4 knockdown or knockout in murine macrophages. NA6 inhibited murine norovirus (MNV) replication with an EC50 of 1.5 μM in RAW264.7 cells. It also lowered viral RNA titer in a human hepatocellular carcinoma Huh7-derived cell line harboring a human norovirus subgenomic replicon. The antiviral activity of NA6 was mainly attributed to IFN-β produced through the TLR4-TRIF signaling pathway. NA6-induced TNF-α, which had little effect on norovirus replication per se, primed macrophages to mount greater antiviral innate immune responses when IFN signaling was activated. NA6 boosted the induction of IFN-β in MNV-infected RAW264.7 cells and upregulated IFN-regulatory factor-1, an IFN-stimulated gene. NA6 induced IFN-β expression in the distal ileum with Peyer's patches and oral administration of NA6 reduced MNV loads through activation of TLR4 signaling, highlighting its potential contribution to protective antiviral innate immunity against norovirus.

Estrogen promotes the metastasis of non‑small cell lung cancer via estrogen receptor β by upregulation of Toll‑like receptor4 and activation of the myd88/NF‑κB/MMP2 pathway.

Estrogen promotes non‑small cell lung cancer (NSCLC) metastasis via estrogen receptorβ (ERβ)‑mediated invasiveness‑associated matrix metalloprotease2 (MMP2) upregulation. However, how ERβ increases the aggressiveness of NSCLC cells remains unclear. Recently, MMP2 was found to be upregulated by Toll‑like receptor4 (TLR4) signaling activation and to promote NSCLC metastasis. Our present study aimed to examine the role of ERβ in the activation of TLR4 signaling and in tumor progression and metastasis, and to explore the synergistic metastatic effect of a combination of ERβ and TLR4 activation on human NSCLC cells invitro and invivo. Here, we found that ERβ is associated with TLR4 in metastatic lymph nodes. Western blot analysis and immunofluorescence revealed that ERβ overexpression upregulated TLR4 protein expression and activated downstream targets, myeloid differentiation primary response88 (myd88)/nuclear factor (NF)‑κB/MMP2, enhancing NSCLC cell migration and invasion invitro. A novel ERβ‑TLR4 interaction in cell plasma was identified by co‑immunoprecipitation and confocal immunofluorescence. The combination of estradiol and specific TLR4 agonist lipopolysaccharide (LPS) synergistically promoted metastatic behaviors in NSCLC cells. In cell culture and murine lung metastasis models, exposure to estradiol and LPS induced increased matrix degradation and accelerated invadopodia and metastasis formation in NSCLC cells compared with that in cells treated with estradiol or LPS alone. Together, we showed that estrogen promoted NSCLC metastasis via ERβ by upregulating TLR4 and activating its downstream signaling axis myd88/NF‑κB/MMP2. The combined targeting of ERβ and TLR4 may be a novel therapeutic strategy against advanced metastatic lung cancer.

Western diet (WD) activates pancreatic Toll‐like receptor 4 (TLR4) signaling in female rats: a potential mechanism of WD‐induced insulin resistance

BackgroundOverconsumption of a WD, a high‐fat and high‐sugar diet, has plagued the United States contributing to the widespread prevalence of type 2 diabetes (T2DM). Insulin resistance (IR) plays a key role in the development of T2DM and is a consistent finding among T2DM patients. The pathogenesis of IR, however, is not well understood. Based on recent evidence that inflammation precedes IR, and previous findings from our lab showing involvement of Toll‐like receptor 4 (TLR4) signaling in diabetic vascular inflammation, we hypothesize that WD leads to pancreatic inflammation through TLR4 signaling activation, contributing to IR.MethodsOur lab has established a model of a WD‐induced metabolic syndrome and IR in female rats. Eight‐week old female Wistar rats were randomized into two experimental groups: Control (n = 9) and WD group (n = 16). The control group received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], and 24.1% protein), while the WD group received a WD (40% fat, 43% carbohydrates [34% sucrose], 17% protein), for 28 weeks. Body weight and food consumption were measured weekly. Lipid and glucose metabolism, and intraperitoneal glucose tolerance tests (IPGTT) were measured. Quantitative IR was assessed using Homeostasis Model Assessment of Insulin Resistance (HOMA‐IR), while functional beta cell capacity was assessed via HOMA‐B calculation. At the terminal experiments, pancreas’ were isolated for molecular (TLR4 signaling) and histological (hematoxylin and eosin, H&E) analysis.ResultsWD group exhibited greater body weight (415.1 ± 30.9 vs. 323.0 ± 24.0 g controls, p<0.01), and increased daily caloric consumption (130.5 ± 7.36 vs.110.7 ± 7.0 kcals controls, p<0.05) in spite of a significant reduction in daily food intake (23.7 ± 1.5 vs. 31.5 ± 1.8 g controls, p<0.01). WD negatively interfered in the glucose metabolism as evidenced by increased blood glucose area under the curve during IPGTT (17531.8 ± 574.3 vs. 12381.25 ± 1480.6 a.u. controls, p<0.001). IR in the WD group was confirmed by increased HOMA‐IR (8.09 ± 1.90 vs. 2.05 ± .042 a.u. controls, p<0.001), which was accompanied by hyperinsulinemia (0.93 ± 0.1 vs. 0.30 ± 0.09 mg/dL controls, p<0.01). No changes were observed in the HOMA‐B, suggesting that β cells remain functional. Moreover, no evidence of vacuolation, irregular outlining and size of the Langerhans islets, which are all markers of β cell dysfunction, were observed in the H&E stained pancreatic section from the WD group. Pancreatic inflammation in the WD group was confirmed by a 42% increase in TNF‐α expression (p<0.05), an inflammatory marker. Strikingly, pancreatic TLR4 (1.8‐fold increased, p<0.05) and its downstream molecule TNF‐receptor associated factor 6 (TRAF6) (3.1‐fold increased, p<0.001) were markedly upregulated in the WD group in comparison to controls, indicating TLR4/TRAF6 signaling activation in the pancreas.ConclusionOur findings suggest that a WD can induce IR along with activation of pancreatic TLR4 signaling‐related inflammation. These results can provide new insights into how high fat and fat sugar diet choices can causes inflammation in the pancreas.Support or Funding InformationNIH

Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P>0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P>0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P<0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.Support or Funding InformationSupported in part by NIH/NIBIB R01‐EB2006.

HMGB1 is a Potential Mediator of Astrocytic TLR4 Signaling Activation following Acute and Chronic Focal Cerebral Ischemia.

Limited, and underutilized, therapeutic options for acute stroke require new approaches to treatment. One such potential approach involves better understanding of innate immune response to brain injury such as acute focal cerebral ischemia. This includes understanding the temporal profile, and specificity, of Toll-like receptor 4 (TLR4) signaling in brain cell types, such as astrocytes, following focal cerebral ischemia. This study evaluated TLR4 signaling, and downstream mediators, in astrocytes, during acute and chronic phases post transient middle cerebral artery occlusion (MCAO). We also determined whether high mobility group box 1 (HMGB1), an endogenous TLR4 ligand, was sufficient to induce TLR4 signaling activation in astrocytes in vivo and in vitro. We injected HMGB1 into normal cortex, in vivo, and stimulated cultured astrocytes with HMGB1, in vitro, and determined TLR4, and downstream mediator, expression by immunohistochemistry. We found that expression of TLR4, and downstream mediators, such as inducible nitric oxide synthase (iNOS), occurs in penumbral astrocytes in acute and chronic phases after focal cerebral ischemia, but was undetectable in cortical astrocytes in the contralateral hemisphere. In addition, cortical injection of recombinant HMGB1 led to a trend towards an almost 2-fold increase in TLR4 expression in astrocytes surrounding the injection site. Consistent with these results, in vitro stimulation of the DI TNC1 astrocyte cell line, with recombinant HMGB1, led to increased TLR4 and iNOS message levels. These findings suggest that HMGB1, an endogenous TLR4 ligand, is an important physiological ligand for TLR4 signaling activation, in penumbral astrocytes, following acute and chronic ischemia and HMGB1 amplifies TLR4 signaling in astrocytes.

Activation of TLR4 signaling inhibits progression of osteosarcoma by stimulating CD8-positive cytotoxic lymphocytes.

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis of advanced cases is still poor. Recently, there have been several reports suggesting the relationship between innate immunity and OS, but the detailed mechanism is unknown. We demonstrate the relationship between OS and Toll-like receptor 4 (TLR4) which is one of the most important factors in innate immunity. We established a syngenic mouse tumor model using C3H/HeN, C3H/HeJ mouse and a highly metastatic OS cell line, LM8. TLR4 activation with lipopolysaccharide (LPS) was performed on both mice and its influence on the progression of OS was evaluated. We also performed CD8 + cells depletion to examine the influence on TLR4 activation effects. Tumor volume of C3H/HeN mice was significantly smaller and overall survival of C3H/HeN mice was significantly longer than C3H/HeJ mice. We found more CD8+ cells infiltrating in lung metastases of C3H/HeN mice and depletion of CD8+ cells canceled the antitumor effects of LPS. TLR4 activation by LPS increased CD8+ cells infiltrating into lung metastases and suppressed OS progression in the mouse model. TLR4 activation may suppress the progression of OS via stimulating CD8+ cells and can be expected as a novel treatment for OS.

Inflammation-dependent downregulation of miR-532-3p mediates apoptotic signaling in human sarcopenia through targeting BAK1.

Inflammation and apoptosis are considered as two major pathological causes of human sarcopenia. The current understanding based on different models recognizes that apoptosis does not trigger inflammation, while emerging evidence indicates that inflammation can induce apoptosis. Here, we provide solid evidence to suggest that the inflammation-dependent downregulation of miR-532 causes apoptosis through targeting a proapoptotic gene BAK1 (BCL2 antagonist/killer 1). To identify miRNAs and genes that are aberrantly expressed in the muscle tissues of sarcopenia patients, we conducted two independent microarray analyses. In total, we identified 53 miRNAs and 69 genes with differential expression levels. Of these aberrantly expressed miRNAs, miR-532-3p showed the most obvious changes in sarcopenia tissues, and more importantly, it can be repressed by the well-known inflammatory inducer lipopolysaccharide (LPS) in vitro. According to gene-based microarray results and the predicted targets of miR-532-3p, we presumed that BAK1 was a putative target of miR-532-3p. Further in vitro and in vivo analyses verified that miR-532-3p could directly bind to the three prime untranslated region (3'-UTR) of BAK1 through the seed sequence CUCCCAC. In addition, we found that NFKB1 (also known as p50), a subunit of the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), could specifically bind to the promoter region of miR-532-3p and repress its expression. Further analysis revealed that the activation of TLR4 (Toll-like receptor 4) signaling led to the translocation of p50 from the cytoplasm to the nucleus, where it repressed miR-532-3p expression and thus led to an increase of BAK1. The accumulated BAK1 activated its downstream apoptotic signaling pathways and resulted in apoptosis, eventually causing the pathogenesis underlying sarcopenia. Overall, our results uncovered a new mechanism by which the inflammation-dependent downregulation of miR-532-3p contributed to the pathogenesis of sarcopenia through mediating BAK1 expression.

Δ42PD-1 inhibitor acts as a PD-1-independent immunotherapy against human hepatocellular carcinoma

Abstract Pathogen infection-induced chronic inflammation is a key contributor to both carcinogenesis and progression of hepatocellular carcinoma (HCC). Persistent toll-like receptor 4 (TLR4) signaling activation can lead to inflammatory responses and chronic inflammation in the tumor microenvironment. Previously, Δ42PD1, a PD1 isoform, has been shown to directly interact with its cognate receptor TLR4 and contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Here, we aim to determine whether such interaction may also occur in HCC to promote TLR4 signaling activation and in turn induce inflammatory responses, contributing to HCC progression. We studied the relationship between TLR4, Δ42PD1, pro-inflammatory cytokines and tumor development in humanized mouse models and HCC patients. In consistence with others’ findings, TLR4 signal was shown to be associated with chronic inflammation and HCC tumor progression. Furthermore, we demonstrated that Δ42PD1 activated TLR4-mediated inflammatory responses in HCC cells via cJUN MAPK transcriptional activation. Notably, Δ42PD1-TLR4 pathway activation likely formed a feedback loop to induce Δ42PD1-expression CD3+ T cells, a new phenotype of T cell exhaustion, further contributing to tumor development. Importantly, administration of a Δ42PD1-specific monoclonal antibody CH101 interrupted Δ42PD1-TLR4 pathway and displayed therapeutic efficacy in both subcutaneous and orthotopic HCC models. Together, our results demonstrated the importance of Δ42PD1-TLR4-mediated inflammatory responses in promoting HCC and provided conclusive evidence that targeting Δ42PD1-TLR4 pathway reduced inflammatory responses to arrest HCC development.

Western diet increases HMGB1 levels and activates cardiac and hepatic Toll‐like receptor 4 (TLR4) signaling in male mice

BackgroundOverconsumption of a Western diet (WD), characterized by a high amount of fats and carbohydrates, is a major contributor to the epidemic of non‐alcoholic liver and cardiovascular disease in the United States. Our laboratory has recently demonstrated that WD causes endothelial dysfunction via vascular TLR4 signaling activation, a key component of innate immunity linked to inflammation. Based on these findings, we hypothesized that WD triggers inflammatory responses in the livers and hearts of male mice via activation of TLR4 signaling. We also investigated whether WD elevates high‐mobility group box 1 protein (HMGB1) levels, a pro‐inflammatory factor known to be an endogenous ligand for TLR4.MethodsAdult male C57BL/J mice were randomized in two experimental groups: Control Group received a regular chow diet and WD group received WD (40% fat, 43% carbohydrates, and 17% protein) for 28 weeks. Prior to terminal experiments, body weights were measured weekly. After euthanasia with isoflurane (via nasal 5% in 100% O2), livers, hearts, and adipose tissue were removed and processed for further molecular assays. Blood samples were also obtained for biochemical analysis.ResultsAs expected, WD group exhibited increased body weight (31.1 ± 3.2 vs. 25.2 ± 1.4g, p<0.01), adipocyte size (7724.2 ± 1745.8 vs. 2727.3 ± 927.1 μm2, p<0.05) and elevated serum free fatty acids (0.20 ± 0.02 vs. 0.14 ± 0.024 mM, p<0.05) compared to controls. Next, to test the hypothesis that WD activates TLR4 signaling in hepatic and cardiac tissue, Western blot analysis was performed. Hepatic expression of TLR4 (2.8 fold increase vs. controls, p<0.001) and its downstream protein TNF receptor‐associated factor 6 (TRAF6) (1.8 fold increase vs. controls, p<0.01) were markedly elevated in the WD group. Similarly, cardiac expression of TLR4 (2.2 fold increase vs. controls, p<0.01) and TRAF6 (2.8 fold increase vs. controls, p<0.001) were increased in the WD group. Strikingly, we found that serum HMGB1 expression was significantly increased in WD group in comparison to the controls (70% elevation vs control, p<0.05).ConclusionOur results suggest that upregulation of HMGB1 may contribute to TLR4/TRAF6 signaling activation in hepatic and cardiac tissue of western diet fed mice. To expand this investigation, we will utilize pharmacological and molecular approaches to target HMGB1 and confirm its role in WD‐induced inflammation.Support or Funding InformationIn‐house Grant NYITThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iron oxide nanoparticles promote macrophage autophagy and inflammatory response through activation of toll-like Receptor-4 signaling

Nanoparticle-induced autophagy is crucial for its metabolism, cytotoxicity and therapy potency, but little is known about how the host immune system would respond to it. In this study, we demonstrated that two clinically used superparamagnetic iron oxide nanoparticles (SPIONs) specifically induced macrophage autophagy through activation of TLR4, followed by phosphorylation of p38 and nucleus translocation of Nrf2, leading to upregulation of p62/SQSTM1 and macrophage scavenger receptor SR-AI mRNA expression. Overexpressed p62 conjugated with LC3 to form aggresome-like induced structures (ALIS) and then fused with SPIONs containing endosomes and lysosomes to form autolysosomes for degradation of endocytosed nanoparticles. More importantly, SPIONs also could promote macrophage autophagy in mouse liver which is their imaging target. We also discovered that SPIONs could stimulate the expression of inflammatory cytokines through activation of TLR4 signaling in macrophage. In general, our findings indicate that SPIONs would interact with TLR4 on the macrophage membrane and trigger its downstream signaling pathway, independent of the classic autophagic p62 reduction pathway. The observed autophagy and induced inflammatory responses in macrophages provide unique and novel perspectives in optimizing imaging/therapy nanoparticle performance in addition to analysis by traditional biochemical evaluation methods. It also enriches our understanding of NP/macrophage interaction mechanisms in reticular endothelial system (RES) organs.

Intraperitoneal injection of 4-hydroxynonenal (4-HNE), a lipid peroxidation product, exacerbates colonic inflammation through activation of Toll-like receptor 4 signaling

Human and animal studies have shown that the colonic concentrations of lipid peroxidation products, such as 4-hydroxynonenal (4-HNE), are elevated in inflammatory bowel disease (IBD). However, the actions and mechanisms of these compounds on the development of IBD are unknown. Here, we show that a systemic treatment of low-dose 4-HNE exacerbates dextran sulfate sodium (DSS)-induced IBD in C57BL/6 mice, suggesting its pro-IBD actions in vivo. Treatment with 4-HNE suppressed colonic expressions of tight-junction protein occludin, impaired intestinal barrier function, enhanced translocation of lipopolysaccharide (LPS) and bacterial products from the gut into systemic circulation, leading to increased activation of Toll-like receptor 4 (TLR4) signaling in vivo. Furthermore, 4-HNE failed to promote DSS-induced IBD in Tlr4-/- mice, supporting that TLR4 signaling contributes to the pro-IBD effects of 4-HNE. Together, these results suggest that 4-HNE exacerbates the progression of IBD through activation of TLR4 signaling, and therefore could contribute to the pathogenesis of IBD.

An anti-asthmatic activity of natural Toll-like receptor-4 antagonist in OVA-LPS-induced asthmatic rats.

Toll-like receptor-4 (TLR4) is a key component of the innate immune system and activation of TLR4 signaling has a significant role in the pathogenesis of asthma. Therefore, our objective was to identify the natural TLR4 antagonist and evaluate its activity in experimentally induced asthma. Soya lecithin origin phosphatidylcholine (soya PC) was identified as a natural TLR4 antagonist by computational study. Based on the computational study, TLR4 antagonist activity of soya PC was confirmed in in vitro lipopolysaccharide (LPS)-induced neutrophil adhesion assay. In the in vivo study, rats were sensitized with ovalbumin (OVA) (100μg/kg, i.p.) on the 7th, 14th and 21st days and challenged intranasally with OVA (100μg/100μL) and LPS (10ng/100μL), 4days/wk for 3weeks. At the end of the experiment, we performed lung function parameters (respiratory rate, tidal volume, airflow rate), inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13), total and differential leukocytes in blood as well as bronchoalveolar lavage fluid (BALf) and histological examinations. The computational study indicated that TLR4 antagonist activity of soya PC is due to linoleic acid (18:2) fatty acid chain. Soya PC significantly suppressed the LPS-induced neutrophil adhesion in a concentration-dependent manner to 1μg/mL. The treatment of soya PC (5 and 10mg/kg, 18days, i.p.) significantly improved the lung function parameters, total and differential leukocyte counts in blood and BALf in asthmatic rats. This efficacy of soya PC was in extent similar to dexamethasone (2.5mg/kg, 18days, i.p.). However, soya PC was superior to dexamethasone in terms of benefits. The protective action of soya PC may be due to TLR4 antagonist activity and linoleic acid composition.