Activation Of TLR4 Signaling Pathway Research Articles

Inhibition of TLR4/NF-κB pathway and endoplasmic reticulum stress by overexpressed S100A4 ameliorates retinal ischemia-reperfusion injury of mice.

Retinal ischemia exists in various ischemic retinopathies including glaucoma, contributing to the death of retinal neurons. Calcium binding protein S100A4 is important in tumors, and our previous study found that S100A4 protects retinal ganglion cells (RGCs) against retinal ischemia-reperfusion (I/R) injury. This study was aimed to further discuss the neuroprotection and mechanisms of S100A4 in retinal I/R of mice. The rAAV-EF1α-s100a4-EGFP-WPRE or rAAV-EF1α-EGFP-WPRE-Pa was injected intravitreally 4 weeks before I/R. S100A4, molecules in TLR4 signaling pathway and endoplasmic reticulum (ER) stress branches, inflammatory molecules, and surviving RGCs and cholinergic amacrine (ChAT) cells were determined by quantitative PCR, western blot, or immunofluorescent staining. The apoptosis and necrosis of retinal neurons induced by I/R were inhibited by overexpressed S100A4. RGCs, ChAT cells, and the retinal function were preserved by S100A4 overexpressing 7 days after I/R. Mechanistically, the beneficial effects of S100A4 may be mediated by inhibiting the activation of TLR4 signaling pathway and alleviating ER stress, leading to the attenuation of inflammatory response of the retina after I/R. Our findings indicated that S100A4 has neuroprotective effectagainst retinal I/R injury, and promoting S100A4 expression may be an effective strategy to inhibit retinal neurons from degeneration in ischemic retinopathy.

Abstract 3611: PAUF induces migration of human pancreatic cancer cells exclusively via the TLR4/MyD88/NF-κB signaling pathway

Abstract Background: Pancreatic adenocarcinoma up-regulated factor (PAUF) is a cancer-secreted protein which is overexpressed on pancreatic cancer (PC) cells and promotes tumor growth in an autocrine way. However, the receptor of PAUF on PC cells has yet to be identified. Meanwhile, it is known that PAUF, as a tumor microenvironment (TME) modulator, plays a role in immune evasion and suppression through toll-like receptors notably TLR4 which are mainly expressed on immune cells. Previous studies show that TLR4 is also highly expressed in PC tumor tissues, compared to normal tissues, and activation of TLR4 signaling pathway induces the migration and invasion of PC cells. Objective: This study aimed to investigate if TLR4 expressed on cancer cells can act as a receptor for PAUF to mediate its tumor promoting effects. Methods and Results: We tested the TLR4 expression in six PC cell lines and one normal pancreatic cell line. And confirmed that TLR4 is expressed in all PC cell lines at variable levels, but not in the normal pancreatic cell line.Next, we found that a chemical TLR4-specific inhibitor TAK-242 significantly reduced the migration of PC cells with high TLR4 expression (BxPC-3), but it did not impact the migration of PC cells with low TLR4 expression (Panc-1). This experiment suggests that some endogenous ligands of TLR4 may have mediated PC cell migration.Using TLR4 overexpressed and knockout PC cell lines, we showed that treatment with recombinant PAUF increased the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. Likewise, treatment of anti-PAUF antibody reduced the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. These results shows that PAUF’s migration and invasion promoting effects of PC cells is TLR4 dependent.To further understand the molecular mechanism of PAUF-induced metastasis (migration/invasion)-promoting effects via TLR4, we conducted a series of in vitro studies including immunoprecipitation (IP), western blot analysis, flow cytometry, and luciferase reporter assay. We demonstrated that PAUF activates TLR4/MyD88 signaling pathway, but not TLR4/TRIF pathway. Conclusions: In conclusion, for the first time this study demonstrates that TLR4 expressed on PC cell surfaces functions as a receptor of PAUF to mediate its metastasis-promoting effects, which are exclusively through the MyD88/NF-κB signaling pathway. This study also suggests TLR4 as a potential biomarker for identification of optimal patients, and a new therapeutic target to treat PC. Anti-PAUF antibody used in this study is currently evaluated by clinical trials in France, Spain, and US FDA. Citation Format: Fen Jiang, So Eun Youn, Da Eun Hong, Tae Heung Kang, Hye Yun Won, Yun Yong Park, Sang Seok Koh. PAUF induces migration of human pancreatic cancer cells exclusively via the TLR4/MyD88/NF-κB signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3611.

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Promotion of angiogenesis in vitro by Astragalus polysaccharide via activation of TLR4 signaling pathway.

During the implantation of functional tissue-engineered constructs for treating bone defects, a functional vascular network is critical for the survival of the construct. One strategy to achieve rapid angiogenesis for this application is the co-culture of outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs) within a scaffold prior to implantation. In the present study, we aim to investigate whether Astragalus polysaccharide (APS) promotes angiogenesis or vascularization via the TLR4 signaling pathway in a co-culture of OECs and POBs. The co-cultures were treated with various concentrations of APS for 24 h and, subsequently, another 7 days, followed by CD31 staining and analysis of micro-vessel-formation areas using software. Additionally, APS (0.4mg/ml for 24 h) was added to monocultures of OECs or POBs for evaluating proliferation, apoptosis, angiogenesis, osteogenesis, TLR4 signaling pathway, and inflammatory cytokine release. We found that APS promoted angiogenesis in the co-culture at the optimal concentration of 0.4mg/ml. TLR4 activation by APS up-regulated the expression level of TLR4/MyD88 and enhanced angiogenesis and osteogenesis in monocultures of OECs and POBs. The levels of E-selectin adhesion molecules, three cytokines (IL-6, TNF-α, and IFN-γ), and VEGF and PDGF-BB, which can induce angiogenesis, increased significantly (p < .05) following APS treatment. Therefore, APS appears to promote angiogenesis and ossification in the co-culture system via the TLR4 signaling pathway. PRACTICAL APPLICATIONS: This study demonstrates that APS may promote angiogenesis and osteocyte proliferation in OEC and POB co-culture systems through the MyD88-dependent TLR4 signaling pathway. APS might represent a potential therapeutic strategy in tissue-engineered bone implantation for the treatment of large bone defects; additionally, it has the advantage of safety, as it exhibits low or no side effects. In the future, it is expected to be used in vitro for the construction of tissue-engineered bone and in vivo after implantation in patients with bone defects for promoting rapid vascularization and ossification of tissue-engineered bone and early fusion with the recipient's bone. In addition, as a food additive, Astragalus membranaceus can be used as a tonic material in patients recovering from a fracture for promoting blood-vessel formation at the fracture site and fracture recovery. Combining traditional Chinese medicine with tissue engineering can provide further strategies for promoting the development of regenerative medicine.

S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.

Effect of Berberine on the Inflammatory Response of ANA-1 Cells Induced by Angiotensin Ⅱ Based on TLR4 Signaling Pathway

Effect of Berberine on the Inflammatory Response of ANA-1 Cells Induced by Angiotensin Ⅱ Based on TLR4 Signaling Pathway

Critical Role of TLR4 on the Microglia Activation Induced by Maternal LPS Exposure Leading to ASD-Like Behavior of Offspring.

Objective: To investigate the role of TLR4 on the microglia activation in the pre-frontal cortex, which leads to autism-like behavior of the offspring induced by maternal lipopolysaccharide (LPS) exposure.Methods: Pregnant TLR4−/− (knockout, KO) and WT (wild type, WT) dams were intraperitoneally injected with LPS or PBS, respectively. The levels of TNFα, IL-1β, and IL-6 in the maternal serum and fetal brain were assessed with ELISA following LPS exposure. The gestation period, litter size and weight of the offspring were evaluated. Three-chamber sociability test, open field test and olfactory habituation/dishabituation test were used to assess the offspring's autism-like behavior at 7 weeks of age. Western blotting was performed to examine the levels of TLR4, Phospho-NFκB p65, IKKα, IBA-1, iNOS, Arg-1, C3, CR3A, NMDAR2A, and Syn-1 expression in the pre-frontal cortex. The morphological changes in the microglia, the distribution and expression of TLR4 were observed by immunofluorescence staining. Golgi-Cox staining was conducted to evaluate the dendritic length and spine density of the neurons in 2-week-old offspring.Results: Maternal LPS stimulation increased serum TNFα and IL-6, as well as fetal brain TNFα in the WT mice. The litter size and the weight of the WT offspring were significantly reduced following maternal LPS treatment. LPS-treated WT offspring had lower social and self-exploration behavior, and greater anxiety and repetitive behaviors. The protein expression levels of TLR4 signaling pathways, including TLR4, Phospho-NFκB p65, IKKα, and IBA-1, iNOS expression were increased in the LPS-treated WT offspring, whereas Arg-1 was decreased. Maternal LPS treatment resulted in the significant reduction in the levels of the synaptic pruning-related proteins, C3 and CR3A. Moreover, the neuronal dendritic length and spine density, as well as the expression levels of the synaptic plasticity-related proteins, NMDAR2A and Syn-1 were reduced in the WT offspring; however, gestational LPS exposure had no effect on the TLR4−/− offspring.Conclusion: Activation of TLR4 signaling pathway following maternal LPS exposure induced the abnormal activation of microglia, which in turn was involved in excessive synaptic pruning to decrease synaptic plasticity in the offspring. This may be one of the reasons for the autism-like behavior in the offspring mice.

A novel ML domain-containing protein (SpMD2) functions as a potential LPS receptor involved in anti-Vibrio immune response

The myeloid differentiation protein 2 (MD2)-related lipid-recognition (ML) proteins display diverse biological functions in host immunity and lipid metabolism by interacting with different lipids. Human MD2, an indispensable accessory protein in TLR4 signaling pathway, specifically recognizes lipopolysaccharides (LPS), thereby leading to the activation of TLR4 signaling pathway to produce many effectors that participate in inflammatory and immuneresponses against Gram-negative bacteria. Toll and immune deficiency (IMD) pathways are first characterized in Drosophila and are reportedly present in crustaceans, but the recognition and activation mechanism of these signaling pathways in crustaceans remains unclear. In the present study, a novel ML protein was characterized in mud crab (Scylla paramamosain) and designated as SpMD2. The complete SpMD2 cDNA sequence is 1114 bp long with a 465 bp open reading frame; it encodes a protein that contains 154 amino acids (aa). In the deduced protein, a signal peptide (1–21 aa residues) and a ML domain (43–151 aa residues) were predicted. SpMD2 shared a similar three-dimensional structure and a close evolutionary relationship with human MD2. SpMD2 was highly expressed in gills, hemocytes, intestine, and hepatopancreas and was upregulated in gills and hemocytes after challenges with bacteria, thereby suggesting its involvement in antibacterial defense. Western blot assay showed that SpMD2 possesses strong binding activities to different bacteria and two fungi. ELISA demonstrated that SpMD2 exhibits binding abilities to LPS, lipid A, peptidoglycan (PGN), and lipoteichoic acid (LTA). Its binding ability to LPS and lipid A were stronger than to PGN or LTA, implying that SpMD2 was an important LPS-binding protein in mud crab. Bacterial clearance assay revealed that the pre-incubation of Vibrio parahemolyticus with SpMD2 facilitates bacterial clearance in vivo and that knockdown of SpMD2 dramatically suppresses the bacterial clearance and decreases the expression of several antimicrobial peptides (AMPs). Furthermore, SpMD2 overexpression could enhance the promoter activity of SpALF2. These results revealed that SpMD2 affects bacterial clearance by regulating AMPs. Thus, by binding to LPS and by regulating AMPs, SpMD2 may function as a potential receptor, which is involved in the recognition and activation of a certain immune signaling pathway against Gram-negative bacteria. This study provides new insights into the diverse functions of ML proteins and into the antibacterial mechanisms of crustaceans.

Effects of Actigen, a second-generation mannan rich fraction, in antibiotics-free diets on growth performance, intestinal barrier functions and inflammation in weaned piglets

This study aimed to investigate the effects of Actigen™, a second-generation mannan rich fraction, on growth performance, intestinal morphology and permeability, and intestinal inflammatory response in weaned piglets. A total of 150 healthy weaned piglets were randomly assigned to the Control, Antibiotics and Actigen groups and received 1 of 3 dietary treatments: a basal antibiotics-free diet to which 250 mg/kg antibiotics (100 mg/kg colistin sulfate, 100 mg/kg olaquindox, and 50 mg/kg Kitasamycin) or 800 mg/kg Actigen were added. Body weight and feed intake were recorded. On day 28 of experimentation, five female and five male piglets per treatment were selected to collect blood, small intestinal segments and mucosa samples. Intestinal morphology and goblet cell number were determined. Expression of tight junction proteins and TLR4 signaling were detected. d-lactic acid (DLA) and inflammatory cytokines in serum were also measured. Compared with the Control group, Actigen and antibiotics supplemented diets reduced incidence of diarrhea (P < 0.05), with no effect on growth performance. Intestinal morphology revealed that antibiotics decreased (P < 0.01), while Actigen increased the villus height and the ratio of villus height to crypt depth (P < 0.01). PAS staining demonstrated that the goblet cell number was elevated in jejunum of the Actigen fed piglets compared with the Antibiotics group (P < 0.05). In addition, the use of antibiotics decreased the expression of tight junction proteins (P < 0.05), while the use of Actigen increased it (P < 0.05). Accordingly, the intestinal permeability was elevated by antibiotics use, with the increased serum DLA (P < 0.05). Furthermore, Actigen fed piglets had lower serum proinflammatory cytokine TNF-α (P < 0.05) and higher serum anti-inflammatory cytokine IL-10 (P < 0.05). In contrast, antibiotics use led to an increase of serum IL-1β (P < 0.05). Moreover, the expression of TLR4, Myd88, and IKKβ phosphyorlation were enhanced by antibiotics use (P < 0.05), suggesting the activation of TLR4 signaling pathway. However, Actigen supplemented diet had no effect on the TLR4 signaling pathway. In conclusion, compared with the Control group, Actigen supplemented diet had no effects on ADG, ADFI and FCR, but reduced the incidence of diarrhea and mortality to the similar level as the Antibiotics group. Actigen also improved the intestinal morphology and permeability, reduced intestinal inflammatory response compared with the Antibiotics group. These findings suggested the potential application of Actigen in weaned piglets production.

CD14 dictates differential activation of mesenchymal stromal cells through AKT, NF-κB and P38 signals.

Mesenchymal stromal cells (MSCs) widely exist in many tissues and have multiple differentiation potential and immunomodulatory capacities. Recently, MSCs have become promising tools for the treatment of various degenerative disorders and autoimmune diseases. The properties of MSCs could be modified in different microenvironments. Thus, it is important to explore the factors controlling MSC function. The presence of Toll-like receptors (TLRs) in MSCs was demonstrated according to previous studies. Consistently, we also illustrated the expression of TLRs in both murine and human MSCs, and displayed that the expression patterns of TLRs in MSCs from different sources. Furthermore, we explored the role of TLR and TLR signaling pathway in MSCs. Interestingly, activation of TLR4-induced expression of cytokines and some specific genes in MSCs. However, MSCs retained much lower mRNA level compared with macrophages. We explored the expression of CD14 in MSCs from different sources, which played a vital role in TLR4 signaling pathway, and found that MSCs are almost negative for CD14. Moreover, only partial activation of TLR4 signaling pathway was observed in MSCs, with no activation of AKT, NF-κB and P38. Here, in the study we defined TLR expression, function and activation in MSCs, which is critical for designing MSC-based therapies.

Glycyrrhizic acid and silymarin alleviate the neurotoxic effects of aluminum in rats challenged with fructose-induced insulin resistance: possible role of toll-like receptor 4 pathway

Aluminum is implicated in the etiology of different neurodegenerative diseases, diabetes and cancer. The current study was conducted to evaluate the protective effects of glycyrrhizic acid (GAM) and silymarin (SLY) on AlCl3-induced neurotoxicity in insulin resistant rats. Insulin resistance (IR) was induced by fructose (10%) in drinking water for 18 weeks. Rats received AlCl3 (34 mg/kg/day) with or without fructose, GAM (40 mg/kg/day), or SLY (100 mg/kg/day). The administration of GAM or SLY suppressed AlCl3-induced memory deficit, oxidative stress, and neuroinflammation in brain tissue of IR rats. Both agents inhibited AlCl3-induced activation of TLR4 signaling pathway including the downstream activation of NF-κB. The results show that IR can partly exacerbate AlCl3-induced neurotoxicity, particularly memory deficit and neuroinflammation. In addition, GAM and SLY showed promising neuroprotective effect against AlCl3-induced brain damage in IR rats. The neuroprotection induced by these natural products might be mediated through their antioxidant and anti-inflammatory effects. The latter effect seems to be mediated via inhibition of TLR4 signaling pathway providing new insights on the mechanisms implicated in AlCl3-induced neurotoxicity and the neuroprotection afforded by GAM and SLY.

Progress in studies on TLR4 signaling pathway and major depressive disorder

TLR4 signaling pathway plays an important role in regulation of the innate immune response and the adaptive immune response. Studies have shown that TLR4 signaling pathway is closely associated with the immune inflammatory process in major depressive disorder, but the underlying mechanisms are not clear. The pathophysiological process of depression involves multiple molecule mechanisms and signal pathways. The change of TLR4 signaling pathways exists in the peripheral circulation system or the central nervous system of depressive patients and depression animal models. The activation of TLR4 signaling pathways is associated with "stress-inflammation-depression" approach and "leaky gut" hypothesis, however, the relationship of peripheral and central TLR4 signaling pathways is not clear yet. TLR4 signaling pathway may be potential targets for the anti-inflammatory treatment of depression.

A cell‐based approach to quantifying endotoxin in human plasma samples

Poor dietary choices are thought to contribute to the risk of developing a variety of chronic diseases, in part, through promoting a state of low‐grade inflammation. For example, there are multiple research reports indicating that high‐fat meals promote a transient post‐prandial inflammatory state. Several investigators have hypothesized that high fat meals promote the absorption of microbial endotoxin across the gut wall and that once in the circulation this promotes inflammation through the activation of TLR4 signaling pathway on circulating innate immune cells (e.g., monocytes). Direct assessment of endotoxin in human blood samples is problematic. Reliance on the limulus amebocyte lysate (LAL) test for quantifying endotoxin in a protein‐rich matrix, such as human plasma, requires that samples be heated prior to the analyses. Recovery of endotoxin spiked into plasma samples prior to heating is remarkably low, typically less than 1%. This phenomena, commonly referred to as neutralization, raises grave doubts about the suitability of the LAL test for quantifying endotoxin in the human circulation. Many researchers have long recognized the need for a more robust and accurate method for quantifying low levels of endotoxin in human circulation. In 1995, a cell‐based bioassay approach referred to as monocyte activation test (MAT) was developed and marketed as an alternative to LAL test. Unfortunately, the MAT requires that blood samples are processed within a few hours of collection, which greatly limits the utility of this alternative approach. We have developed and tested a cell‐based bioassay (CBA) for quantifying endotoxin in human plasma following storage at −80°C by using THP‐1 cells (i.e., a human monocytic cell line) stably‐transfected with a luciferase reporter driven by multiple NF‐kB promoters. Our CBA provides reliable and reproducible quantitation of endotoxin in unheated human plasma samples with a limit of detection approaching 0.05 endotoxin units (EU)/mL of plasma. Sensitivity and precision of our CBA compares well with the performance of a recently released commercial cell line (InvivoGen). In conclusion, both of these CBAs appear to provide the research community with a reliable alternative to the LAL test for quantifying endotoxin in human plasma.Support or Funding InformationThis research was supported in part by Grant Number P50AT006273 from the National Center for Complementary and Integrative Health (NCCIH), the Office of Dietary Supplements(ODS), and the National Cancer Institute (NCI). Additional funding was provided by the Food‐for‐the‐21st Century Program ‐Nutrition Cluster, which is part of the College of Agriculture, Food and Natural Resources at the University of Missouri ‐ Columbia. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NCCIH, ODS, NCI, or the National Institutes of Health.

Extracellular polysaccharide from Bordetella species reduces high glucose-induced macrophage apoptosis via regulating interaction between caveolin-1 and TLR4

Microphage apoptosis is a critical event in atherosclerotic lesions in patients with diabetes. In the present investigation, high glucose treatment inhibited Akt phosphorylation and activated caspase 3 in primary peritoneal macrophage, leading to cell apoptosis. Hypoxia prolonged macrophage survival in high glucose condition. Extracellular polysaccharide from Bordetella species (EPS) further decreased cell apoptosis in response to high glucose during hypoxia. Under high glucose and hypoxic condition, EPS treatment promoted caveolin-1 phosphorylation by recognizing TLR4. Caveolin-1 phosphorylation elevated membrane Glut1 level to accelerate glucose consumption, which should be the reason for protective effect of EPS on macrophage exposed to high glucose. Further investigation demonstrated that TLR4-dependent caveolin-1 phosphorylation induced by EPS promoted association of caveolin-1 with TLR4, which should be critical for activation of TLR4 signaling pathway.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. None.

TLR4 signaling pathway in mouse Lewis lung cancer cells promotes the expression of TGF-β1 and IL-10 and tumor cells migration

The signaling pathways that trigger tumor cell escape from immune surveillance are not understood completely. Toll-like receptors (TLRs) are considered to be expressed in both immune cells and tumor cells. By detecting TLRs expression in mouse Lewis lung cancer (LLC) before and after co-culture with mouse lymphocytes, the authors concluded that LLC cells constitutively expressed TLR1, TLR2, TLR3, TLR4, TLR5, TLR6 and TLR9. Meanwhile, TLR4 expression in LLC cells was the strongest after co-culture with mouse lymphocytes. To investigate the possible roles of TLR4 signaling pathway in LLC, the concentrations of TGF-β1 and IL-10 protein in LLC cells supernatant were detected by ELISA, and the migration of LLC cells were detected by transwell assay after lipopolysaccharide (LPS) stimulation. TLR4 protein expression in LLC cells was also detected after LPS stimulation by FCM. The results indicated that both levels of TGF-β1 and IL-10 protein were significantly increased after LPS stimulation and reached to a maximum at 24 h and 10 μg/mL of LPS. The migrated LLC cells with LPS stimulation were significantly increased and reached to a maximum at 10 μg/mL of LPS. The expression of TLR4 protein was significantly enhanced after 10 μg/mL of LPS stimulation. These results suggest that the activation of TLR4 signaling pathway in lung cancer cells may be involved in tumor escape and progression by promoting the expression of TGF-β1 and IL-10 and tumor cells migration.

Lipopolysaccharide Recognition via Toll-Like Receptor Signaling Complexes: A Large-Scale Simulation Study

Toll-like receptors (TLRs) are type I transmembrane glycoproteins that have emerged as key pathogen sensors in the mammalian innate immune response. TLR4 is of particular biomedical interest, because it mediates the response to bacterial lipopolysaccharide (LPS), a potent early indicator of microbial infection and the primary inducer of fatal septic shock syndrome. Unfortunately, subtle alterations in the structure of LPS derivatives can profoundly alter the resultant immunological response, hampering the rational design of TLR4 immunomodulators. To unravel the associated structure-activity relationships, we have performed long-timescale, all-atom molecular dynamics simulations of the signaling-active receptor complex. The system, which consists of two TLR4 molecules bound to two MD-2 lipid recognition domains, constitutes approximately half a million atoms in solvent. The conformational dynamics of the hetero-oligomer, and essential conserved loops, are highly responsive over timescales of hundreds of nanoseconds in a manner dependent upon the presence of a variety of bound “activating” or inactivating ligands, including e.g., hexa-acylated lipid A, the main inducer of the immunological response to LPS; its tetra-acylated precursor lipid IVa, which acts as an agonist in mice but as an antagonist in humans; and Eritoran, a strong synthetic antagonist currently undergoing clinical trials. This helps to rationalize how specificity is achieved in the activation of TLR4 signaling pathways. In addition, a novel, spontaneously collapsed conformation of the MD-2 fold has been identified in the lipid-free complex, consistent with previously hypothesized clamshell-like gating motions in homologous mite allergen proteins. Supported by further simulations of the isolated MD-2 domain, this provides a possible mechanism for ligand gating and the prevention of TLR4 pathway stimulation in the absence of pathogenic signals.

Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).

AbstractAbstract 1482Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Mediates Lipopolysaccharide-Induced Inflammation Via p38 MAPK and NF-Kappab.

Abstract 3598Poster Board III-535Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/−3.9% of TNF-alpha, 68%+/−6.9% of IL-1beta, 50%+/−4.3% of IL-6 and 40%+/−3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.8∼2.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease. Disclosures:No relevant conflicts of interest to declare.

TLR4 signaling in cancer cells promotes chemoattraction of immature dendritic cells via autocrine CCL20

Toll-like receptors (TLRs) are involved in the production of inflammatory mediators upon specific ligands stimuli. Chemokines are important inflammatory mediators capable of chemoattracting diverse immune cells. In addition to normal immune cells, the expression of TLRs and chemokines has been detected in various tumor cells. However, the roles of TLRs and chemokines expressed by tumor cells in the processes of tumor progression and immune escape have not been fully elucidated. Here we report that TLR4 ligation by lipopolysaccharide (LPS) significantly promotes CT-26 colon cancer cells to produce chemokine CCL20 via activation of TLR4 signaling pathways. We find that LPS treatment of CT-26 cells can significantly increase the chemoattraction of immature dendritic cells (DC) by the autocrine CCL20. Our studies suggest that TLR4 expressed by tumor cells may be involved in the induction of chemokines like CCL20, providing a potential linkage between chronic inflammation and tumor immune escape.