LPS-TLR4 Signaling Research Articles

Mechanism of Salvia miltiorrhiza Bunge extract to alleviate Chronic Sleep Deprivation-Induced cognitive dysfunction in rats

BackgroundBidirectional communication between the gut microbiota and the brain may play an essential role in the cognitive dysfunction associated with chronic sleep deprivation(CSD). Salvia miltiorrhiza Bunge (Danshen, DS), a famous Chinese medicine and functional tea, is extensively used to protect learning and memory capacities, although the mechanism of action remains unknown. PurposeThe purpose of this research was to explore the efficacy and the underlying mechanism of DS in cognitive dysfunction caused by CSD. MethodsDS chemical composition was analyzed by UPLC-QTOF-MS/MS. Forty rats were randomly assigned to five groups (n = 8): control (CON), model (MOD), low- (1.35 g/kg, DSL), high-dose (2.70 g/kg, DSH) DS group, and Melatonin(100 mg/kg, MT) group. A CSD rat model was established over 21 days. DS's effects and the underlying mechanism were explored using the open-field test(OFT), Morris water-maze(MWM), tissue staining(Hematoxylin and Eosin Staining, Nissl staining, Alcian blue-periodic acid SCHIFF staining, and Immunofluorescence), enzyme-linked immunosorbent assay, Western blot, quantitative real-time polymerase chain reaction(qPCR), and 16S rRNA sequencing. ResultsWe demonstrated that CSD caused gut dysbiosis and cognitive dysfunction. Furthermore, 16S rRNA sequencing demonstrated that Firmicutes and Proteobacteria were more in fecal samples from model group rats, whereas Bacteroidota and Spirochaetota were less. DS therapy, on the contrary hand, greatly restored the gut microbial community, consequently alleviating cognitive impairment in rats. Further research revealed that DS administration reduced systemic inflammation via lowering intestinal inflammation and barrier disruption. Following that, DS therapy reduced Blood Brain Barrier(BBB) and neuronal damage, further decreasing neuroinflammation in the hippocampus(HP). Mechanistic studies revealed that DS therapy lowered lipopolysaccharide (LPS) levels in the HP, serum, and colon, consequently blocking the TLR4/MyD88/NF-κB signaling pathway and its downstream pro-inflammatory products(IL-1β, IL-6, TNF-α, iNOS, and COX2) in the HP and colon. ConclusionDS treatment dramatically improved spatial learning and memory impairments in rats with CSD by regulating the composition of the intestinal flora, preserving gut and brain barrier function, and reducing inflammation mediated by the LPS-TLR4 signaling pathway. Our findings provide novel insight into the mechanisms by which DS treats cognitive dysfunction caused by CSD.

Microbial lipopolysaccharide-induced inflammation contributes to cognitive impairment and white matter lesion progression in diet-induced obese mice with chronic cerebral hypoperfusion.

White matter lesions (WMLs) are involved in the pathological processes leading to cognitive decline and dementia. We examined the mechanisms underlying the exacerbation of ischemia-induced cognitive impairment and WMLs by diet-induced obesity, including lipopolysaccharide (LPS)-triggered neuroinflammation via toll-like receptor (TLR) 4. Wild-type (WT) and TLR4-knockout (KO) C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD), and subjected to bilateral carotid artery stenosis (BCAS). Diet groups were compared for changes in gut microbiota, intestinal permeability, systemic inflammation, neuroinflammation, WML severity, and cognitive dysfunction. In WT mice, HFD induced obesity and increased cognitive impairment and WML severity compared with LFD-fed mice following BCAS. HFD caused gut dysbiosis and increased intestinal permeability, and plasma LPS and pro-inflammatory cytokine concentrations. Furthermore, HFD-fed mice had higher LPS levels and higher neuroinflammatory status, including increased TLR4 expression, in WMLs. In TLR4-KO mice, HFD also caused obesity and gut dysbiosis but did not increase cognitive impairment or WML severity after BCAS. No difference was found between HFD- and LFD-fed KO mice for LPS levels or inflammatory status in either plasma or WMLs. Inflammation triggered by LPS-TLR4 signaling may mediate obesity-associated exacerbation of cognitive impairment and WMLs from brain ischemia.

Peroxisome proliferator-activated receptors-alpha and gamma synergism modulate the gut-adipose tissue axis and mitigate obesity

AimTo evaluate the effects of single PPARα or PPARγ activation, and their synergism (combined PPARα/γ activation) upon the gut-adipose tissue axis, focusing on the endotoxemia and upstream interscapular brown adipose tissue (iBAT) function in high-saturated fat-fed mice. Methods: Male C57BL/6 mice received a control diet (C, 10% lipids) or a high-fat diet (HF, 50% lipids) for 12 weeks. Then, the HF group was divided to receive the treatments for four weeks: HFγ (pioglitazone, 10 mg/kg), HFα (WY-14643, 3.5 mg/kg), and HFα/γ (tesaglitazar, 4 mg/kg). Results: The HF group exhibited overweight, oral glucose intolerance, gut dysbiosis, altered gut permeability, and endotoxemia, culminating in iBAT whitening. The downregulation of LPS-Tlr4 signaling underpinned reduced inflammation and improved lipid metabolism in iBAT in the HFα/γ group, the unique to show normalized body mass and increased energy expenditure. Conclusion: PPARα/γ synergism treated obesity by ameliorating the gut-adipose tissue axis, where restored gut microbiota and permeability controlled endotoxemia and rescued iBAT whitening through favored thermogenesis.

Dietary d-Lactate Intake Facilitates Inflammatory Resolution by Modulating M1 Macrophage Polarization.

Given the d-lactate dehydrogenase (D-LDH) deficiency, L- but not d-lactate is assumed to be the physiological isomer in mammals. Paradoxically, many fermented foods (e.g., yogurt, sauerkraut, cheeses) often contain substantial amounts of d-lactate. In the present study, dietary d-lactate may be a previously unrecognized nutrient aiding in inflammatory resolution is hypothesized. The anti-inflammatory properties of d-lactate are evaluated in experimental colitis and endotoxemia. Oral administration of d-lactate favorably affects acute inflammation in two different mouse models. Analysis of lactate-the lactate receptor (the hydroxycarboxylic acid receptor 1 HCA1, formerly GPR81) signal axis in inflammation is performed in primary peritoneal macrophages and wild-type (WT) or GPR81 knockout (KO) mice. GPR81 KO mice are susceptible to endotoxic shock than WT mice, while d-lactate exerts its anti-inflammatory activities in a GPR81-dependent manner. Mechanistically, the activation of lactate-GPR81 axis may suppress LPS-TLR4 signaling to modulate M1 macrophage polarization. Although D-LDH deficiency in mammals impairs d-lactate clearance, it might prolong its plasma terminal half-life, and thus provide a pharmacokinetic advantage of d-lactate over l-lactate. This study highlights housekeeping function of the lactate-GPR81 axis in inflammation control, and suggests that dietary intake of d-lactate may underlie Metchnikoff's probiotic yogurt theory of life prolongation.

Association of Lipopolysaccharide-Toll-Like Receptor 4 Signaling and Microalbuminuria in Patients with Type 2 Diabetes Mellitus.

PurposeIntestinal flora imbalance has been implicated in the activation of innate immunity in the kidneys. However, little is known about the potential links between lipopolysaccharide (LPS)-toll-like. receptor 4 (TLR4) signaling activated by intestinal barrier dysfunction and microalbuminuria in type 2 diabetes mellitus (T2DM).Patients and Methods61 patients with T2DM were stratified based on the absence (n=32) or presence (n=29) of microalbuminuria. There were also 28 control subjects. Urinary albumin excretion rate (UAER), serum levels of LPS, D-lactic acid (DLA), diamine oxidase (DAO), fasting blood glucose (FBG), interleukin-6 (IL-6), glycosylated hemoglobin A1 (HbA1c), and high-sensitivity C-reactive protein (hs-CRP), and TLR4 expression in peripheral blood mononuclear cells (PBMCs) were measured.Resultshs-CRP, IL-6, LPS, DLA, DAO, and TLR4 were markedly increased in subjects with T2DM compared to the controls (P < 0.05 for all). Moreover, LPS was positively correlated with FBG, HbA1c, hs-CRP, IL-6, UAER, DLA, DAO, and TLR4 (P < 0.05 for all). In addition, TLR4 was positively correlated with UAER, hs-CRP, FBG, DLA, HbA1c, and LPS (P < 0.05 for all). In regression analyses, TLR4, LPS, HbA1c, and hs-CRP were independently associated with UAER (P < 0.05 for all), while FBG, LPS, TLR4, and hs-CRP (P < 0.05 for all) were found to be risk factors for microalbuminuria in T2DM.ConclusionIntestinal integrity is compromised in subjects with T2DM, and the activation of LPS-TLR4 signaling might play an important role in the development of microalbuminuria in T2DM.

Schisantherin A alleviates non-alcoholic fatty liver disease by restoring intestinal barrier function

BackgroundNon-alcoholic fatty liver disease (NAFLD) is intricately linked to dysregulation of the gut–liver axis, and correlated with intestinal inflammation and barrier disruption.ObjectivesTo investigate the protective effects and possible molecular mechanism of Schisantherin A (Sin A) in a high-fat diet (HFD) induced NAFLD mouse model.MethodsHFD-fed NAFLD mice were treated with the vehicle and 80 mg/kg Sin A every day for 6 weeks. The gut permeability of the NAFLD mice was assessed by intestinal permeability assays in vivo and transepithelial electrical resistance (TEER) measurements in vitro were also used to evaluate the function of the gut barrier. TLR4 inhibitor was then used to investigate the impact of Sin A in the LPS- TLR4 signaling pathway. Alternatively, the composition of the microbiome was assessed using 16S rRNA amplification. Finally, the experiment of antibiotic treatment was performed to elucidate the roles of the gut microbiome mediating Sin A induced metabolic benefits in the NAFLD mice.ResultsWe found that Sin A potently ameliorated HFD-induced hepatic steatosis and inflammation, alleviated gut inflammation, and restored intestinal barrier function. We also observed that Sin A improved gut permeability and reduced the release of lipopolysaccharide (LPS) into circulation and further found that Sin A can suppress LPS-TLR4 signaling to protect against HFD-induced NAFLD. Sin A treatment altered the composition of the microbiome in NAFLD mice compared to vehicle controls.ConclusionsSin A is an effective and safe hepatoprotective agent against HFD-induced NAFLD by partly ameliorating gut inflammation, restoring intestinal barrier function, and regulating intestinal microbiota composition.

A complex proinflammatory cascade mediates the activation of HSCs upon LPS exposure in vivo

AbstractInfections are a key source of stress to the hematopoietic system. While infections consume short-lived innate immune cells, their recovery depends on quiescent hematopoietic stem cells (HSCs) with long-term self-renewal capacity. Both chronic inflammatory stress and bacterial infections compromise competitive HSC capacity and cause bone marrow (BM) failure. However, our understanding of how HSCs act during acute and contained infections remains incomplete. Here, we used advanced chimeric and genetic mouse models in combination with pharmacological interventions to dissect the complex nature of the acute systemic response of HSCs to lipopolysaccharide (LPS), a well-established model for inducing inflammatory stress. Acute LPS challenge transiently induced proliferation of quiescent HSCs in vivo. This response was not only mediated via direct LPS-TLR4 conjugation on HSCs but also involved indirect TLR4 signaling in CD115+ monocytic cells, inducing a complex proinflammatory cytokine cascade in BM. Downstream of LPS-TLR4 signaling, the combined action of proinflammatory cytokines such as interferon (IFN)α, IFNγ, tumor necrosis factor-α, interleukin (IL)-1α, IL-1β, and many others is required to mediate full HSC activation in vivo. Together, our study reveals detailed mechanistic insights into the interplay of proinflammatory cytokine-induced molecular pathways and cell types that jointly orchestrate the complex process of emergency hematopoiesis and HSC activation upon LPS exposure in vivo.

A High-Fat High-Fructose Diet Dysregulates the Homeostatic Crosstalk Between Gut Microbiome, Metabolome, and Immunity in an Experimental Model of Obesity.

Ample evidence supports the prominent role of gut-liver axis in perpetuating pathological networks of high-fat high-fructose (HFF) diet induced metabolic disorders, however, the molecular mechanisms are still not fully understood. Herein, this study aims to present a holistic delineation and scientific explanation for the crosstalk between the gut and liver, including the potential mediators involved in orchestrating the metabolic and immune systems. An experimental obesity-associated metaflammation rat model is induced with a HFF diet. An integrative multi-omics analysis is then performed. Following the clues illustrated by the multi-omics discoveries, putative pathways are subsequently validated by RT-qPCR and Western blotting. HFF diet leads to obese phenotypes in rats, as well as histopathological changes. Integrated omics analysis shows that there exists a strong interdependence among gut microbiota composition, intestinal metabolites, and innate immunity regulation in the liver. Some carboxylic acids may contribute to gut-liver communication. Moreover, activation of the hepatic LPS-TLR4 pathway in obesity is confirmed. HFF-intake disturbs gut flora homeostasis. Crosstalk between gut microbiota and innate immune system mediates hepatic metaflammation in obese rats, associated with LPS-TLR4 signaling pathway activation. Moreover, α-hydroxyisobutyric acid and some other organic acids may play a role as messengers in the liver-gut axis.

Hemorrhagic Transformation in Patients with Large-Artery Atherosclerotic Stroke is Associated with Gut Microbiota and LPS-TLR4 Signaling Pathway

Hemorrhagic Transformation in Patients with Large-Artery Atherosclerotic Stroke is Associated with Gut Microbiota and LPS-TLR4 Signaling Pathway

Differential regulation of lipopolysaccharide-induced IL-1β and TNF-α production in macrophages by palmitate via modulating TLR4 downstream signaling

Diabetic patients are susceptible to infectious diseases. Bacterial invasion activates immune cells such as macrophages through interaction between LPS and TLR4, and induces the expression of inflammatory mediators, including IL-1β and TNF-α, which play key roles in the elimination of infections. Unregulated overproduction or underproduction of these cytokines has been reported as a major factor in the development of septic shock, immune deficiency, and autoimmunity. Recent studies found that metabolic abnormalities of diabetes, such as hyperglycemia and dyslipidemia, played a major role in modulating the immune response. In this study, we studied the effects of palmitic acid (PA) pretreatment on LPS-induced IL-1β and TNF-α production and LPS-TLR4 signaling in macrophages. Compared with control, PA pretreatment significantly increased LPS-induced TNF-α production and secretion in macrophages. In contrast, LPS-induced IL-1β production and secretion was significantly suppressed by PA pretreatment. PA pretreatment did not affect the expression levels of TLR4 or Myd88, or the endocytosis of TLR4 in macrophages. However, PA pretreatment significantly suppressed the phosphorylation level and nuclear translocation of NF-κB, and the phosphorylation level of ERK1/2, whereas increased the phosphorylation levels of p38 and JNK. The activation of IKK which was upstream of NF-κB and ERK1/2 was attenuated, while the activation of TAK1 which was upstream of JNK and p38 was augmented by PA pretreatment. Inhibitors of NF-κB, MEK1/2, and p38 significantly decreased IL-1β expression, while JNK and p38 pathway inhibitors significantly inhibited TNF-α expression. The differential regulation of LPS-induced TNF-α and IL-1β production by PA was associated with cellular metabolism of PA, because inhibiting metabolism of PA with etomoxir or pretreatment with Br-PA which cannot be metabolized reversed these effects. We also showed that PA treatment increased acetylated IKK level which might contribute to the suppressed activation of IKK. The present study showed that LPS-induced production of TNF-α and IL-1β was regulated by different TLR4 downstream pathways in macrophages. PA differentially affected LPS-induced production of TNF-α and IL-1β in macrophages through differentially modulating these pathways. Further experiments will be needed to determine how these phenomena lead to the impaired immune response in patients with diabetes.

Fecal microbiota transplantation protects rotenone-induced Parkinson\u2019s disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis

BackgroundParkinson’s disease (PD) is a prevalent neurodegenerative disorder, displaying not only well-known motor deficits but also gastrointestinal dysfunctions. Consistently, it has been increasingly evident that gut microbiota affects the communication between the gut and the brain in PD pathogenesis, known as the microbiota-gut-brain axis. As an approach to re-establishing a normal microbiota community, fecal microbiota transplantation (FMT) has exerted beneficial effects on PD in recent studies. Here, in this study, we established a chronic rotenone-induced PD mouse model to evaluate the protective effects of FMT treatment on PD and to explore the underlying mechanisms, which also proves the involvement of gut microbiota dysbiosis in PD pathogenesis via the microbiota-gut-brain axis.ResultsWe demonstrated that gut microbiota dysbiosis induced by rotenone administration caused gastrointestinal function impairment and poor behavioral performances in the PD mice. Moreover, 16S RNA sequencing identified the increase of bacterial genera Akkermansia and Desulfovibrio in fecal samples of rotenone-induced mice. By contrast, FMT treatment remarkably restored the gut microbial community, thus ameliorating the gastrointestinal dysfunctions and the motor deficits of the PD mice. Further experiments revealed that FMT administration alleviated intestinal inflammation and barrier destruction, thus reducing the levels of systemic inflammation. Subsequently, FMT treatment attenuated blood-brain barrier (BBB) impairment and suppressed neuroinflammation in the substantia nigra (SN), which further decreased the damage of dopaminergic neurons. Additional mechanistic investigation discovered that FMT treatment reduced lipopolysaccharide (LPS) levels in the colon, the serum, and the SN, thereafter suppressing the TLR4/MyD88/NF-κB signaling pathway and its downstream pro-inflammatory products both in the SN and the colon.ConclusionsOur current study demonstrates that FMT treatment can correct the gut microbiota dysbiosis and ameliorate the rotenone-induced PD mouse model, in which suppression of the inflammation mediated by the LPS-TLR4 signaling pathway both in the gut and the brain possibly plays a significant role. Further, we prove that rotenone-induced microbiota dysbiosis is involved in the genesis of PD via the microbiota-gut-brain axis.6NusmmVQuAXh_sD82L334KVideo abstract

Ginsenoside Rk3 Suppresses Hepatocellular Carcinoma Development through Targeting the Gut-Liver Axis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Our previous reports showed that ginsenoside Rk3 provided excellent efficacy in alleviating the intestinal inflammatory response and protecting the liver, but its mechanism for HCC prevention remains to be explored. Here, the results suggested that Rk3 displayed potent antitumor effects against a dimethyl nitrosamine- and CCl4-induced HCC mouse model. Results revealed that Rk3 application inhibited liver injury, fibrosis, and cirrhosis. In parallel, Rk3 lowered the inflammatory response by decreasing the expression of inflammatory cytokines, inducing apoptosis, and blocking the cell cycle. Meanwhile, Rk3 effectively ameliorated the gut microbiota dysbiosis. Furthermore, correlation analysis revealed that the LPS-TLR4 signaling pathway, which was inhibited by Rk3, plays a key role in preventing HCC. To conclude, our research provides valuable insights into how Rk3 application targets the gut-liver axis and suppresses HCC development, suggesting that Rk3 might be a promising candidate for clinical treatment of HCC.

Centrally administered orexin prevents lipopolysaccharide and colchicine induced lethality via the vagal cholinergic pathway in a sepsis model in rats

Orexins are neuropeptides implicated in several physiological functions. Accumulating findings suggest a relationship between orexin and sepsis. A recent study demonstrated that orexin acts centrally to improve conditions in sepsis. The present study aims to clarify the precise mechanisms by which central orexin could induce a protective action against septic conditions. We established a new septic model by treating rats with lipopolysaccharide (LPS) and colchicine and used this to examine the effect of brain orexin on survival. Observation of survival was stopped three days after the chemicals injection or at death. We established a lethal model (rats died within 24 h) by injecting subcutaneously a combination of 1 mg/kg LPS and 1 mg/kg colchicine. A Toll-like receptor 4 (TLR4) inhibitor completely blocked lethality, suggesting a vital role of LPS-TLR4 signaling in the process. Intracisternal orexin-A dose-dependently reduced lethality in the sepsis model while neither intracisternal orexin-B nor intraperitoneal orexin-A changed the mortality rate. Vagal stimulation with carbachol or 2-deoxy-D-glucose improved survival and atropine potently blocked the protection by carbachol or 2-deoxy-D-glucose. The orexin-A-induced reduction of lethality was significantly blocked by atropine or surgical vagotomy. Intracisternal injection of an OX1 receptor antagonist blocked the improvement of survival by intracisternal injection of orexin-A, carbachol, or 2-deoxy-D-glucose. These results suggest that orexin acts centrally to reduce the lethality in our septic model treated (LPS and colchicine). Activation of the vagal cholinergic pathway may mediate the action of orexin, and the OX1 receptor in the brain might play a role in the process. Since the efferent vagus nerve mediates anti-inflammatory mechanisms, we speculate that the vagal cholinergic anti-inflammatory pathway is implicated in the mechanisms of septic lethality reduction by brain orexin.

TBK1/IKKε Negatively Regulate LPS-Induced Neutrophil Necroptosis and Lung Inflammation.

Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that controls cell release of inflammatory mediators from innate immune cells, such as polymorphonuclear neutrophils (PMNs), and critically regulates the progress of inflammation. Cell necroptosis features receptor-interacting protein (RIPK) 1 activation and necroptosome formation. This leads to loss of plasma membrane integrity, the release of cell contents into the extracellular space, and subsequent increased inflammation. Here, we report an intra-PMN mechanism of negative regulation of necroptosis mediated through TBK1/IKKε. Using an in vivo mouse model of intratracheal injection (i.t.) of LPS and in vitro LPS stimulation of mouse PMN, we found that LPS-TLR4 signaling in PMNs activates and phosphorylates TBK1 and IKKε, which in turn suppress LPS-induced formation of the RIPK1-RIPK3-MLKL (necrosome) complex. TBK1 dysfunction by knockdown or inhibitor significantly increases the phosphorylation of RIPK1 (∼67%), RIPK3 (∼68%), and MLKL (∼50%) and promotes RIPK1-RIPK3 and RIPK3-MLKL interactions and increases PMN necroptosis (∼83%) in response to LPS, with subsequent augmented lung inflammation. These findings suggest that the LPS-TLR4-TBK1 axis serves as a negative regulator for PMN necroptosis and might be a therapeutic target for modulating PMN death and inflammation.

Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p&amp;lt;0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p&amp;lt;0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p&amp;lt;0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p&amp;lt;0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p&amp;lt;0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p&amp;lt;0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p&amp;lt;0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p&amp;lt;0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p&amp;lt;0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Acute Colchicine Poisoning Causes Endotoxemia via the Destruction of Intestinal Barrier Function: The Curative Effect of Endotoxin Prevention in a Murine Model

BackgroundColchicine binds to intracellular tubulin and prevents mitosis. Colchicine is also used as an anti-inflammatory drug. Meanwhile, excess administration of medication or accidental ingestion of colchicine-containing plants can cause acute colchicine poisoning, which initially results in gastrointestinal effects that may be followed by multiorgan dysfunction. However, the mechanism of colchicine poisoning remains unclear, and there are no standard therapeutic strategies.AimsWe focused on intestinal barrier function and attempted to reveal the underlying mechanism of colchicine poisoning using an animal model.MethodsColchicine was orally administered to C57Bl/6 mice. Then, we performed histopathological analysis, serum endotoxin assays, and intestinal permeability testing. Additionally, the LPS-TLR4 signaling inhibitor TAK-242 was intraperitoneally injected after colchicine administration to analyze the therapeutic effect.ResultsWe observed villus height reduction and increased numbers of apoptotic cells in the gastrointestinal epithelium of colchicine-treated mice. Both intestinal permeability and serum endotoxin levels were higher in colchicine-treated mice than in control mice. Although colchicine-poisoned mice died within 25 h, those that also received TAK-242 treatment survived for more than 48 h.ConclusionColchicine disrupted intestinal barrier function and caused endotoxin shock. Therapeutic inhibition of LPS-TLR4 signaling might be beneficial for treating acute colchicine poisoning.

TLR4 increases the stemness and is highly expressed in relapsed human hepatocellular carcinoma.

Toll‐like receptor 4 (TLR4) plays an essential role in cancer progress. Here, we find that the expression of TLR4 in relapsed human hepatocellular carcinoma (HCC) clinical samples is higher than that in the non‐relapsed ones, which leads us to explore the role of TLR4 in cancer stemness. We reported that TLR4‐AKT signaling pathway was activated by lipopolysaccharides (LPS) in HCC cell lines to enhance the cancer stemness capacity, which was reflected by the increased percentage of CD133+ CD49f+ population and side population, enhanced sphere formation, and the upregulation of stemness marker gene‐SOX2. Downregulation of SOX2 attenuated the enhanced HCC stemness induced by LPS, indicating SOX2 as a downstream mediator of LPS‐TLR4 signaling. The role of LPS‐TLR4 signaling in inducing HCC stemness was further confirmed by tumor xenograft experiment in vivo. Taken together, our findings provide a novel therapeutic target to prevent the recurrence of HCC.

LPS-опосредованное воспаление при сердечно-сосудистых заболеваниях

Возникновение и развитие сердечной недостаточности как проявление атеросклеротического процесса или первичного поражения кардиомиоцитов при кардиомиопатиях может быть связано с накоплением в крови эндотоксинов - компонентов бактериальных клеток, в частности липополисахарида (ЛПС) клеточной стенки грамотрицательных бактерий. ЛПС при связывании с TOLL-подобными рецепторами (TLR) запускают сигнальные пути, вследствие чего происходит синтез провоспалительных цитокинов, выработка активных форм кислорода и азота, развитие окислительного стресса и воспаления в месте атеросклеротического поражения. Активация ЛПС-TLR4-сигнальных путей в тромбоцитах способствует развитию тромбоза. При ишемии миокарда и сердечной недостаточности наблюдается дисбаланс между окислительным стрессом и антиоксидантными механизмами. Миокард имеет эндогенные восстановительные механизмы, в том числе системы тиоредоксина (Trx) и глутатиона (GSH), которые направлены на удаление активных форм кислорода и восстановление окисленных белков, часть из которых участвует в ЛПС-TLR4-сигнальных путях. Например, GSH способен модифицировать адапторный белок MyD88, а Trx является прямым ингибитором протеинкиназы ASK1, способствуя подавлению проапоптотических сигнальных путей во время острого воспаления в кардиомиоцитах. Кроме того, Trx предотвращает дисфункцию митохондрий, увеличивает производство АТФ, ингибирует апоптоз и тем самым оказывает кардиопротекторное действие. Emergence and development of heart failure as a manifestation of atherosclerotic process or primary lesion to cardiomyocytes in cardiomyopathies can be caused by blood accumulation of endotoxins, components of bacterial cells, such as lipopolysaccharide (LPS) of gram-negative bacterial cell wall. LPS binding to TOLL-like receptors (TLR) triggers signaling pathways to induce synthesis of pro-inflammatory cytokines, production of reactive oxygen and nitrogen species, and development of oxidative stress and inflammation at the site of atherosclerotic lesion. Activation of LPS-TLR4-signaling pathways in platelets contributes to development of thrombosis. An imbalance between oxidative stress and antioxidant mechanisms is observed in myocardial ischemia and heart failure. The myocardium has endogenous restorative mechanisms, including the systems of thioredoxin (Trx) and glutathione (GSH) designed for removing active oxygen species and reducing oxidized proteins, some of which are involved in LPS-TLR4 signaling pathways. For example, GSH is able to modify the adaptor protein MyD88 while Trx is a direct inhibitor of protein kinase ASK1, to facilitate suppression of proapoptotic signaling pathways during acute inflammation in cardiomyocytes. In addition, Trx prevents mitochondrial dysfunction, increases ATP production, inhibits apoptosis and, thereby, exerts a cardioprotective effect.

Butyrate upregulates the TLR4 expression and the phosphorylation of MAPKs and NK-κB in colon cancer cell in vitro.

Microbiota and its induced inflammation in colorectal mucosa have been considered risk factors for the development of colorectal carcinogenesis. Previous studies demonstrated that the coexisting elements of microbiota in the gut, such as short chain fatty acids (SCFAs) and lipopolysaccharides (LPS), which exhibited regulatory effects on the intestinal epithelial cells individually. Unfortunately, the association between butyrate and the toll-like receptor (TLR) signaling pathway in the development of colon cancer is not fully elucidated. In the present study, by culturing human colon cancer SW480 cells or mouse colon cancer CT26 cells with butyrate and/or TLR4 ligand LPS in vitro, it was identified that butyrate suppressed the growth and promoted apoptosis of these cancer cells. Notably, the expression levels of TLR4 and CD14 were markedly increased on these butyrate-treated cells, but not on LPS-alone treated cells. Additionally, butyrate treatment induced the phosphorylation of extracellular signal-regulated kinase, tumor protein 38, c-Jun NH2-terminal kinase and nuclear factor-κB (NF-κB) p65, and then promoted the pro-inflammatory cytokine tumor necrosis factor-α, but not interleukin 6 secretion in SW480 and CT26 cells. Therefore, butyrate treatment regulates the expression of TLR4, mitogen-activated protein kinase and NF-κB signal pathway activation and pro-inflammatory response in vitro. Although the exact mechanisms have not been fully explored, these results suggested that butyrate and LPS-TLR4 signaling mediated innate immunity in colon cancer cells through two distinct but inter-regulated pathways. Thus, butyrate can further initiate innate immunity against tumor cells by upregulating the TLR4 expression and activation to preserve intestinal homeostasis.

P204 Is Required for Canonical Lipopolysaccharide-induced TLR4 Signaling in Mice

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204−/− mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-β and pro-inflammatory cytokines. The serum levels of IFN-β and pro-inflammatory cytokines were also significantly reduced in p204−/− mice following LPS challenge. In addition, p204−/− mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases.