Lectin-type Oxidized LDL Receptor Research Articles

Antihypertensive effects of CBD are mediated by altered inflammatory response: A sub-study of the HYPER-H21-4 trial

Despite the abundance of preclinical data, cardiovascular effects of cannabidiol (CBD) in humans remain controversial. HYPER-H21-4 trial was designed as a randomized, placebo-controlled, crossover trial aimed to explore the effects of chronic CBD dosing on ambulatory blood pressure (BP) and vascular stiffness in hypertensive individuals. In this pre-specified analysis of the trial we aimed to elucidate the anti-inflammatory effects of CBD by measuring the dynamic of serum cytokines. Specifically, interleukin 1β (IL-1β), IL-8, IL-6, IL-10, IL-18, Plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor alpha (TNF-α) and lectin-type oxidized LDL receptor 1 (LOX-1) were measured. A total of 65 patients with primary hypertension were included. After five weeks of oral CBD administration, but not of placebo, serum concentrations of IL-8, IL-10 and IL-18 were significantly lower in comparison to baseline concentrations (p = 0.044, p < 0.001 and p < 0.001, respectively). Serum levels of other cytokines showed no CBD-associated dynamic. Higher IL-8 and IL-10 baseline levels heralded higher diastolic BP reduction (r = -0.478, p < 0.001 and r = -0.265, p = 0.034, respectively), whereas the extent of reduction in IL-8 and IL-10 serum levels correlated with the extent of reduction in diastolic BP (r = 0.434, p < 0.001 and r = 0.594, p < 0.001, respectively). Overall, the results of the present analysis imply that the antihypertensive effects of CBD in patients with primary hypertension are accompanied by changes in serum concentrations of multiple cytokines.

Metabolite profiling, hypolipidemic, and anti-atherosclerosis activity of mixed vegetable fermentation extract

Although positive association between fermented vegetables intake with the risk of coronary heart disease (CHD) has increased attention nowadays, the metabolite profiling and the mechanism of action are still elusive. This study designed to investigate the secondary metabolites, hypolipidemic, and anti-atherogenic effect of mixed vegetable fermentation extract (MVFE). The metabolite screening of the MVFE was assessed using the Liquid Chromatography Tandem Mass Spectrophotometer (LC-MS/MS) method. The result of LC-MS/MS was used as ligands to inhibit the binding of oxidized LDL (oxLDL) and Cluster Differentiation 36 (CD36), Scavenger Receptor A1 (SRA1), Lectin-type oxidized LDL receptor 1 (LOX1). This work was performed with molecular docking using Discovery Studio 2021, PyRx 0.9, and Autodock Vina 4.2 followed by analyzing Network Pharmacology, Protein Protein Interaction (PPI) using Cytoscape 3.9.1 and String 2.0.0. Finally, the clinical effect of MVFE was evaluated using in vivo study. Twenty rabbits were assigned to normal, negative control, and MVFE group that were fed with standard diet, high fat diet (HFD), HFD supplemented with MVFE 100, 200 mg/kg BW, respectively. The serum level of Total Cholesterol (TC) and Low-Density Lipoprotein (LDL-c) were detected at the end of week 4.The LC-MS/MS analysis identified 17 compounds categorized as peptides, fatty acids, polysaccharides, nucleoside, flavonoids, flavanols, and phenolic compounds. Based on the docking study, more negative binding affinity was observed in the interaction between metabolites with the scavenger receptors (SR) than simvastatin. The number of nodes and edges based on Network Pharmacology analysis were 268 and 482, respectively. The PPI network showed that MVFE metabolites exerts its athero-protective effect by modulating various cellular processes including inflammation, improvement of endothelial function, and modulation of lipid metabolism. Blood TC and LDL-c concentrations in the negative control (458.82 ± 82.03; 191.87 ± 92.16 mg/dL) were higher significantly compared to the normal group (87.03 ± 29.27; 43.33 ± 5.75 mg/dL). The MVFE administration decreased the TC (100, 200 mg/kg BW MVFE: 269.96 ± 85.34; 130.17 ± 45.02 mg/dL) and LDL-c level (100, 200 mg/kg BW MVFE = 87.24 ± 22.85; 41.82 ± 11.08 mg/dL) dose-dependently (p < 0,001).The secondary metabolites derived from fermented mixed vegetables extract might be developed as a potential strategy to prevent CHD by targeting the multiple pathways in atherosclerosis.

LOX-1: Implications in atherosclerosis and myocardial ischemia.

Understanding the pathophysiology of atherosclerosis is fundamental to the practice of cardiovascular medicine. Atherosclerosis is a multi-step cascade of accumulation of lipids and downstream changes that lead to a fibro-fatty plaque formation in the arterial intima. Multiple biochemical stimuli, cellular receptors and intra-cellular signals are implicated in this complex mechanism. Lectin-type oxidized LDL receptor-1 or LOX-1 is a type II membrane glycoprotein receptor which has emerged as an important effector of atherosclerosis. Hence, LOX-1 modification and its clinical consequences are of much interest in recent times.

Lectin-type oxidized LDL receptor 1 modulates matrix metalloproteinase 2 production in peri-implantitis.

Peri-implantitis is a disease in which inflammatory lesions that affect the surrounding soft and hard tissues develop. Matrix metalloproteinase 2 (MMP2) is hypothesized to be involved in this destructive process. However, the regulatory mechanism of action of MMP2 in the peripheral tissues of the implant are not fully understood. To determine the expression of MMP2 in peri-implantitis, peri-implant crevicular fluid (PICF) was collected from patients with peri-implantitis. The healthy volunteers' peripheral blood human macrophages infected with Porphyromonas gingivalis (P. gingivalis) were used as a cell model to explore the regulatory mechanism of MMP2 regarding dental implants. Western blotting, reverse transcription-quantitative PCR and immunofluorescence staining were used to measure the expression of MMP2 in the present study. MMP2 expression was increased in the PICF of the patients with peri-implantitis and in human macrophages infected with P. gingivalis. Lectin-type oxidized LDL receptor 1 (LOX-1) mediated the expression of MMP2 in human macrophages upon infection of P. gingivalis, whereas dendritic cell-associated c-type lectin-1 did not appear to be involved in this regulatory process. However, JNK and ERK1/2 were involved in P. gingivalis induced MMP2 expression. The results of this study showed that MMP2 was involved in peri-implantitis. MMP2 was upregulated by LOX-1 in a JNK and ERKk1/2 signaling dependent manner in the cell model. The LOX-1/MMP2 signaling pathway may thus be a potential target for management of peri-implantitis.

Polymorphonuclear myeloid-derived suppressor cells link inflammation and damage response after trauma.

Elimination of the posttraumatic inflammatory response and recovery of homeostasis are crucial for the positive prognosis of trauma patients. Myeloid-derived suppressor cells (MDSCs) are known to play a regulatory role in the posttraumatic immune response in mice, but their induction source and involved potential mechanism are poorly understood. Here, we report that polymorphonuclear MDSCs (PMN-MDSCs) are activated after trauma and are closely associated with the progression of the posttraumatic inflammatory response. In humans, lectin-type oxidized LDL receptor 1 (LOX1) was used to specifically characterize LOX1+ PMN-MDSCs. Trauma patients showed high intracellular reactive oxygen species (ROS) production, as well as activation of LOX1+ PMN-MDSCs. These MDSCs contribute to the anti-inflammatory immune response by regulating the Treg/Th17 and Th2/Th1 balances after trauma, increasing the levels of anti-inflammatory factors, and decreasing the levels of proinflammatory factors. The number of LOX1+ PMN-MDSCs was positively correlated with the positive clinical prognosis of trauma patients with infection. Activation of LOX1+ PMN-MDSCs is mediated by NF-κB signal, and TGF-β1 may be as an important inducer for LOX1+ PMN-MDSCs in the posttraumatic cytokine environment. In a pseudofracture trauma mouse model, we also observed the activation of PMN-MDSCs, accompanying high levels of intracellular ROS production, NF-κB phosphorylation, and changes in the inflammatory environment, in particularly by regulating the Treg/Th17 and Th2/Th1 balance. And more significantly, posttraumatic inflammation was alleviated in mice after transferring trauma-derived PMN-MDSCs, but aggravated after injecting with Gr1 agonistic antibody. These findings provide evidence for the specific role of PMN-MDSCs in the regulation of posttraumatic inflammation.

Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients

AbstractReplacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

Pelargonic acid vanillylamide and rosuvastatin protect against oxidized low-density lipoprotein-induced endothelial dysfunction by inhibiting the NF-κB/NLRP3 pathway and improving cell-cell junctions

Oxidized low-density lipoprotein (ox-LDL) not only causes hyperlipidemia and contributes to atherosclerosis but also induces the endothelial dysfunction that leads to cardiovascular diseases. The nuclear factor-kappa B (NF-κB) pathway plays a key role in many chronic disorders and is a transcriptional factor in various inflammatory responses. The present study aimed to investigate the synergistic effects of pelargonic acid vanillylamide (PAVA) and rosuvastatin (RSV) on ox-LDL-induced inflammatory responses in human vascular endothelial cells (HUV-EC-C). HUV-EC-C were pretreated with PAVA or RSV and their combination for 2 h followed by ox-LDL for 24 h. The MTT assay was used to measure mitochondrial function. The DCFH-DA assay was used to evaluate oxidative phosphorylation, and western blotting was used to measured NF-κB/NLRP3 and related signaling pathways in HUV-EC-C. Ox-LDL induced lectin-type oxidized LDL receptor 1 (LOX-1) expression, NADPH oxidase 4 activation, and the overexpression of reactive oxygen species, which were inhibited by pretreatment with the combination of PAVA and RSV. Moreover, PAVA and RSV inhibited ox-LDL-induced NF-κBp65 activation. Ox-LDL induced NF-κB/NLRP3 pathway activation by inducing C-reactive protein expression, NLRP3 activation, caspase-1 activation, and IL-1β secretion, which were inhibited by pretreatment with the combination of PAVA and RSV. The combination of PAVA and RSV reduced ox-LDL-induced recruitment of monocytes to the site of inflammation, inhibited activation of the NLRP3 inflammasome, and ameliorated the impairment of cell-cell junctions through the NF-κB pathway. Our results suggest that the synergistic effects of PAVA and RSV provide a novel mechanism for the treatment of cardiovascular diseases.

Synergistic effects of electronegative-LDL- and palmitic-acid-triggered IL-1β production in macrophages via LOX-1- and voltage-gated-potassium-channel-dependent pathways

Electronegative LDL (LDL(-)) and free fatty acids (FFAs) are circulating risk factors for cardiovascular diseases (CVDs) and have been associated with inflammation. Interleukin-1 beta (IL-1β) represents a key cytokine in the development of CVD; however, the initial trigger of IL-1β in CVD remains to be explored. In this study, we investigated the combined effects of LDL(-) from the plasma of ST-segment elevation myocardial infarction (STEMI) patients or diet-induced hypercholesterolemic rabbits and bovine serum albumin bound palmitic acid (PA-BSA) on IL-1β production in macrophages. Macrophages derived from THP-1 cells or human peripheral blood mononuclear cells were independently treated with LDL(-), PA-BSA or cotreated with LDL(-) and PA-BSA. The results showed that nLDL and/or PA-BSA had no effect on IL-1β, and LDL(-) slightly increased IL-1β; however, cotreatment with LDL(-) and PA-BSA resulted in abundant secretion of IL-1β in macrophages. Rabbit LDL(-) induced the elevation of cellular pro-IL-1β and p-Iκ-Bα, but PA-BSA had no effect on pro-IL-1β or p-Iκ-Bα. In potassium-free buffer, LDL(-)-induced IL-1β reached a level similar to that induced by cotreatment with LDL(-) and PA-BSA. Moreover, LDL(-) and PA-BSA-induced IL-1β was inhibited in lectin-type oxidized LDL receptor-1 (LOX-1) knockdown cells and by blockers of voltage-gated potassium (Kv) channels. LDL(-) from diet-induced hypercholesterolemic rabbit had a similar effect as STEMI LDL(-) on IL-1β in macrophages. These results show that PA-BSA cooperates with LDL(-) to trigger IL-1β production in macrophages via a mechanism involving the LOX-1 and Kv channel pathways, which may play crucial roles in the regulation of inflammation in CVD.

Circulating CD15+ LOX-1+ PMN-MDSCs are a potential biomarker for the early diagnosis of non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the most common clinical lung cancer. Polymorphonuclear-myeloid derived suppressor cells (PMN-MDSCs), which are the major population of MDSCs, are involved in NSCLC progression. Recently, it was found that lectin-type oxidized LDL receptor 1 (LOX-1) could identify human PMN-MDSCs. However, the role of CD15+ LOX-1+ PMN-MDSCs in NSCLC early diagnosis has not been revealed. Here, we tried to confirm the application of the newly identified CD15+ LOX-1+ PMN-MDSCs in the early diagnosis of NSCLC. Flow cytometry (FCM) was used to detect the proportion of CD15+ LOX-1+ PMN-MDSCs in the peripheral blood (PB) of healthy controls (HC) and NSCLC patients. The correlation of CD15+ LOX-1+ PMN-MDSC frequency with levels of cytokeratin 19-fragments (CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen 125 (CA125) was analysed. Receiver operating characteristic (ROC) curve was used to estimate the diagnostic efficacy of CD15+ LOX-1+ PMN-MDSCs for NSCLC. Additionally, the association of CD15+ LOX-1+ PMN-MDSC frequency with NSCLC prognosis/recurrence after surgery was explored. The proportion of CD15+ LOX-1+ PMN-MDSCs increased in PB of NSCLC patients. CD15+ LOX-1+ PMN-MDSC proportion was positively correlated with levels of CEA, CA125 and CYFRA21-1. Detection of PMN-MDSC percentage in PB owed high sensitivity and specificity for NSCLC diagnosis. The proportion of CD15+ LOX-1+ PMN-MDSCs decreased in patients after surgery. The frequency of CD15+ LOX-1+ PMN-MDSCs was lower in NSCLC patients without recurrence compared to those with recurrence after surgery. Circulating CD15+ LOX-1+ PMN-MDSCs are a potential diagnostic marker for NSCLC, and are associated with NSCLC prognosis and recurrence after surgery.

Azilsartan ameliorates ox-LDL-induced endothelial dysfunction via promoting the expression of KLF2.

Background: Oxidized LDL(Ox-LDL) mediated endothelial dysfunction is involved in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Azilsartan is a potent agent for the treatment of hypertension as the antagonist of the angiotensin II receptor. This study will investigate whether Azilsartan possesses a beneficial effect against endothelial cell dysfunction induced by ox-LDL and explore the underlying preliminary mechanism.Methods: Ox-LDL was applied to construct an in vitro endothelial dysfunction model in human umbilical vascular endothelial cells (HUVECs). The expression of lectin-type oxidized LDL receptor 1 (LOX-1), endothelial nitric oxide synthase (eNOS), tight junction protein occludin, and transcriptional factor Krüppel-like factor 2 (KLF2) was detected using qRT-PCR and Western blot. ELISA and qRT-PCR were utilized to evaluate the production of chemokine monocyte chemotactic protein 1 (MCP-1) and chemokine (C-X-C motif) Ligand 1 Protein (CXCL1) in treated HUVECs. The generation of nitro oxide (NO) was determined using DAF-FM DA staining assay. KLF2 was silenced by transfecting the cells with specific Small interfering RNA (siRNA). FITC-dextran permeation assay was used to check the endothelial monolayer permeability of treated HUVECs.Results: Firstly, the elevated expressions of LOX-1, MCP-1, and CXCL-1 induced by stimulation with ox-LDL were significantly suppressed by Azilsartan. The downregulated eNOS and reduced production of NO induced by ox-LDL were reversed by the introduction of Azilsartan. Secondly, enlarged endothelial monolayer permeability and decreased expression of occludin stimulated with ox-LDL were greatly reversed by treatment with Azilsartan but were abolished by silencing the expression of KLF2. Lastly, the inhibited expression of KLF2 induced by ox-LDL was significantly elevated by the introduction of Azilsartan.Conclusion: Azilsartan might ameliorate ox-LDL-induced endothelial damage via elevating the expression of KLF2.

Emergence of immunosuppressive LOX-1+ PMN-MDSC in septic shock and severe COVID-19 patients with acute respiratory distress syndrome.

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive properties. In cancer patients, the expression of lectin‐type oxidized LDL receptor 1 (LOX‐1) on granulocytic MDSC identifies a subset of MDSC that retains the most potent immunosuppressive properties. The main objective of the present work was to explore the presence of LOX‐1+ MDSC in bacterial and viral sepsis. To this end, whole blood LOX‐1+ cells were phenotypically, morphologically, and functionally characterized. They were monitored in 39 coronavirus disease‐19 (COVID‐19, viral sepsis) and 48 septic shock (bacterial sepsis) patients longitudinally sampled five times over a 3 wk period in intensive care units (ICUs). The phenotype, morphology, and immunosuppressive functions of LOX‐1+ cells demonstrated that they were polymorphonuclear MDSC. In patients, we observed the significant emergence of LOX‐1+ MDSC in both groups. The peak of LOX‐1+ MDSC was 1 wk delayed with respect to ICU admission. In COVID‐19, their elevation was more pronounced in patients with acute respiratory distress syndrome. The persistence of these cells may contribute to long lasting immunosuppression leaving the patient unable to efficiently resolve infections.

Pyrogallol-Phloroglucinol-6 6-Bieckol on Attenuates High-Fat Diet-Induced Hypertension by Modulating Endothelial-to-Mesenchymal Transition in the Aorta of Mice.

Endothelial-to-mesenchymal transition (EndMT), which is involved in the development of various cardiovascular diseases, is induced by dyslipidemia or obesity. In dyslipidemia, the increased levels of oxidized low-density lipoproteins (oxLDL) upregulated the lectin-type oxidized LDL receptor 1 (Lox-1), which then upregulated the down signaling pathways of PKC-α/MMPs/TGF-β/SMAD2 or 3 and increased the EndMT. In this study, we investigated the effect of pyrogallol-phloroglucinol-6,6-bieckol (PPB), which is a compound of Ecklonia cava (E. cava), on decreased blood pressure (BP) by attenuating the EndMT in a high-fat diet- (HFD-) fed animal model. We also investigated PPB's attenuation effect on EndMT in oxLDL-treated mouse endothelial cells as an in vitro model. The results indicated that, in the aorta or endothelial cells of mice, the HFD or oxLDL treatment significantly increased the expression of Lox-1/PKC-α/MMP9/TGF-β/SMAD2/SMAD3. The PPB treatment significantly decreased its expression. In contrast, the HFD or oxLDL treatment significantly decreased the expression of the EC markers (PECAM-1 and vWF) while the PPB treatment significantly increased them. Moreover, the HFD or oxLDL treatment significantly increased the expression of the mesenchymal cell markers (α-SMA and vimentin) while PPB treatment significantly decreased them. PPB decreased the intima-media thickness and extracellular matrix amount of the aorta and attenuated the BP, which was increased by the HFD. In conclusion, PPB attenuated the upregulation of Lox-1/PKC-α/MMP9/TGF-β/SMAD2 and 3 and restored the EndMT in HFD-fed animals. Moreover, PPB showed a restoring effect on HFD-induced hypertension.

Accumulation of LOX-1+ PMN-MDSCs in nasopharyngeal carcinoma survivors with chronic hepatitis B might permit immune tolerance to epstein-barr virus and relate to tumor recurrence.

Chronic hepatitis B (CHB) has been reported to be associated with impaired prognosis for patients with nasopharyngeal carcinoma (NPC). However, the latent mechanism is unclear. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) induce immune suppression in CHB and promote the development of hepatocellular carcinoma. Lectin-type oxidized LDL receptor-1 (LOX-1) was recently identified as a specific marker for PMN-MSDC. We found NPC survivors with CHB had high levels of LOX-1+ PMN-MDSCs. LOX-1+ PMN-MDSCs significantly reduced T cell proliferation and activation. Endoplasmic reticulum stress was induced in LOX-1+ PMN-MDSCs. In addition, LOX-1+ PMN-MDSCs increased their expression of NOX2, a key reactive oxygen species (ROS)-related genes, and levels of ROS illustrated by the DCFDA test. The ROS inhibitor N-acetylcysteine abrogated the suppression of LOX-1+ PMN-MDSCs on T cell activation. The EBV DNA-positivity rate was higher in NPC survivors with CHB than in NPC patients without CHB. Those presenting with positive EBV DNA displayed higher LOX-1+ PMN-MDSC levels. LOX-1+ PMN-MDSCs suppressed the CD8+ T cell response against EBV. This study revealed LOX-1+ PMN-MDSC accumulation and activation in NPC survivors with CHB. LOX-1+ PMN-MDSCs might suppress the host immune response to EBV through ER stress/ROS pathway. These results explained the association of CHB with unfavorable NPC prognosis.

Colchicine inhibits the prothrombotic effects of oxLDL in human endothelial cells

BackgroundTissue Factor (TF) plays a pivotal role in coronary thrombosis. Oxidized low-density lipoproteins (oxLDL) are crucial in development of atherosclerosclerosis. Moreover, oxLDL are known to induce TF expression on several cell types including endothelial cells. The lectin-type oxidized LDL receptor 1 (LOX-1) represent the oxLDL receptor. Colchicine is an anti-mitotic drug recently proven to have beneficial effects in cardiovascular disease via unknown mechanisms. Thus, we aim at investigating colchicine effects on TF expression in oxLDL stimulated human vascular endothelial cells (HUVEC). Some molecular mechanism(s) potentially involved were investigated. MethodsHUVEC were pre-incubated with colchicine 10 μM for 1 h and then stimulated with oxLDL (50 μg/mL). TF gene (RT-PCR), protein (western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. TF translocation to cell surface was investigated by immunofluorescence. NF-κB/IκB axis was examined by western blot analysis and translocation assay. Finally, LOX-1 expression was also investigated. ResultsColchicine significantly reduced TF gene and protein expression as well as its procoagulant activity in oxLDL-treated HUVEC. These effects seem to be related mainly to action of colchicine on microtubules that, in turn, modulate TF trafficking in the cytoplasm, NF-κB/IκB pathway and LOX-1 expression. ConclusionsData of the present study, although in vitro, indicate that one of the hypothetical mechanisms by which colchicine exert protective cardiovascular effects might be its ability to inhibit the pro-thrombotic activity of oxLDL.

Inflammasome Activation in Human Macrophages Induced by a LDL (-) Mimetic Peptide.

The inflammasome is responsible for maturation of interleukin-1β (IL-1β) and interleukin-18 (IL-18) contributing to the inflammatory process in atherosclerosis. It is shown here that an electronegative low-density lipoprotein [LDL (-)] apoB-100 mimetic peptide can activate the transcriptional and posttranslational signs needed for complete inflammasome activation. This peptide, named p2C7, can activate the Toll-like receptor 4 (TLR4) that induces NF-κB activation and the transcription of inflammasome components. After blocking TLR4 with a neutralizing antibody, inflammasome component (NLRP3, CASP1, and ASC) and IL1b and IL18 gene downregulation occurred in human-derived macrophages stimulated with p2C7 or LDL (-). Moreover, the posttranslational signal was activated by the interaction between p2C7 and the lectin-type oxidized LDL receptor 1 (LOX-1), as demonstrated by the induction of caspase-1 cleavage in macrophages. The blockage of either TLR4 or LOX-1 decreased IL-1β and IL-18 secretion by human-derived macrophages as both pathways are necessary for complete inflammasome activation. These findings suggest a mechanism by which macrophages transduce the pro-inflammatory signal provided by LDL (-) ApoB-100 and its mimetic peptides to activate the inflammasome protein complex what may be relevant for the inflammatory process in atherosclerosis.

LOX-1 is involved in TLR2 induced RANKL regulation in peri-implantitis

PurposeTo explore whether receptor activator of nuclear factor kappa-B ligand (RANKL) is involved in the nosogenesis of peri-implantitis and to reveal the regulatory mechanism in Porphyromonas gingivalis induced RANKL production. MethodsTherefore, we collected peri-implant crevicular fluid (PICF) and gingival tissues from healthy implants and peri-implantitis patients. The expression of RANKL in samples was tested by ELISA, Western blot and immunofluorescence staining. The production of RANKL in THP-1 macrophages stimulated with P. gingivalis was detected by qRT-PCR and Western blot. Then macrophages were pre-treated with neutralizing antibodies of Toll-like receptor 2 (TLR2) or lectin-type oxidized LDL receptor 1 (LOX-1) and inhibitors of TLR2, LOX-1 or Erk1/2 before P. gingivalis stimulation to evaluate the roles of TLR2, LOX-1 and Erk1/2 in RANKL production by qRT-PCR and Western blot. ResultsThe protein level of RANKL was higher in PICF of peri-implantitis patients than healthy implants. We observed increased RANKL expression in P. gingivalis infected macrophages compared to controls. RANKL induced by P. gingivalis stimulation was mediated by TLR2 and Erk1/2 signaling pathway in THP-1 macrophages. LOX-1 is involved in TLR2 induced RANKL expression. ConclusionRANKL was involved in peri-implantitis, and regulated by TLR2, LOX-1 and Erk1/2 signaling against P. gingivalis infection. As the novel inflammation pathway triggers, TLR2 and LOX-1 which mediate RANKL production seems to be potential drug targets of peri-implantitis.

Electronegative LDL from Rabbits Fed with Atherogenic Diet Is Highly Proinflammatory.

Electronegative low-density lipoprotein (LDL(-)) has been found in the plasma of familial hypercholesterolemia and acute myocardial infarction and has been implicated in atherosclerosis and cardiovascular disease. However, less is known about the involvement of LDL(-) in atherosclerosis-related inflammation. This study aims at investigating the inducibility of LDL(-) by atherogenic diet in rabbits and at exploring the proinflammatory potential of the diet-induced LDL(-) in macrophages. Rabbits were fed with an atherogenic diet; LDL was isolated from plasma by NaBr density gradient ultracentrifugation and was then resolved into nLDL and LDL(-) by anion-exchange chromatography. Isolated nLDL and LDL(-) were directly used or incubated with 10 μM CuSO4 for 24 h to produce copper- (Cu-) ox-nLDL and Cu-ox-LDL(-). The effects of these LDLs on inflammation were evaluated in THP-1-derived macrophages. Macrophages were treated with nLDL, LDL(-), and extensively oxidized LDL (ox-LDL), then the levels of interleukin- (IL-) 1β, IL-6, and tumor necrosis factor- (TNF-) α in a culture medium were determined by ELISA, and the levels of total and phosphorylated IκB, p65, p38, JNK, and ERK in cell lysates were determined by Western blotting. The LDL(-) induced significantly higher levels of IL-1β, IL-6, and TNF-α in the medium. The levels of phosphorylated/total IκB, p65, p38, JNK, and ERK were also upregulated by LDL(-). In contrast, nLDL, Cu-ox-nLDL, and Cu-ox-LDL(-) exhibited much less effect. Knockdown of lectin-type oxidized LDL receptor- (LOX-) 1 resulted in significant reduction in LDL(-)-induced IL-1β, IL-6, and TNF-α. In addition, these LDL(-) effects were also markedly attenuated by inhibition of NF-κB and ERK1/2. The data suggested that LDL(-) induced inflammation through LOX-1-, NF-κB-, and ERK1/2-dependent pathways. Taken together, our results show that rabbits fed with atherogenic diet produce a highly proinflammatory LDL(-) that is more potent in inducing inflammation than nLDL and extensively oxidize LDL in macrophages. The results thus provide a novel link between diet-induced hypercholesterolemia and inflammation.

LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression.

Background/aimsPatients with glioblastoma multiforme (GBM) that is the most common brain cancer in adults have a rather poor prognosis. The accumulation of immune suppressive myeloid-derived suppressor cell (MDSC) is negatively associated with clinical outcomes in various cancers. A recent study identified that lectin-type oxidized LDL receptor 1 (LOX-1) may serve as a specific marker of human polymorphonuclear neutrophil (PMN)-MDSC. Thus, herein we focused on exploring the role of LOX-1+ PMN-MDSC in GBM progression.MethodsLOX-1, IFN-γ, dichlorodihydrofluorescein diacetate (DCFDA), CD15, CD4 and CD8 expression levels were examined by flow cytometry. ARG1 and iNOS expression levels in PMN were examined by quantitative real-time PCR. LOX-1 and CD15 expression levels in tumor tissue were determined by immunofluorescent microscopy. T cell proliferation was determined by 3H-thymidine incorporation.ResultsWe identified a protumorigenic subset of PMN, which constitutively expressed LOX-1 and accumulated in the peripheral blood of GBM patients. Compared to LOX-1− PMN, the LOX-1+ PMN exhibited a PMN MDSC profile, with a significant increase in the expression of DCFDA, ARG1 and iNOS, and the capacity of inhibiting the CD3+ T cell proliferation in a dependent-ARG1/iNOS way. Additionally, we found that LOX-1+ PMN negatively correlated with effector immune cells in GBM patients, accumulated in GBM tissues, and was related to early recurrence and disease progression tightly.ConclusionOur study revealed that LOX-1+ PMN-MDSC inhibited the T cell proliferation to enhance immune suppression, which may play a key role in driving the GBM progression.

ATF4 Involvement in TLR4 and LOX-1-Induced Host Inflammatory Response to Aspergillus fumigatus Keratitis.

Purpose Activating transcription factor 4 (ATF4) is induced by various stressors. Here, we investigated the expression of ATF4 in the host inflammatory response to Aspergillus fumigatus (A. fumigatus) keratitis. Methods A. fumigatus keratitis mouse models developed by intrastromal injection as well as corneal epithelium scratching were examined daily with a slit lamp microscope for corneal opacification and ulceration. Subsequent in vitro experimentation was carried out in human corneal epithelial cells (HCECs) as well as THP-1 macrophages infected with A. fumigatus. Inhibitors, including CLI-095, Poly (I), SCH772984, and SP600125, were used to assess the role of proteins like toll-like receptor 4 (TLR4), lectin-type oxidized LDL receptor 1 (LOX-1), extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal kinase (JNK) in ATF4 expression as a response to A. fumigatus infection. This assessment was made in both mouse models and HCECs using western blot. Results Compared to the controls, ATF4 was increased in corneas from two kinds of A. fumigatus keratitis models at 3 days after infection. ATF4 expression was upregulated with A. fumigatus conidia both in HCECs and THP-1 macrophages 16 hours after stimulation. Furthermore, ATF4 expression in response to A. fumigatus infection was shown to be dependent on TLR4 and LOX-1 expression, and ERK1/2 and JNK contributed to the expression of ATF4 in response to A. fumigatus. Conclusion Our results clearly indicate that ATF4 was involved in the host antifungal immune response to A. fumigatus keratitis; expression was found to be dependent on TLR4, LOX-1 expression, and MAPKs pathway.

Fenretinide Inhibits Neutrophil Recruitment and IL-1β Production in Aspergillus fumigatus Keratitis.

Purpose:Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. Fenretinide, a derivative of vitamin A, has been shown to suppress inflammation in a multitude of diseases. In this study, we aimed to characterize the effect of fenretinide in Aspergillus fumigatus keratitis of the eye in a mouse model.Methods:In vivo and in vitro experiments were performed in mouse models and THP-1 macrophage cell cultures infected with A. fumigatus, respectively. Experimental subjects were first pretreated with fenretinide, and then the effect of the compound was assessed with clinical evaluation, neutrophil staining, myeloperoxidase assay, quantitative polymerase chain reaction (qRT-PCR), and western blot.Results:We confirmed that fenretinide contributed to protection of corneal transparency during early mouse A. fumigatus keratitis by reducing neutrophil recruitment, decreasing myeloperoxidase (MPO) levels and increasing apoptosis. Compared with controls, fenretinide impaired proinflammatory cytokine interleukin 1 beta (IL-1β) production in response to A. fumigatus exposure with contributions by lectin-type oxidized LDL receptor 1 (LOX-1) and c-Jun N-terminal kinase (JNK).Conclusions:Together, these findings demonstrate that fenretinide may suppress inflammation through reduced neutrophil recruitment and inflammatory cytokine production in A. fumigatus keratitis.