Presence Of Lipopolysaccharide Stimulation Research Articles

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.

Inflammatory stimuli promote oxidative stress in pancreatic acinar cells via Toll-like receptor 4/nuclear factor-κB pathway.

The Toll‑like receptor 4/nuclear factor‑κB (TLR4/NF‑κB) pathway is vital to the pathogenesis of acute pancreatitis (AP). The aim of the present study was to identify the mechanism of the activation of the TLR4/NF‑κB signaling pathway in the viability of primary pancreatic cells. The cells were stimulated with lipopolysaccharide (LPS) for the activation of NF‑κB signaling. Next, the reactive oxygen species (ROS) level was evaluated by detecting the concentration of malondialdehyde and glutathione peroxidase. Cell viability was measured by Cell Counting Kit‑8 and MTT assays, while the percentage of apoptosis was detected by flow cytometry. Quantitative polymerase chain reaction was used to detect TLR4, B‑cell lymphoma 2 (Bcl2), Bcl2‑associated X protein (Bax) and phorbol‑12‑myristate‑13‑acetate‑induced protein 1 (PMAIP1) expression levels. Western blot assay was also conducted to detect TLR4 protein expression, while the activity of NF‑κB signaling was measured by determining the p65 and phosphorylated p65 protein levels. In addition, the effect of TLR4 overexpression or treatment with TLR4 antagonists in the presence of LPS stimulation was investigated. The results revealed that ROS levels were increased and cell viability was decreased in LPS‑stimulated pancreatic acinar cells. TLR4, Bax and PMAIP1 levels were increased, Bcl2 expression was decreased and NF‑κB signaling was activated in LPS‑stimulated pancreatic acinar cells. Furthermore, pancreatic cells with TLR4 overexpression exhibited increased ROS level and decreased viability. Finally, the effect caused by LPS stimulation was partially reversed by treatment of pancreatic acinar cells with TLR4 antagonists. In conclusion, the current study investigated a novel regulatory mechanism of the TLR4/NF‑κB pathway in LPS‑stimulated pancreatic cells, which may contribute to pancreatitis. The damage of these cells due to increased ROS levels was observed to occur through activation of the TLR4/NF‑κB pathway.

Experimental study on 1,25(OH)2D3 amelioration of oral lichen planus through regulating NF‐κB signaling pathway

To explore the protective function of vitamin D (VD)/vitamin D receptor (VDR) on the development of oral lichen planus (OLP) and elaborate the underling mechanism of it. H&E staining, myeloid peroxidase (MPO) assays, quantitative PCR (qPCR), Western blotting, and Elisa were used to test the human biopsies and serum. QPCR, Western blotting, Elisa, and siRNA transfection were also performed in LPS-induced keratinocytes to observe the functions of vitamin D and VDR. The lack of VDR in the diseased biopsies from OLP patients was associated with activated helper T-cell type 1 (Th1)-driven inflammatory response. Importantly, the status of serum 25-hydroxyvitamin D of OLP patients was reduced consistently. In a cultured cell model, 1,25(OH)2 D3 could downregulate excessive production of pro-inflammatory factors induced by lipopolysaccharide (LPS) in keratinocyte HaCat cells. Mechanistically, even though LPS-induced cytokines in keratinocytes were inhibited both by nuclear factor-κB (NF-κB) inhibitor and by activator protein 1 (AP-1) inhibitor, VDR-dependent 1,25(OH)2 D3 blocked the activation of phosphorylated-NF-κB p65 rather than c-Jun/c-Fos in the presence of LPS stimulation. These results suggest that 1,25(OH)2 D3 plays an anti-inflammatory role in OLP by mediating NF-κB signaling pathway but not AP-1 signaling pathway with a VDR-dependent manner, predicting vitamin D supplement may be a potential strategy for the OLP management.

Low Glucose Concentrations Induce a Similar Inflammatory Response in Monocytes from Type 2 Diabetic Patients and Healthy Subjects.

This study aims to assess the proinflammatory interleukin 1β (IL-1β) and anti-inflammatory IL-10 production by monocytes from 38 patients with type 2 diabetes and 31 controls in different glucose concentrations. Monocytes were incubated in low (2.5 mmol/L)-, normal (5.0 mmol/L)-, and high (20 mmol/L)-glucose conditions in the presence and absence of lipopolysaccharide (LPS). Monocytes from both patients and controls only produced a significant increase in IL-1β in low-glucose conditions (p < 0.01), and this phenomenon was amplified in the presence of LPS, while it was not seen in normal- or high-glucose conditions, not even in the presence of LPS stimulation. There was no increase in IL-10 production by monocytes from either diabetic patients or controls using whatever glucose concentrations, except when treated with LPS in normal-glucose conditions. These findings seem to suggest that low-glucose conditions induce an inflammatory response in monocytes in all individuals, as an intrinsic capacity of this cell line. On the other hand, monocytes only retain their anti-inflammatory ability in response to known inflammatory stimuli such as LPS, under normal-glucose concentrations. In conclusion, human monocytes express an inflammatory pattern in low-glucose conditions in vitro. This response could contribute to explaining the higher cardiovascular risk induced by hypoglycemia in diabetic patients.

MicroRNA-155 Deficiency Results in Decreased Macrophage Inflammation and Attenuated Atherogenesis in Apolipoprotein E–Deficient Mice

microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E-deficient (apoE(-/-)) and miR155(-/-)/apoE(-/-) (double knockout) mice fed a Western diet. Compared with apoE(-/-) mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6C(high) cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE(-/-) mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role. miR155 deficiency attenuates atherogenesis in apoE(-/-) mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.

Rhein Inhibits the Expression of Vascular Cell Adhesion Molecule 1 in Human Umbilical Vein Endothelial Cells with or without Lipopolysaccharide Stimulation

Reducing the expression of endothelial cell adhesion molecules (ECAMs) is known to decrease inflammation-induced vascular complications. In this study, we explored whether rhein can reduce the inflammation-induced expression of ECAMs in human umbilical vein endothelial cells (HUVECs) with or without lipopolysaccharide (LPS) stimulation. HUVECs were treated with different concentrations of rhein with or without 2.5 μg/ml LPS stimulation. Cell viability was assayed using the MTT method. Real-time PCR and Western blot analysis were used to measure the transcription and expression levels of ECAMs, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-SELECTIN and related signaling proteins. The results indicated that rhein (0-20 μmol/L) and LPS (0-10 μg/ml) had no effect on the viability of HUVECs. LPS could promote the expression of VCAM-1, ICAM-1 and E-SELECTIN. Rhein appeared to target VCAM-1, ICAM-1 and E-SELECTIN, with the transcription and expression of all three factors being reduced by the rhein treatment (10 and 20 μmol/L). The transcription and expression of VCAM-1 were also reduced by treatment with rhein (10 and 20 μmol/L) in the presence of LPS stimulation. In conclusion, rhein treatment reduced the expression of VCAM-1 in HUVECs via a p38-dependent pathway.

Angiotensin II inhibits interleukin-6 mRNA expression of LPS-stimulated macrophages through down-regulating calcium signaling

BackgroundThe renin-angiotensin system plays a key role in the regulation of blood pressure following hemorrhage and shock. Recent studies also suggest renin-angiotensin system regulates inflammatory mediator production although the amechanism is largely unknown. This purpose of the study was to examine the effect of angiotensin II on macrophage (MØ) IL-6 messenger RNA (mRNA) expression induced by lipopolysaccharide (LPS) and on the alterations in the calcium influx. MethodsJ774A.1 cells, a mouse MØ cell line, were exposed to E. coli LPS (1 or 10 μg/ml) in the presence of angiotensin II (10 nM to 1 μM). IL-6 mRNA expression was determined by the reverse transcription polymerase chain reaction technique. IL-6 protein production was measured by ELISA. To examine the involvement of calcium signaling in IL-6 mRNA expression, MØ were exposed to various calcium agonists and antagonists in the presence of LPS stimulation. Changes of intracellular [Ca2+] by LPS stimulation and angiotensin II treatment were determined by a fura-2 fluorescence ratio method. ResultsLPS stimulation increased MØ IL-6 mRNA expression, which was inhibited by Angiotensin II in a dose-dependent fashion. Both thapsigargin and A23187 augmented the IL-6 mRNA levels induced by LPS stimulation, but only thapsigargin was able to induce IL-6 mRNA directly. TMB-8 but not verapamil inhibited LPS-stimulated MØ IL-6 mRNA. Finally, angiotensin II significantly altered the changes in intracellular [Ca2+] levels induced by LPS stimulation by reducing both the peak and slope of calcium spikes. ConclusionsOur data show that calcium signaling is closely related to IL-6 mRNA expression. Angiotensin II inhibits IL-6 mRNA expression of LPS-stimulated MØ. The inhibitory effects of angiotensin II appear, at least in part, to be mediated through down regulating calcium dependent pathways.

Three‐Tier Model for Inflammasome Expression and a New Concept of Inflammation Privilege

We examined a new hypothesis that the tissue expressions of TLRs/NLRs/inflammasome/caspase‐1/IL‐1β pathway are differentially regulated. We made several findings: (1) Vascular tissues and heart express fewer types of TLRs and NLRs than immune tissues; and (2) Based on the expression data of inflammasomes (NALP1, NALP3 and IPAF), the tissues can be classified into three tiers: the first tier tissues including blood and thymus express inflammasome(s) in constitutive status; the second tier tissues have inflammasome(s) in nearly‐ready expression status (with the requirement of upregulation of one component); the third tier tissues, like heart, require upregulation of at least two components to assemble functional inflammasomes. We examined the mRNA levels of inflammasome components in human aortic endothelial cells after stimulated with lipopolysaccharide (LPS). In the presence of LPS stimulation, the mRNA levels of the main inflammasome component including ASC, caspase‐1, NALP‐1 and NALP‐3 are up‐regulated, which supports our three‐tier model. Our original model of three‐tier expression of inflammasomes would further suggest a new concept of tissues’ inflammation privilege, which provides an important insight to the differences among tissues in initiating acute inflammation and upregulation of inflammasomes in inflammation‐privileged tissues in response to chronic metabolic stimuli.

Effect of 6-Gingerol on Pro-Inflammatory Cytokine Production and Costimulatory Molecule Expression in Murine Peritoneal Macrophages

Pro-inflammatory cytokines produced primarily by macrophages are key elements in many surgical conditions including sepsis, ischemia-reperfusion injury, and transplant rejection. Herbal products are being used as alternative treatments in such inflammatory conditions. Ginger is known for its ethno-botanical applications as an anti-inflammatory agent. 6-gingerol is one of the active ingredients of ginger that imparts ginger with its anti-inflammatory properties. We hypothesized that the anti-inflammatory effect of 6-gingerol is because of inhibition of macrophage activation, more specifically by an inhibition of pro-inflammatory cytokines and antigen presentation by lipopolysaccharide (LPS) activated macrophages. To study the effect of 6-gingerol on pro-inflammatory cytokines, we measured the liberation of TNF-alpha, IL-1beta, and IL-12 by murine peritoneal macrophages exposed to several doses of 6-gingerol in the presence of LPS stimulation. We also studied the effect of 6-gingerol on the cell surface expression of B7.1, B7.2, and MHC II. Finally, we examined the APC function of the 6-gingerol treated macrophages by a primary mixed lymphocyte reaction. 6-gingerol inhibited the production of pro-inflammatory cytokines from LPS stimulated macrophages but had no effect on the LPS-induced expression of B7.1, B7.2, and MHC II. The APC function of LPS stimulated macrophages was also unaffected by 6-gingerol treatment. Our data indicate that 6-gingerol selectively inhibits production of pro-inflammatory cytokines from macrophages but does not affect either the APC function or cell surface expression of MHC II and costimulatory molecules. We, thus, provide a mechanistic insight into the anti-inflammatory properties of 6-gingerol that may be useful to treat inflammation without interfering with the antigen presenting function of macrophages.