Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis.

Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldlr-/-Nod1/2-/-) which were subsequently analyzed regarding experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition. Compared to Ldlr-/- mice, Ldlr-/-Nod1/2-/- mice showed reduced plasma lipids and increased hepatic expression of the scavenger receptor LDL receptor-related protein 1 after feeding a high-fat diet for 12 weeks. Furthermore, intestinal cholesterol and its bacterial degradation product coprostanol were elevated in Ldlr-/-Nod1/2-/- mice, correlating with the increased abundance of Eubacterium coprostanoligenes as assessed by 3rd generation sequencing of the gut microbiota. Atherosclerotic plaques of Ldlr-/-Nod1/2-/- mice exhibited less lipid deposition and macrophage accumulation. Moreover, macrophages from Ldlr-/-Nod1/2-/- mice showed higher expression of the cholesterol efflux transporters Abca1 and Abcg1 and accordingly reduced foam cell formation. Deficiency of Nod1 and Nod2 led to reduced plaque lipid deposition and inflammatory cell infiltration in atherosclerotic plaques. This might be explained by diminished plasma lipid levels and foam cell formation due to altered expression of key regulators of the hepatic cholesterol pathway as well as differential intestinal cholesterol metabolism and microbiota composition.

Suppressor of Cytokine Signaling 1 is Involved in Gene Regulation Which Controls the Survival of Ly6Clow Monocytes in Mice.

BACKGROUND/AIMS: Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6Chigh) and to a lesser extent on patrolling monocytes (Ly6Clow). Our previous study revealed an accumulation of Ly6Chigh monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6Chigh and Ly6Clow monocytes. METHODS: In monocyte subsets from SOCS-1competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA). RESULTS: mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators. CONCLUSION: Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6Clow monocytes.

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration.

Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2-/- knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.

Anti-Inflammatory Effects of C1q/Tumor Necrosis Factor-Related Protein 3 (CTRP3) in Endothelial Cells.

The C1q/TNF-related protein 3 (CTRP3) represents a pleiotropic adipokine reciprocally associated with obesity and type 2 diabetes mellitus and exhibits anti-inflammatory properties in relation to lipopolysaccharides (LPS)-mediated effects in adipocytes, as well as monocytes/macrophages. Here, we focused on the influence of CTRP3 on LPS-mediated effects in endothelial cells in order to expand the understanding of a possible anti-inflammatory function of CTRP3 in a setting of endotoxemia. An organ- and tissue-specific expression analysis by real-time PCR revealed a considerable Ctrp3 expression in various adipose tissue compartments; however, higher levels were detected in the aorta and in abundantly perfused tissues (bone marrow and the thyroid gland). We observed a robust Ctrp3 expression in primary endothelial cells and a transient upregulation in murine endothelial (MyEND) cells by LPS (50 ng/mL). In MyEND cells, CTRP3 inhibited the LPS-induced expression of interleukin (Il)-6 and the tumor necrosis factor (Tnf)-α, and suppressed the LPS-dependent expression of the major endothelial adhesion molecules Vcam-1 and Icam-1. The LPS-induced adhesion of monocytic cells to an endothelial monolayer was antagonized by CTRP3. In C57BL/6J mice with an LPS-induced systemic inflammation, exogenous CTRP3 did not affect circulating levels of TNF-α, ICAM-1, and VCAM-1. In conclusion, we characterized CTRP3 beyond its function as an adipokine in a setting of vascular inflammation. CTRP3 inhibited LPS-induced endothelial expression of adhesion molecules and monocyte cell adhesion, indicating an important vascular anti-inflammatory role for CTRP3 in endotoxemia.

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

Variety matters: Diverse functions of monocyte subtypes in vascular inflammation and atherogenesis.

Monocytes are important mediators of the innate immunity by recognizing and attacking especially bacterial pathogens but also play crucial roles in various inflammatory diseases, including vascular inflammation and atherosclerosis. Maturation, differentiation and function of monocytes have been intensively explored for a long time in innumerable experimental and clinical studies. Monocytes do not represent a uniform cell type but could be further subdivided into subpopulations with distinct features and functions. Those subpopulations have been identified in experimental mouse models as well as in humans, albeit distinguished by different cell surface markers. While Ly6C is used for subpopulation differentiation in mice, corresponding human subsets are differentiated by CD14 and CD16. In this review, we specifically focused on new experimental insights from recent years mainly in regard to murine monocyte subpopulations and their roles in vascular inflammation und atherogenesis.

Anti-tumor necrosis factor-α therapy increases plaque burden in a mouse model of experimental atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is critically fueled by vascular inflammation through oxidized lipids and inflammatory cytokines such as tumor necrosis factor (TNF)-α. Genetic disruption of Tnf-α reduces atherosclerosis in experimental mouse models. However, less is known about the therapeutic potential of Tnf-α blockage by pharmacological inhibitors such as monoclonal antibodies, which are already approved for several inflammatory disorders in patients. Therefore, we investigated the effect of pharmacological TNF-α inhibition on plaque development in experimental atherosclerosis. RESULTS: 10 week old male Ldlr-/- mice were divided into 4 groups (n = 7-10) and fed a high fat, high cholesterol diet for 6 and 12 weeks. Simultaneously, the mouse-specific anti-Tnf-α monoclonal antibody CNTO5048 (CNT) or a control IgG was administered. RESULTS: CNT reduced circulating inflammatory markers without affecting body weight and glucose metabolism. Unexpectedly, CNT treatment increased plasma triglyceride levels and pro-atherogenic very-low-density lipoprotein (VLDL) cholesterol as well as plaque burden in the thoracoabdominal aorta and in the aortic root. In addition, we observed decreased smooth muscle cell content in the lesions and a trend towards reduced collagen deposition upon Tnf-α inhibition. Furthermore, inflammatory gene expression in the aortic arch was increased following Tnf-α inhibitor treatment. CONCLUSIONS: Although up to 12-week pharmacological inhibition of TNF-α in Ldlr-/- mice diminishes systemic inflammation, experimental plaque burden and vascular inflammatory gene expression are increased, while markers of plaque stability decrease. These observations may be explained by the development of a pro-atherogenic plasma lipid profile.

Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion.

OBJECTIVE: It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab-which is approved for several inflammatory disorders-on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions. METHODS AND RESULTS: Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice. CONCLUSION: Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.

Lipocalin (LCN) 2 Mediates Pro-Atherosclerotic Processes and Is Elevated in Patients with Coronary Artery Disease.

BACKGROUND: Lipocalin (LCN) 2 is associated with multiple acute and chronic inflammatory diseases but the underlying molecular and cellular mechanisms remain unclear. Here, we investigated whether LCN2 is released from macrophages and contributes to pro-atherosclerotic processes and whether LCN2 plasma levels are associated with the severity of coronary artery disease progression in humans. METHODS AND RESULTS: In an autocrine-paracrine loop, tumor necrosis factor (TNF)-α promoted the release of LCN2 from murine bone-marrow derived macrophages (BMDM) and vice versa. Moreover, LCN2 stimulation of BMDM led to up-regulation of M1 macrophage markers. In addition, enhanced migration of monocytic J774A.1 cells towards LCN2 was observed. Furthermore, LCN2 increased the expression of the scavenger receptors Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as well as scavenger receptor class A-1 (SRA-1) and induced the conversion of macrophages to foam cells. In atherosclerotic lesions of low density lipoprotein receptor-deficient (ldlr-/-) mice fed a high fat, high cholesterol diet, LCN2 was found to be co-localized with macrophages in the shoulder region of the atherosclerotic plaque. In addition, LCN2 plasma levels were significantly increased in plasma samples of these mice. Finally, LCN2 plasma levels correlated with the severity of coronary artery disease (CAD) in patients as determined by coronary angiography. CONCLUSIONS: Here we demonstrated that LCN2 plays a pivotal role in processes involved in atherogenesis by promoting polarization and migration of monocytic cells and development of macrophages towards foam cells. Moreover, LCN2 may be used as a prognostic marker to determine the status of CAD progression.