Transcription Of Mediators Research Articles

BET bromodomain inhibitors diminish IL‐1B‐induced transcription of NF‐κB target genes

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of pancreatic β-cells, thought to be a result of chronic inflammation of pancreatic islets. Elevated proinflammatory cytokine levels in the islet results in increased nitric oxide production through increased transcription of inducible nitric oxide synthase (iNOS), reduced glucose responsiveness, and ultimately selective destruction of insulin-producing β-cells. The exact causes of T1D are poorly understood and although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins suggests an, as yet undetermined role for epigenetic factors. Recent studies have shown that epigenetic regulatory proteins contribute to the onset and progression of autoimmune diabetes. The bromodomain and extraterminal domain (BET) family of proteins (Brd2, Brd3, Brd4 and BrdT) have emerged as promising drug targets for a wide variety of diseases. Bromodomain-containing proteins are epigenetic “readers” of histone acetylation and we hypothesize they regulate proinflammatory cytokine induced transcription in β-cells. Uniquely, the BET family proteins contain two bromodomains (BD1 and BD2), which affords BET proteins the capacity to simultaneously bind two acetylated histones and/or other nuclear proteins such as DNA-binding factors to modulate transcription in a cell-type specific manner. Here, we show that BET bromodomain inhibitors decrease the β-cell response to proinflammatory cytokines in insulinoma β-cell line (INS832/13) and primary rat islets. We found that BET inhibitors mitigated cytokine-induced transcription of inflammatory mediators, including iNOS, which is known to damage β-cells. This decrease is mediated through inhibition of the NF-κB pathway by BET inhibitors. Furthermore, using knockdown of BET proteins in insulinoma β-cell lines, we identified key mechanisms through which this altered transcriptional profile occurs. This work provides important mechanistic insights into the disease onset and progression of autoimmune diabetes for which current therapies are ineffective.

Gene Expression of Inhibitory Receptors and Immune Regulatory Molecules in Polarized Macrophages Derived from Early- and Late-Stage BD Patients

Background: Bipolar disorder (BD) has been associated with increased levels of peripheral inflammatory mediators and neuroinflammation. Previously, we observed different immune responses in macrophages of BD patients at different stages of the disorder. Thus, we aimed to further evaluate the regulation of immune response in BD by quantifying the expression of immune checkpoint receptors and respective ligands, as well as molecules involved in regulation and transcription of inflammatory mediators in polarized macrophages of early and late stages individuals with BD. Methods: qRT-PCR was performed to analyze the expression of TLR1, TLR6, PD-1, NFKB1, PD-L1, PD-L2 and TIM-3 in samples of proinflammatory M1 or M(IFNγ+LPS) and anti-inflammatory M2 or M(IL-4) macrophages derived from peripheral blood mononuclear cells (PBMCs) of euthymic BD patients (n=16), classified as early-stage BD (BD-E, n=9) and late-stage BD (BD-L, n=7) - according to Functional Assessment Short Test (FAST) - and healthy controls (HC, n=10). Results: M(IL-4) from BD-E showed higher expression levels of NFKB1 and PD-L1 in comparison to HC (p<0.05), while BD-L only had higher expression levels of PD-L1 compared to HC (p<0.05). No statistical differences were found between groups for the expression levels of TLR1, PD-L2 and TIM-3 in M(IL-4) phenotype, while expression levels remained unchanged in M(IFNγ+LPS) for all markers. TLR6 and PD-1 did not show PCR amplification in both macrophage phenotypes. Conclusion: Our findings suggest an immunological regulation acting as a compensatory mechanism by M2 anti-inflammatory macrophages at an early stage of BD, although decreased regulation in these cells seems to be observed at late stages of the disorder. We hypothesize that such alterations are possibly due to chronic low-grade inflammation during the course of BD, resulting in the ‘exhaustion’ of macrophage response. However, further investigation is required to comprehend better the role of immune regulatory mechanisms in the disorder. Funding Information: This work was supported by the FIPE-HCPA (Project Number 150396) and National Council for Scientific and Technological Development (CNPq, Brazil; Project Number PQ 302382/2019-4); and BA is a scholarship recipient from CNPq, Brazil. Declaration of Interests: None to declare. Ethics Approval Statement: All participants provided written informed consent before their inclusion, and this study was approved by the Institutional Review Board of Hospital de Clinicas de Porto Alegre (HCPA, Project Number 150396).

Th1-Polarized, Dengue Virus-Activated Human Mast Cells Induce Endothelial Transcriptional Activation and Permeability

Dengue virus (DENV), an arbovirus, strongly activates mast cells (MCs), which are key immune cells for pathogen immune surveillance. In animal models, MCs promote clearance of local peripheral DENV infections but, conversely, also promote pathological vascular leakage when widely activated during systemic DENV infection. Since DENV is a human pathogen, we sought to ascertain whether a similar phenomenon could occur in humans by characterizing the products released by human MCs (huMCs) upon direct (antibody-independent) DENV exposure, using the phenotypically mature huMC line, ROSA. DENV did not productively infect huMCs but prompted huMC release of proteases and eicosanoids and induced a Th1-polarized transcriptional profile. In co-culture and trans-well systems, huMC products activated human microvascular endothelial cells, involving transcription of vasoactive mediators and increased monolayer permeability. This permeability was blocked by MC-stabilizing drugs, or limited by drugs targeting certain MC products. Thus, MC stabilizers are a viable strategy to limit MC-promoted vascular leakage during DENV infection in humans.

Microbubble formulation influences inflammatory response to focused ultrasound exposure in the brain

Focused ultrasound and microbubble (FUS + MB)-mediated blood–brain barrier (BBB) permeability enhancement can facilitate targeted brain-drug delivery. While controlling the magnitude of BBB permeability enhancement is necessary to limit tissue damage, little work has attempted to decouple these concepts. This work investigated the relationship between BBB permeability enhancement and the relative transcription of inflammatory mediators 4 h following sonication. Three microbubble formulations, Definity, BG8774, and MSB4, were compared, with the dose of each formulation normalized to gas volume. While changes in the transcription of key proinflammatory mediators, such as Il1b, Ccl2, and Tnf, were correlated to the magnitude of BBB permeability enhancement, these correlations were not independent of microbubble formulation; microbubble size distribution may play an important role, as linear regression analyses of BBB permeability magnitude versus differential gene expression for these proinflammatory mediators revealed significantly greater slopes for MSB4, a monodisperse microbubble with mean diameter of 4 μm, compared to Definity or BG8774, both polydisperse microbubbles with mean diameters below 2 μm. Additionally, the function of an acoustic feedback control algorithm, based on the detection threshold of ultraharmonic emissions, was assessed. While this control strategy was effective in limiting both wideband emissions and red blood cell extravasation, microbubble formulation was found to influence the magnitude of BBB leakage and correlations to acoustic emissions. This work demonstrates that while the initial magnitude of FUS + MB-mediated BBB permeability enhancement has a clear influence on the subsequent inflammatory responses, microbubble characteristics influence these relationships and must also be considered.

Oligomeric amyloid‐beta 1–42 inhibits antigen presentation

AbstractBackgroundBeyond microglia, the involvement of other immune cell types and immune functions in Alzheimer’s disease (AD) is not well understood. Here, we investigated the potential immunomodulatory properties in vivo and in vitro of oligomeric species of beta‐amyloid‐peptide (Aβ) on microglia and other putative antigen presenting cells (APCs).MethodsWe used multicolour flow‐cytometry, confocal microscopy and MSD biochemical analysis to characterise amyloid deposition and brain immunephenotype of APP‐PS1‐dE9 mice of 3, 6, 12 and 20 months of age. Next, we applied a customised in vitro systems (using OT‐II ovalbumin‐specific transgenic CD4+ T cells and ovalbumin as antigen) to study the effect of recombinant, oligomeric preparations of human Aβ1‐42 on antigen presentation by primary dendritic cells. Finally, via RNAseq, we investigated the effect of oligomeric Aβ 1‐42 on the transcriptome of primary dendritic cells.ResultsWe show that, in the APP‐PS1‐dE9 mouse model, the deposition of the first beta‐amyloid plaques at 6 months of age is accompanied by a significant reduction in MHC class II surface levels on brain APCs, suggesting impaired antigen presentation capability induced by beta‐amyloid. This hypothesis was confirmed by our in vitro experiments, using the prototypical antigen presenting cells, primary bone‐marrow derived dendritic cells. Incubation with a preparation containing various forms of oligomeric Aβ1‐42 (but not of scrambled peptide) inhibited antigen presentation by primary dendritic cells, in a dose‐dependent manner. Finally, we found that human oligomeric Aβ 1‐42 alters the transcription of key immune mediators in dendritic cells, including cytokines, chemokines and adhesion molecules.ConclusionsOur results suggest that, beyond their well‐characterised neurotoxic effects, oligomeric forms of Aβ 1‐42 exert hitherto unknown immunomodulatory properties. By inhibiting antigen presentation by cerebral APCs, Aβ 1‐42 might interfere with physiological brain immune surveillance, thus facilitating Aβ 1‐42 and tau spreading in AD.

Enhanced transforming growth factor-beta signaling and fibrosis in the pectoralis major muscle of broiler chickens affected by wooden breast myopathy

Fibrosis has also been recorded as a prominent pathological feature within wooden breast (WB) myopathy of broiler chickens. This study was conducted to evaluate the accumulation of fibril collagen, deposition of the extracellular matrix (ECM) components, and the underlying mechanism mediating the pathogenic fibrotic process in the pectoralis major (PM) muscle of WB-affected birds. Broiler chickens were categorized into the control and WB groups based on the evaluation of myopathic lesions. Results indicated that the total content and area of collagen in cross-sections of the PM muscle, as well as the augmented expression of collagen-I and fibronectin in the ECM, were greatly increased in birds with WB. Wooden breast myopathy upregulated expressions of transforming growth factor-beta (TGF-β) and the phosphorylation of Smad 2 and 3, thereby activating TGF-β-mediated Smad signaling pathway, which further enhanced the transcription of profibrotic mediators. In addition, regulators involved in collagen biosynthesis and cross-linking including prolyl 4-hydroxylase, lysyl oxidase, lysyl hydroxylase, and decorin were increased in the WB muscle. Finally, the expressions of both matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) were increased in the WB muscle, which might be related with reduced ECM remodeling. Overall, WB myopathy induces severe fibrosis by enhancing ECM deposition and collagen cross-linking in the PM muscle of broiler chickens, possibly via the activation of TGF-β signaling and the dysregulation of the MMP and TIMP system.

Chinese Medicine Huzhen Tongfeng Formula Effectively Attenuates Gouty Arthritis by Inhibiting Arachidonic Acid Metabolism and Inflammatory Mediators.

The Chinese herbal medicine, Huzhen Tongfeng Formula (HZTF), derived from traditional Chinese medicine (TCM) practice, has recognized therapeutic benefits for gouty arthritis (GA). HZTF is currently in the late stage of approval process as a new anti-GA drug application. However, the underlying mechanism of HZTF as an antigout medication is unclear. In this study, we combined network pharmacology and experimental validation approaches to elucidate the mechanism of action of HZTF. First, the relative drug-disease target networks were constructed and analyzed for pathway enrichment. Potential pathways were then validated by in vitro and in vivo experiments. We found that 34 compounds from HZTF matched 181 potential drug targets. Topology analysis revealed 77 core targets of HZTF, which were highly related to gout, following screening of KEGG pathway enrichment. Further analysis demonstrated that the arachidonic acid metabolic pathway was the most relevant pathway involved in the mechanism of HZTF. Validation experiments showed that HZTF significantly inhibited the inflammatory cell infiltration into gouty joints, improved the swelling of affected joints, and increased the pain threshold. HZTF significantly reduced the transcription and production of various cytokines and inflammatory mediators in vitro. In particular, cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase were simultaneously downregulated. In conclusion, our study suggests that the antigout mechanism of HZTF is associated with the inhibition of the arachidonic acid pathway, resulting in the suppression of inflammatory cytokines and mediators. These findings extend our understanding of the pharmacological action of HZTF, rationalizing the application HZTF as an effective herbal therapy for GA.

HOPS/Tmub1 involvement in the NF-kB-mediated inflammatory response through the modulation of TRAF6

HOPS/Tmub1 is a ubiquitously expressed transmembrane ubiquitin-like protein that shuttles between nucleus and cytoplasm during cell cycle progression. HOPS causes cell cycle arrest in G0/G1 phase, an event associated to stabilization of p19Arf, an important tumor suppressor protein. Moreover, HOPS plays an important role in driving centrosomal assembly and maintenance, mitotic spindle proper organization, and ultimately a correct cell division. Recently, HOPS has been described as an important regulator of p53, which acts as modifier, stabilizing p53 half-life and playing a key role in p53 mediating apoptosis after DNA damage. NF-κB is a transcription factor with a central role in many cellular events, including inflammation and apoptosis. Our experiments demonstrate that the transcriptional activity of the p65/RelA NF-κB subunit is regulated by HOPS. Importantly, Hops−/− cells have remarkable alterations of pro-inflammatory responses. Specifically, we found that HOPS enhances NF-κB activation leading to increase transcription of inflammatory mediators, through the reduction of IκBα stability. Notably, this effect is mediated by a direct HOPS binding to the E3 ubiquitin ligase TRAF6, which lessens TRAF6 stability ultimately leading increased IKK complex activation. These findings uncover a previously unidentified function of HOPS/Tmub1 as a novel modulator of TRAF6, regulating inflammatory responses driven by activation of the NF-κB signaling pathway. The comprehension on how HOPS/Tmub1 takes part to the inflammatory processes in vivo and whether this function is important in the control of proliferation and tumorigenesis could establish the basis for the development of novel pharmacological strategies.

Oligomeric Forms of Human Amyloid-Beta(1-42) Inhibit Antigen Presentation.

Genetic, clinical, biochemical and histochemical data indicate a crucial involvement of inflammation in Alzheimer's disease (AD), but harnessing the immune system to cure or prevent AD has so far proven difficult. Clarifying the cellular heterogeneity and signaling pathways associated with the presence of the AD hallmarks beta-amyloid and tau in the brain, would help to identify potential targets for therapy. While much attention has been so far devoted to microglia and their homeostatic phagocytic activity, additional cell types and immune functions might be affected in AD. Beyond microglia localized in the brain parenchyma, additional antigen-presenting cell (APC) types might be affected by beta-amyloid toxicity. Here, we investigated potential immunomodulatory properties of oligomeric species of beta-amyloid-peptide (Aβ) on microglia and putative APCs. We performed a comprehensive characterization of time- and pathology-dependent APC and T-cell alterations in a model of AD-like brain beta-amyloidosis, the APP-PS1-dE9 mouse model. We show that the deposition of first beta-amyloid plaques is accompanied by a significant reduction in MHC class II surface levels on brain APCs. Furthermore, taking advantage of customized in vitro systems and RNAseq, we demonstrate that a preparation containing various forms of oligomeric Aβ1-42 inhibits antigen presentation by altering the transcription of key immune mediators in dendritic cells. These results suggest that, beyond their neurotoxic effects, certain oligomeric Aβ forms can act as immunomodulatory agents on cerebral APCs and interfere with brain antigen presentation. Impaired brain immune surveillance might be one of the factors that facilitate Aβ and tau spreading in AD.

2117-P: BET Bromodomain Inhibitors Mitigate Cytokine-Induced Transcription in ß-Cells via Inhibition of Nf-κB

Type 1 diabetes is an autoimmune disease characterized by chronic inflammation of pancreatic islets, mediated by proinflammatory cytokines, leading to reduced response to glucose and ultimately selective destruction of insulin-producing β-cells. In common with many autoimmune diseases, the exact causes are poorly understood. Although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins suggest an, as yet undetermined, contribution of epigenetic factors. Recent studies have begun to explore the contributions of epigenetic regulatory proteins in onset and progression of autoimmune diabetes. An important class of epigenetic “readers” is the bromodomain and extraterminal domain (BET) family (Brd2, Brd3, Brd4, and Brdt), which has emerged as promising drug targets for a wide variety of diseases. A unique trait of the BET family is that these proteins contain two bromodomains (BD1 and BD2) that afford them the capacity to simultaneously bind two acetylated histones and/or non-histone proteins such as DNA-binding factors to modulate transcription in a cell-type specific manner. Here, we use an insulinoma β-cell line (INS832/13) and primary rat islets to investigate the ability of chemical inhibitors of BET bromodomain proteins to decrease the β-cell response to proinflammatory cytokines. We found that BET inhibitors mitigated cytokine-induced transcription of inflammatory mediators known to damage β-cells. This is mediated through the inhibition of the NF-κB pathway by BET inhibitors. Furthermore, using CRISPR/Cas9 knockout insulinoma β-cell lines of Brd2, Brd3, and Brd4 and BD1 vs. BD2 specific chemical inhibitors, we identified key mechanisms through which this altered transcriptional profile occurs. This work provides important mechanistic insights to disease onset and progression of autoimmune diabetes for which current therapies are ineffective. Disclosure J. Nord: None. S. Wynia-Smith: None. B.C. Smith: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK119359)

Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease.

BackgroundIncreased gene transcription of hypoxia‐induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD).ObjectiveTo characterize hypoxia‐induced profibrotic pathways in naturally occurring CKD in cats.AnimalsTwelve client‐owned cats with CKD and 8 healthy control cats.MethodsIn this prospective, cross‐sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription‐quantitative PCR for characterization of transcript levels of hypoxia‐inducible factor‐1α (HIF1A), matrix metalloproteinases‐2 (MMP2), ‐7 (MMP7), and ‐9 (MMP9), tissue inhibitor of metalloproteinase‐1 (TIMP1), transforming growth factor‐β1 (TGFB1), and vascular endothelial growth factor‐A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys.ResultsKidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P < .001), and lower levels of VEGFA (P = .006) than those from control cats. Transcript levels of MMP7 (P = .05) and TIMP1 (P = .005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P = .002), MMP7 (P = .02), and TIMP1 (P = .02) were positively, whereas those of VEGFA (P = .003) were negatively, associated with histologic score severity.Conclusion and Clinical SignificanceEvaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

Cardiomyoblast caveolin expression: effects of simulated diabetes, α-linolenic acid, and cell signaling pathways.

Caveolins regulate myocardial substrate handling, survival signaling, and stress resistance; however, control of expression is incompletely defined. We test how metabolic features of type 2 diabetes (T2D), and modulation of cell signaling, influence caveolins in H9c2 cardiomyoblasts. Cells were exposed to glucose (25 vs. 5 mM), insulin (100 nM), or palmitate (0.1 mM), individually or combined, and the effects of adenylate cyclase (AC) activation (50 μM forskolin), focal adhesion kinase (FAK) or protein kinase C β2 (PKCβ2) inhibition (1 μM FAK inhibitor 14 or CGP-53353, respectively) or the polyunsaturated fatty acid (PUFA) α-linolenic acid (ALA; 10 μM) were tested. Simulated T2D (elevated glucose + insulin + palmitate) depressed caveolin-1 and -3 without modifying caveolin-2. Caveolin-3 repression was primarily palmitate dependent, whereas high glucose (HG) and insulin independently increased caveolin-3 (while reducing expression when combined). Differential control was evident: baseline caveolin-3 was suppressed by FAK/PKCβ2 and insensitive to AC activities, with baseline caveolin-1 and -2 suppressed by AC and insensitive to FAK/PKCβ2. Forskolin and ALA selectively preserved caveolin-3 in T2D cells, whereas PKCβ2 and FAK inhibition increased caveolin-3 under all conditions. Despite preservation of caveolin-3, ALA did not modify nucleosome content (apoptosis marker) or transcription of proinflammatory mediators in T2D cells. In summary, caveolin-1 and -3 are strongly repressed with simulated T2D, with caveolin-3 particularly sensitive to palmitate; intrinsic PKCβ2 and FAK activities depress caveolin-3 in healthy and stressed cells; ALA and AC activation and PKCβ2 inhibition preserve caveolin-3 under T2D conditions; and caveolin-3 changes with T2D and ALA appear unrelated to inflammatory signaling or extent of apoptosis.

Profibrotic gene transcription in renal tissues from cats with ischemia-induced chronic kidney disease.

To characterize transcription of profibrotic mediators in renal tissues of cats with ischemia-induced chronic kidney disease (CKD). Banked renal tissues from 6 cats with experimentally induced CKD (RI group) and 8 healthy control cats. For cats of the RI group, both kidneys were harvested 6 months after ischemia was induced for 90 minutes in 1 kidney. For control cats, the right kidney was evaluated. All kidney specimens were histologically examined for fibrosis, inflammation, and tubular atrophy. Renal tissue homogenates underwent reverse transcription quantitative PCR assay evaluation to characterize gene transcription of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-β1, and vascular endothelial growth factor A. Gene transcription and histologic lesions were compared among ischemic and contralateral kidneys of the RI group and control kidneys. Ischemic kidneys had greater transcript levels of MMP-7, MMP-9, and transforming growth factor-β1 relative to control kidneys and of MMP-2 relative to contralateral kidneys. Transcription of TIMP-1 was upregulated and that of vascular endothelial growth factor A was downregulated in ischemic and contralateral kidneys relative to control kidneys. Transcription of HIF-1α did not differ among kidney groups. For ischemic kidneys, there were strong positive correlations between transcription of HIF-1α, MMP-2, MMP-7, and TIMP-1 and severity of fibrosis. Transcription of genes involved in profibrotic pathways remained altered in both kidneys 6 months after transient renal ischemia. This suggested that a single unilateral renal insult can have lasting effects on both kidneys.

The route of infection with Leptospira interrogans serovar Copenhageni affects the kinetics of bacterial dissemination and kidney colonization.

The goal of this study was to characterize how natural routes of infection affect the kinetics of pathogenic Leptospira dissemination to blood and kidney. C3H/HeJ mice were sublethally infected with L. interrogans serovar Copenhageni FioCruz L1-130 (Leptospira) through exposure of a dermis wound and through oral and nasal mucosa, in comparison to uninfected mice and to mice infected via standard intraperitoneal inoculation. In striking contrast to oral mucosa inoculation, transdermal and nasal mucosa infections led to weight loss, renal colonization and inflammation, as previously observed for conjunctival and intraperitoneal infections. However, the timing at which Leptospira gained access to blood, as well as Leptospira’ colonization of the kidney and shedding in urine, differed from intraperitoneal infection. Furthermore, a comparative analysis of transcription of pro-inflammatory mediators in kidney and total immunoglobulin isotyping in serum from infected mice, showed increased innate immune response markers (KC, MIP-2, TNF-α) and lower Th1 associated IFN-γ in kidney, as well as lower Th1 associated IgG2a in mice infected through the nasal mucosa as compared to intraperitoneal infection. We conclude that the route of infection affects the timing at which Leptospira gains access to blood for dissemination, as well as the dynamics of colonization and inflammation of the kidney.

NOD Signaling and Cell Death.

Innate immune signaling and programmed cell death are intimately linked, and many signaling pathways can regulate and induce both, transcription of inflammatory mediators or autonomous cell death. The best-characterized examples for these dual outcomes are members of the TNF superfamily, the inflammasome receptors, and the toll-like receptors. Signaling via the intracellular peptidoglycan receptors NOD1 and NOD2, however, does not appear to follow this trend, despite involving signaling proteins, or proteins with domains that are linked to programmed cell death, such as RIP kinases, inhibitors of apoptosis (IAP) proteins or the CARD domains on NOD1/2. To better understand the connections between NOD signaling and cell death induction, we here review the latest findings on the molecular regulation of signaling downstream of the NOD receptors and explore the links between this immune signaling pathway and the regulation of cell death.

2182Sirtuin 5 regulates arterial thrombosis by modulating endothelial plasminogen activator inhibitor-1

Abstract Introduction Arterial thrombosis as a result of plaque rupture or erosion is a crucial event in myocardial infarction and stroke. Oxidative stress and inflammation promote endothelial dysfunction and play a pivotal role in destabilization of the atherosclerotic plaque. Sirtuin 5 (SIRT5) is a member of the sirtuin protein family with function as a NAD+-dependent protein desuccinylase and demalonylase. Being implicated in the regulation of different pathophysiological processes among which production of reactive oxygen species and transcription of inflammatory mediators, SIRT5 plays a role in the development of several cardiovascular diseases such as myocardial infarction and stroke. To date, the possible involvement of SIRT5 as a mediator of arterial thrombosis remains to be investigated. Purpose In this study we investigate the putative role of this protein in arterial thrombosis by using an established in vivo mouse model. The translational value of animal findings as well as the molecular mechanism underlying the observed effect will be investigated also in primary human aortic endothelial cells (HAECs). Methods SIRT5 knockout (KO) as well as SIRT5 transgenic (TG) animals were used for in vivo experiments. HAECs treated with SIRT5 silencing RNA (si-SIRT5) and stimulated with tumor necrosis factor (TNF)-α were used for in vitro assays. Results When compared to WT animals, SIRT5 KO mice display blunted carotid artery thrombus formation as underlined by delayed time to occlusion in a photochemical injury model. Oppositely, in SIRT5 TG mice the formation of an occlusive thrombus is accelerated (Fig 1). Mechanistically, SIRT5 KO and WT animals show no difference in terms of vascular tissue factor (TF) activity, TF concentration in plasma and expression of TF pathway inhibitor (TFPI) in the aorta. In line with the observed reduced thrombogenicity, SIRT5 KO animal express reduced level of the pro-thrombotic plasminogen activator-1 (PAI-1), as assessed by western blot in aorta lysate. Of interest, SIRT5 genetic deletion does not affect platelet aggregation, as assessed by ex-vivo collagen-induced aggregometry. In HAECs, SIRT5-silencing inhibits PAI-1 expression in response to TNF-α. Real-time polymerase chain reaction revealed that inhibition of PAI-1 expression occurs at the mRNA level. This effect is mediated by reduced activation of the MAP kinase Erk 1/2, but not JNK (Fig 1). Conclusions SIRT5 mediates arterial thrombosis by increasing endothelial PAI-1 expression. Hence, SIRT5 may be an effective therapeutic target in the context of atherothrombosis.

Repurposing mosloflavone/5,6,7-trimethoxyflavone-resveratrol hybrids: Discovery of novel p38-α MAPK inhibitors as potent interceptors of macrophage-dependent production of proinflammatory mediators

Herein, we address repurposing hybrids of mosloflavone or 5,6,7-trimethoxyflavone with amide analogs of resveratrol from anticancer leads to novel potent anti-inflammatory chemical entities. To unveil the potent anti-inflammatory molecules, biological evaluations were initiated in LPS-induced RAW 264.7 macrophages at 1 μM concentration. Promising compounds were further evaluated at various concentrations. Multiple proinflammatory mediators were assessed including NO, PGE2, IL-6, TNF-α and IL-1β. Compound 5z inhibited the induced production of NO, PGE2, IL-6, TNF-α and IL-1β at the low 1 μM concentration by 44.76, 35.71, 53.48, 29.39 and 41.02%, respectively. Compound 5z elicited IC50 values as low as 2.11 and 0.98 μM against NO and PGE2 production respectively. Compounds 5q and 5g showed potent submicromolar IC50 values of 0.31 and 0.59 μM respectively against PGE2 production. Reverse docking of compound 5z suggested p38-α MAPK, which is a key signaling molecule within the pathways controlling the transcription of proinflammatory mediators, as the molecular target. Biochemical testing confirmed these compounds as p38-α MAPK inhibitors explaining its potent inhibition of proinflammatory mediators’ production. Collectively, the results presented 5z as a promising compound for further development of anti-inflammatory agents for treatment of macrophages-and/or immune mediated inflammatory diseases.

Exome sequencing in multiple sclerosis families identifies 12 candidate genes and nominates biological pathways for the genesis of disease.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.

SAT-606 Graves' Orbitopathy Treated with Kahaly's Protocol: IV High Dose Glucocorticoids

Graves’ Orbitopathy (GO) is present in approximately 25-50-% of patients with Graves’ hyperthyroidism. Treatment should rely on a thorough assessment of the clinical activity score (CAS), severity of GO and its impact on the patient’s quality of life. Most patients can be managed with non surgical treatments alone, and those requiring surgical intervention often benefit from concurrent medical therapy. We are presenting a case of active and moderate GO successfully treated with high dose glucocorticoids (GCs). 48 yo female presented with history of subclinical hyperthyroidism since age 40 with only complaint of excessive tearing and redness in both eyes for last 1 yr. Physical exam was remarkable for exophthalmos of bilateral eyes with minimal inferior scleral show with no evidence of goiter. Her labs showed TSH of 0.05 (0.27-4.2 microIU/ml), FT4 2.36(0.90-1.7 ng/dL), TPO 502(<9.0 IU/ml), TSI 132(<=122%) and TRAb 12.43(<=1.75IU/L). She was treated with methimazole, currently requiring 2.5 mg daily to maintain euthyroid status. Initially she was treated with artificial tears for 2 weeks followed by tapering dose of oral prednisone for 3 weeks. 3 months later patient presented with double, blurry vision while trying to read. On exam she had exophthalmos, mild lid lag, conjunctival injection, and early pannus formation on left cornea with CAS 3,TSH 1.41,FT4 0.83 and FT3 76.2. Patient was treated with dexamethasone/ neomycin/ polymyxin B eye drops at bedtime with doxycycline 50 mg BID PO for 3 months with improvement in her symptoms and remained symptom free for 2 yrs. 2 yrs later patient presented with bilateral eye pain, redness with tearing, left > right and diplopia with horizontal gaze with CAS of 6. She was again treated with oral prednisone taper over 3 weeks without any improvement. Then she was treated with IV methylprednisolone 500 mg weekly for 6 weeks, followed by 250 mg weekly for another 6 weeks with marked improvement in her symptoms of eye pain, redness of eyelid and conjunctiva and caruncle swelling and mild improvement in eyelid swelling 4 weeks after treatment. Our patient was successfully treated with high dose IV GCs. Corticosteroids activate transcription of anti inflammatory mediators and inhibit the transcription of proinflammatory mediators (ie. cytokines and prostaglandins). IV steroids suppress immune system through genomic pathway and non genomic pathway. IV GCs are more effective and have a lower side effects burden than oral steroids and thus are the preferred mode of administration. Patient should be screened for contraindications to IV GCs, including liver dysfunction, recent viral hepatitis, severe cardiovascular morbidity, uncontrolled HTN and diabetes. High dose IV GCs can be safely used for treatment of active, moderate to severe GO and should be used as firstline treatment in patients with active and moderate to severe GO.

Topical application of cashew gum or chlorhexidine gel reduces overexpression of proinflammatory genes in experimental periodontitis.

This study aimed to explore the effect of topically administering an orabase gel containing cashew gum (CG), a complex polysaccharide from Anacardium occidentale L., on the transcription of important proinflammatory (COX-2, NOS-2, INF-γ, OSCAR, and MYD88) and anti-inflammatory genes (IL-10, IL-4, and TGFβ1) in the gingival tissues of rats with ligature-induced periodontitis, compared to the effect observed upon topically applying a well-known antibiofilm agent (chlorhexidine) under the same experimental conditions. The gene expression profile in the gingival tissues of rats with periodontitis treated with CG did not statistically significantly differ from that observed in the group of animals treated with chlorhexidine. Results showed that CG is able to attenuate general inflammation in the periodontium by reducing the transcription of proinflammatory mediators in a MYD88-independent manner, and not by inducing the expression of anti-inflammatory factors. In conclusion, this study demonstrated that CG and chlorhexidine treatment reduced significantly the gene overexpression (COX-2, NOS-2, INF-γ, OSCAR, and TGFβ1) in the model of ligature-induced periodontitis.