TNF-alpha mRNA Research Articles

Paternal High Fat Diet and Exercise Differentially Regulate Placental Development and Inflammation in a Sex-Specific Manner in C57BL6/J Mice (P19-003-19)

ObjectivesWe have shown that increases in T2D risk in male offspring when the father consumes a high-fat (HF) diet can be normalized when the father also exercises during preconception, and that this protection may occur by epigenetic increases in insulin signaling within offspring skeletal muscle. In our current study, we investigated to determine how paternal HF diet and exercise conditions alter sperm miRNA, fetal weight and placental inflammation. MethodsThree-week old male C57BL/6 mice were fed a normal-fat (NF) diet (16% fat) or a HF diet (45% fat) and assigned to either voluntary wheel running exercise or cage activity for 3 months prior to mating with NF diet fed dams. Sperm samples were collected to determine changes in miRNA that may account for the enhanced offspring skeletal muscle responses that helped normalize paternal HF-induced glucose intolerance. Placentae were collected to determine whether changes in sperm miRNA expression differed by amount of placental inflammation. ResultsSperm expression of miRNA 193b increased with paternal HF and exercise. In F1 males, placental and fetal weight decreased with HF diet while, in F1 female, paternal HF and exercise had no effect on placental and fetal weights. Paternal HF diet decreased placental IL-6 and TNF-alpha mRNA expression in F1 females, while no effects were observed in F1 male placenta. ConclusionsTaken together these data suggest that paternal HF diet has a greater impact on placental development of male fetuses while paternal exercise has greater impact on placental inflammation of female fetuses. For both female and male fetuses, these paternal influences are mediated via sperm miRNA 193b. miR-193b is involved in regulation of the cell cycle and adipogenesis but may have additional functions. Thus, the exact role of sperm miRNA 193b in sex-specific epigenetic transmission of paternal HF diet and exercise on placental and fetal development needs further evaluation. Funding SourcesUSDA Agricultural Research Service Project #3062-51000-052-00D.

Probiotic Supplementation Regulates Placental and Fetal Development in C57BL6/J Mice (P20-008-19)

ObjectivesMaternal high-fat (HF) diet predisposes offspring to metabolic syndrome, in part, by inducing alterations during placental development resulting in abnormal fetal development and offspring birth weight. We previously reported in a rat model that a maternal malnutrition diet increases fetal inflammation, and placental vascularization, and decreases offspring birth weight followed by adult-onset obesity. In the current study, we investigated when during gestational development a maternal HF diet induces changes in placental weight, lipid content, and inflammation. We further investigated whether probiotic supplementation offsets HF-diet induced adverse outcomes. MethodsTwo-month old female C57BL/6 mice were fed diets of 16% fat (normal-fat, NF) or 45% fat (high-fat, HF) for 8 weeks followed by 4 weeks of probiotic (Bifidobacterium animalis subsp. lactis, BB-12) supplementation. Fetuses and placentae were examined mid- (D12) and late- (D19) gestation ResultsPlacental length, width, and weight as well as fetal weight were decreased in the HF group at D12. Probiotic supplementation reversed the HF diet-induced reduction in placental weight at D12. These beneficial effects of probiotic supplementation were absent at D19 as fetal weights from all HF diet fed groups weighed less regardless of probiotic supplementation. Probiotic supplementation reduced placental TNF-alpha mRNA at D12 and IL-10 mRNA at D19. Triacylglyceride concentration was increased at D19 by HF diet, in part, by the preceding increases in lipoprotein lipase (LPL) and plasma membrane-associated fatty-acid binding protein (FABPpm) mRNA expression at D12. ConclusionsThese findings suggest that maternal HF diet alters placental size and weight as well as fetal weight and that probiotic supplementation renders a protective effect against these changes in mid-gestation. Future studies are needed to determine whether the protective mechanism of probiotic supplementation in mid-gestation has beneficial effects on offspring developmental programming. Funding SourcesUSDA Agricultural Research Service Project #3062-51000-052-00D.

Lack of association between 4 key TNF-alpha promoter polymorphisms and hepatitis C virus infection in a population of Egyptian patients

Introduction: With 170 million chronic hepatitis C virus (HCV) cases worldwide, HCV is considered a major life-threatening pathogen. HCV is a crucial causative of liver cirrhosis and hepatocellular carcinoma. Tumor necrosis factor-alpha (TNF-alpha) is thought to be a mediator in the development of viral hepatitis. Because HCV is epidemic in Egypt, this study aimed to characterize the distribution of TNF-alpha gene promoter polymorphisms and their relation to TNF-alpha expression in HCV patients.
 Methods: Four promoter polymorphisms; −1031T/C, −863C/A, −857C/T, and −308G/A, were studied by restriction fragment length polymorphism in a population of Egyptian HCV patients.
 Results: Compared to healthy subjects, none of these polymorphisms were associated with HCV infection. The wild-type −1031T, −863A, −857C, and −308G alleles were highly prevalent in the studied population. Sequencing the promoter region spanning the four studied polymorphisms in some subjects did not reveal any difference in the nucleotide variance pattern, compared with the TNF-alpha reference sequence. Relative TNF-alpha mRNA expressions in HCV patients and healthy subjects were statistically indifferent.
 Conclusion: Since previous studies confirmed an increase in TNF-alpha level in case of viral infections, this study focuses on mechanisms of post-transcriptional and posttranslational modifications of TNF-alpha gene in HCV patients, which decode the genetic factors linked to HCV infection and severity.

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp ( Ctenopharyngodon idella ) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean beta-conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g beta-conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary beta-conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS-generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid-intestine, dietary beta-conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase-3, caspase-8 and caspase-9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease-activating factor-1 [Apaf1] and Bcl-2-associated X protein [BAX]) and mitogen-activated protein kinase (MAPK)-related signal molecules (Jun N-terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho-p38 MAPK. Moreover, in the distal intestine, dietary beta-conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase-3, caspase-8 and caspase-9 activities, death ligand (TNF-alpha) mRNA expression level, and p38 MAPK and phospho-p38 MAPK protein levels. In summary, dietary soybean beta-conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS-mediated MAPK signalling.

Chemical Composition and Biological Activities of Lebanese Pentapera Plant.

BackgroundDue to their chemical constituents and biological properties, plants have long been used to control life-threatening diseases. The flora of Lebanon includes many plants that have already been demonstrated to have medicinal value, and other species, such as Pentapera sicula libanotica, that are yet to be characterized. The present study characterized the chemical composition, anti-oxidant, anti-inflammatory, and anti-proliferative potential of aqueous, ethanol, and methanol extracts derived from the leaves of the Lebanese Pentapera plant.Material/MethodsHigh-performance liquid chromatography (HPLC) was used to determine the chemical composition. Gas chromatography (GC) coupled with mass spectrometry (MS) was applied to determine the content of essential oil. DPPH radical scavenging assay was performed to evaluate the anti-oxidant potential. The anti-inflammatory potential was assessed using quantitative real-time PCR (qRT-PCR) by measuring TNF-α, IL-6, and CCL4 mRNA levels, and we assessed Cox-2 and iNOS proteins levels using Western blot (WB) analysis. MTT assay was carried out to determine the anti-proliferative potential.ResultsWe identified, mainly in the alcoholic (methanol and ethanol) extracts, distinct bioactive compounds with pharmacological relevance. In parallel, with their phytochemical content, these 2 extracts showed significant anti-oxidant, anti-inflammatory and anti-proliferative capacities.ConclusionPentapera sicula libanotica appears to be a promising pharmacological tool.

Abstract # 3078 Contributions of astrocytic IL-1R1 signaling in blast-induced traumatic brain injury

Traumatic Brain Injury (TBI) is a leading cause of disability in the United States with an annual economic cost of $76.5 billion. TBI results in profound astrogliosis, the generation of neuropsychiatric complications and an increased incidence of seizures. TBI increases CNS proinflammatory cytokine expression and recent reports have shown that interleukin-1alpha (IL-1alpha) signaling is a requirement for the generation of neurotoxic astrocytes. We hypothesize that IL-1alpha and interleukin-1beta (IL-1beta) signal through functional type 1 interleukin-1 receptors (IL-1R1’s) on astrocytes post-injury thereby contributing to negative functional outcomes post-TBI. Using a murine model of blast-induced TBI (bTBI), we detected increased mRNA expression and immunoreactivity of the astrocytic marker glial fibrillary acidic protein (GFAP) in the ipsilateral cortex as compared to sham animals, signifying the presence of reactive astrocytes. Increases in il-1beta, il-1alpha and tnf-alpha mRNA were detected post-TBI, indicating increased CNS production of proinflammatory cytokines post-TBI. Mice exposed to bTBI exhibited increased impulsive-like behaviors in the elevated plus maze (EPM) and an increase in the propensity for kainic acid (KA)-induced seizures. Future studies will utilize murine models affording the conditional restoration and elimination of IL-1R1 (Robson et al. 2016) in astrocytes in conjunction with bTBI to study neuroinflammatory profiles and functional outcomes post-injury. Understanding the molecular and temporal requirements for the negative consequences of IL-1R1 signaling post-injury may lead to therapeutic options for those with TBI.

Binding Interface of Gapdh to the Au Rich Elements from TNF-Alpha mRNA Revealed by Hydrogen Deuterium Exchange Coupled with Mass Spectrometry

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous protein primarily known for its glycolytic function. More recent studies have revealed GAPDH as a multifaceted, multifunctional protein involved in various cellular processes including iron homeostasis, heme transport, apoptosis, tumerogenesis, oxidative stress response, chromatin structure among many others. As part of its multifunctional purpose, GAPDH has been shown to bind to various cellular RNAs and has been classified as a non-canonical RNA binding protein. In particular, it has been shown to bind AU-rich element of mRNA transcripts and consequently affecting translation. However, the detailed structural characterization of the RNA bound complex has remained elusive. The RNA binding site remains unknown thereby restricting potential therapeutic strategies. Our previous studies established that GAPDH binds the AU rich element of TNF-α mRNA transcript via a two-step model. In addition, we have shown that a T229K mutant of GAPDH displayed altered RNA and NAD+ binding properties, potentially signaling to a linkage between RNA binding and the cofactor. Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) revealed that the T229K mutant induced conformational changes into distal grooves along the P-axis of the tetrameric protein. In this study, we have mapped the binding interfaces of three GAPDH constructs displaying varying NAD+ binding properties with the AU rich element from TNF-α mRNA using HDX-MS. A largely common pattern of localized protection from exchange pointed to the grooves along the P-axis as the RNA binding interfaces, as hypothesized. Additionally, some variations in HDX protection point to potential conformational basis for the linkage with NAD+ binding.

Isoprenoid Depletion Increases Cytokine mRNA Levels in Primary Human Monocytes

Mevalonate Kinase Deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase (MVK) gene, which produces an enzyme responsible for the production of isoprenoids. Previous research in various model systems has demonstrated that depletion of isoprenoids leads to inflammasome activation and increased levels of secreted IL‐1B on a post‐translational level. However, patient data indicates that MKD is a multi‐cytokine disease with increased plasma levels of cytokines such as TNF‐alpha, IL‐6, and IFN‐gamma. In order to study the mechansims responsible for these changes, the mevalonate pathway was inhibited with lovastatin in peripheral blood mononulcear cells (PBMCs) and monocytes isolated from the blood of healthy donors followed by stimulation with lipopolysaccharide (LPS) to induce an inflammatory response. Lovastatin treatment resulted in significantly increased levels of TNF‐alpha, IL‐6, IL‐12p40, and IFN‐gamma mRNA in both PBMCs and monocytes following LPS stimulation as compared to control cells. Lovastatin treatment did not alter IL‐1B, CD14, or IL‐18 mRNA levels either before or after LPS stimulation. Addition of mevalonate to the cells treated with lovastatin prevented the increased cytokine mRNA levels indicating that these changes are due to inhibition of the mevalonate pathway. These results indicate that isoprenoid depletion alters the expression of multiple cytokines on the mRNA level in primary human monocytes, which can be used as a model system ex vivo to study these changes.Support or Funding InformationDickinson College Faculty Start‐up and Research & Development Committee FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy

Experimental aristolochic acid nephropathy is characterized by transient acute proximal tubule necrosis and inflammatory cell infiltrates followed by interstitial fibrosis and tubular atrophy. The respective role of T-cell subpopulations has never been studied in the acute phase of the mouse model, and was heretofore exclusively investigated by the use of several depletion protocols. As compared to mice injected with aristolochic acids alone, more severe acute kidney injury was observed after CD4+ or CD8+ T-cells depletion. TNF-alpha and MCP-1 mRNA renal expressions were also increased. In contrast, regulatory T-cells depletion did not modify the severity of the aristolochic acids induced acute kidney injury, suggesting an independent mechanism. Aristolochic acids nephropathy was also associated with an increased proportion of myeloid CD11bhighF4/80mid and a decreased proportion of their counterpart CD11blowF4/80high population. After CD4+ T-cell depletion the increase in the CD11bhighF4/80mid population was even higher whereas the decrease in the CD11blowF4/80high population was more marked after CD8+ T cells depletion. Our results suggest that CD4+ and CD8+ T-cells provide protection against AA-induced acute tubular necrosis. Interestingly, T-cell depletion was associated with an imbalance of the CD11bhighF4/80mid and CD11blowF4/80high populations.

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

Significant Cytokine mRNA Expression Changes Immediately after Initiation of Cardiopulmonary Resuscitation.

Introduction The purpose of this study was to evaluate immediate immunological changes following cardiopulmonary resuscitation (CPR). mRNA expression levels of selected immunomodulatory cytokines in out-of-hospital cardiac arrest (OHCA) survivors were detected and correlated to clinical parameter. Methods OHCA survivors with sustained unconsciousness after return of spontaneous circulation (ROSC) were included. PAXgene whole blood samples were drawn immediately after initiation of CPR and subsequently after 6 h, 12 h, 24 h, 48 h, and 72 h. TNF-alpha, IL-8, IL-10, and IL-1ra mRNA levels were quantified by RT-qPCR and compared to multiple organ failure, 30-day survival, and the induction of therapeutic hypothermia (TH). Results 25 patients (63 ± 15 years) were enrolled presenting a characteristic time-dependent cytokine profile in the early postresuscitation period. High initial TNF-alpha and IL-8 mRNA levels were followed by a significant decrease. IL-1ra mRNA levels significantly increased beginning after 6 h. Nonsurvivors showed significantly higher IL-8 mRNA levels immediately after CPR. TH induced significantly higher IL-1ra mRNA levels compared to normothermia. Conclusion Significant mRNA cytokine expression changes are already detectable immediately after initiation of CPR. These expressional changes are significantly different depending on 30-day survival. TH seems to attenuate proinflammatory immune reaction by a significant increase of IL-1ra mRNA levels. This trial is registered with DRKS00012940.

Protective effects arising from inhibition of TRPC1/5 ion channel permeation in hypercholesterolaemic mice

Transient Receptor Potential Canonical (TRPC) proteins assemble to form calcium-permeable non-selective cationic channels. We previously showed that TRPC1 and TRPC5 are expressed by adipocytes and that their inhibition increased the concentration of circulating adiponectin, which is an important anti-inflammatory and insulin-sensitising adipokine (Sukumar et al 2012, Circ Res 111, 191). The aim of this study was to investigate if the channels are relevant in hypercholesterolaemia. ApoE-knockout mice with conditional global expression of dominant-negative ion pore mutant TRPC5 (DNT5) were used to suppress ion permeation in the channels in vivo. Mice were fed with western-style diet and male litter-mates were compared with and without DNT5. Mice expressing DNT5 had significantly lower body weight compared to controls after 6 weeks and 12 weeks of treatment. Histological analysis showed significant reduction in adipocyte size. Quantitative RT-PC R analysis of adipose tissue showed that adiponectin mRNA was more abundant in the DNT5 group. By contrast, TNF-alpha and interleukin-6 mRNA were less abundant. No obvious adverse effects of DNT5 were evident. The data suggest that ion flux through heteromeric TRPC1/TRPC5 channels is a driver for weight gain, suppression of adiponectin gene expression and increased inflammation in hypercholesterolaemic mice. Therefore, inhibitors of the ion channels could be a strategy for protecting against obesity, insulin-resistance and inflammation. Supported by the Medical Research Council

Doxorubicin inhibits muscle inflammation after eccentric exercise.

BackgroundDoxorubicin, a widely used anti‐tumour drug, is known to cause muscle loss in cancer patients.MethodsFollowing an acute dose of doxorubicin injection (2.5 mg/kg per body weight), we examined macrophage distribution in rat soleus muscle challenged by eccentric exercise (downhill running). Long‐term doxorubicin treatment (one injection every 3 days) on muscle mass and survival were also determined.ResultsUnder non‐exercised condition, increased tumour necrosis factor (TNF)‐alpha mRNA and decreased IL‐10 mRNA were observed in soleus muscle of doxorubicin‐treated rats, compared with saline‐treated control rats. However, increases in inflammation score (leukocyte infiltration), nitrotyrosine level, and M1 macrophage (CD68+) invasion in exercised soleus muscle were absent in doxorubicin‐treated rats, whereas increased M2 macrophage (CD163+) localization in exercised muscle was less affected by doxorubicin. Despites coenzyme Q (Q10) supplementation significantly elevated TNF‐alpha mRNA, nitrotyrosine, and anti‐oxidant gamma‐glutamylcysteine synthetase (GCS) levels in non‐exercised soleus muscle, these pro‐inflammatory responses were also abolished in doxorubicin‐treated rats. Results from long‐term doxorubicin treatment show a significant muscle loss followed by an accelerated death, which cannot be reversed by Q10 supplementation.Conclusions(i) Doxorubicin impairs inflammation mechanism by depleting M1 macrophage in exercised skeletal muscle; (ii) Muscle loss and accelerated death during prolonged doxorubicin treatment cannot be reversed by Q10 supplementation.

Abstract # 1824 Healthy smokers’ peripheral blood lymphocytes exhibit differential inflammatory gene expression to glucocorticoid and lipopolysaccharide exposure

Cigarette smoking is linked to elevated inflammation and development of chronic inflammation-related conditions such as asthma, autoimmune diseases, and chronic bronchitis. Among these patient populations, those who smoke often have poorer symptom management/relief via usual treatments, including glucocorticoids, suggesting glucocorticoid resistance. Even less is understood about the early changes in smokers’ immune function especially those who have not yet developed a clinical disease. Thus, we recruited healthy smokers (e.g., no clinical diagnoses or current medication use) and age-, sex-, and BMI-matched never smokers to examine glucocorticoid sensitivity. Following a 12-h fast, 47 adults (30.5 +/−8.1 yrs; 27 males) provided a blood sample and answered several psychosocial questionnaires. Using a repeated-measures ANOVA, dexamethasone significantly reduced all peripheral blood lymphocytes’ (PBLs) IL-6, TNF-alpha, and IFN-gamma mRNA expression response to lipopolysaccharide (LPS; p p p = .07) compared to smokers. Thus, our data suggest that even prior to chronic disease development and elevated systemic inflammation (i.e., CRP levels

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model.

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

Abstract # 1732 Exposure to a social stressor results in changes to microbial-produced short chain fatty acids and increased TNF-alpha production by colonic epithelial cells

There is increasing evidence that stressor exposure results in significant community-wide changes in the composition of the colonic microbiota. However, whether stressor exposure also changes the metabolic activities of the microbiota is not yet known. Microbes in the colon produce short chain fatty acids (SCFA), primarily butyric, acetic, and propionic acids. These SCFAs have multiple effects on host physiology, and have been proposed to play an important part in the pathogenesis of intestinal diseases. To determine whether stressor exposure would change the levels of microbial-produced SCFAs, mice were exposed to repeated social defeat for 2 h a day on 6 consecutive days. Colonic contents were then removed and levels of butyric, acetic, and propionic acids measured using gas chromatography-mass spectrometry. In addition, SCFA receptor expression was measured and cytokine mRNA was measured in colonic epithelial cells. Exposure to the SDR Stressor resulted in significant reductions in butyric and acetic acid levels in the colon. Interestingly, propionic acid levels were significantly increased in stressor-exposed mice. In addition, exposure to the stressor significantly increased TNF-alpha mRNA in colonic epithelial cells. These data indicate that levels of microbial-produced metabolites are significantly changed in the colon during stressor exposure. Because SCFAs strongly affect cytokine production by colonic epithelial cells, it is possible that stressor-induced changes in SCFAs exacerbate colonic inflammation. Supported by NIH Grant AT006552-01A1.

Abstract 614: TRAIL Deletion in Mice Promotes Vascular Insulin Resistance

TNF-related apoptosis-inducing ligand (TRAIL) is protective in cardiovascular disease and diabetes; whether it regulates insulin resistance and inflammation in the vessel wall is unknown. Wildtype and Trail -/- mice were placed on a high fat diet (HFD) for 12 w. Plasma chemistries were assessed; glucose and insulin tolerance tests performed. Histological GLUT4 expression in skeletal muscle was assessed. Aortic inflammatory marker expression and downstream insulin signaling was also measured; myography was performed. In response to a HFD for 12 w, wildtype mice had increased aortic IL-1beta, MCP-1, IL-6, TNF-alpha and TRAIL mRNA, with reduced circulating TRAIL levels. Compared to 12 w HFD-fed wildtype mice, Trail -/- mice had significantly increased plasma glucose, insulin, and cholesterol levels. Insulin intolerance was worse with TRAIL deletion, with reduced GLUT4 expression and glucose uptake in skeletal muscle. In response to insulin, Trail -/- aortas displayed impaired vasorelaxation and reduced aortic p-Akt expression. Importantly, aortic insulin resistance was associated with > 20-fold increased aortic mRNA expression for IL-1beta, IL-6, and TNF-alpha. We show that TRAIL protects against insulin resistance and type-2 diabetes, in part by regulating downstream insulin signals and inflammation in the vessel wall. These findings suggest that TRAIL may offer a novel therapeutic solution to combat inflammation and insulin resistance in diabetic vascular diseases.

2015 AHA Late-Breaking Basic Science Abstracts.

### 23060 ### Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent Cohorts Antoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupon1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autonoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, Spain Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a\_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a\_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). …

Glia Maturation Factor-Gamma Regulates the Intracellular Growth of Salmonella Via Modulation of Ferroportin in Murine Macrophages

Salmonella is an intracellular bacterial pathogen that replicates within membrane-bound compartment and alters host iron metabolism for its own survival. Persistent survival and replication within phagocytes is central to the pathogenesis of Salmonella infections. Macrophages play a critical role in regulating iron metabolism for securing body iron sufficiency and controlling the availability of iron for intracellular proliferation of pathogens. However, the relationship of Salmonella-induced changes of macrophage iron metabolism to the survival and replication mechanism of this pathogen within macrophages remains poorly understood. Thus, it is critical to identify the host factors involved in the intracellular survival and replication of Salmonella in order to design more-efficient antimicrobial therapeutics. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously demonstrated that GMFG negatively regulate TLR4-induced proinflammatory signaling, but its function in macrophage response to intracellular bacteria infection remains unclear. In this study, we investigated the role of GMFG in Salmonella-infected murine macrophages by using small-interfering RNA (RNAi) techniques to knockdown GMFG. We found that knockdown of GMFG significantly enhanced the numbers of intracellular Salmonella growth (>3-fold, p< 0.008) at 24 hr postinfection compared with control siRNA transfected Raw264.7 macrophages. However, there was no significant difference in growth numbers of bacteria observed at 4 hr postinfection, indicating that GMFG does not influence bacterial phagocytosis. Immunofluorescence microscopy also revealed an accumulation of Salmonella, in GMFG knockdown macrophages at 24 hr postinfection. Knockdown of GMFG results in marked decreased the iron exporter ferroportin protein levels and increased iron storage ferritin-L protein levels in Raw264.7 macrophages. Further, the intracellular iron content was elevated in GMFG-knockdown macrophages compared with control macrophages (1.9-fold, p< 0.05). These observations indicate that regulatory impact of GMFG in Salmonella intracellular growth may be through modulation of macrophage iron metabolism. Moreover, consistent with previous studies, we found that Raw264.7 macrophage infections with Salmonella increase the expression of the iron transporter ferroportin and ferritin, indicating this is the host defense strategy against infection with intracellular microbes by limiting their access to iron. Although there was no marked altered in GMFG protein level after 24hr infection with Salmonella, GMFG knockdown macrophage infected with Salmonella displayed increased the intracellular iron content and iron storage protein ferritin compared with control macrophages. Further analysis of cytokines expression in Salmonella-infected GMFG-knockdown macrophage revealed enhanced the proinflammatory TNF-alpha mRNA (1.86-fold, p<0.05) and anti-inflammatory IL-10 mRNA (2.2-fold, p< 0.0) at 24 hr postinfection, which is paralleled with increased intracellular Salmonella replication. Our results suggest that GMFG modulation of crucial pathways of macrophage iron metabolism and immune function in murine macrophages infected with Salmonella. DisclosuresNo relevant conflicts of interest to declare.