GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

PPAR-gamma agonists, mainly 15d-PGJ(2), reduce eosinophil recruitment following allergen challenge

We evaluate the immunomodulation of Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists 15d-PGJ(2) and rosiglitazone (RGZ) in a model of chronic eosinophilia. 15d-PGJ(2) and RGZ significantly reduce eosinophil migration into the peritoneal cavity and down-regulate the eosinopoiesis. The synthesis of IL-5 was decreased after the treatment with 15d-PGJ(2) and RGZ corroborating with the eosinophil migration inhibition. However, IgE was decreased only after the administration of 15d-PGJ(2) in part due to B-cell inhibition. We also observed a decrease in the synthesis of IL-33, IL-17 and IL-23, suggesting that besides the modulation of Th2 pattern, there is a modulation via IL-23 and IL-17 suggesting a role of these cytokines in the eosinophil recruitment. In fact IL-17(-1-) mice failed to develop an eosinophilic response. Altogether, the results showed that PPAR-gamma agonists mainly 15d-PGJ(2), have therapeutic efficacy in eosinophil-induced diseases with an alternative mechanism of control, via IL-23/IL-17 and IL-33. (C) 2011 Elsevier Inc. All rights reserved.

Endurance exercise training increases APPL1 expression and improves insulin signaling in the hepatic tissue of diet-induced obese mice, independently of weight loss

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1a), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3 beta) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1a association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3 beta phosphorylation levels and glycogen content at 24?h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss. J. Cell. Physiol. 227: 29172926, 2012. (C) 2011 Wiley Periodicals, Inc.

Major involvement of mTOR in the PPAR gamma-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)gamma agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPAR gamma agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPAR gamma activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPAR gamma activation.-Blanchard, P-G., W. T. Festuccia, V. P. Houde, P. St-Pierre, S. Brule, V. Turcotte, M. Cote, K. Bellmann, A. Marette, and Y. Deshaies. Major involvement of mTOR in the PPAR gamma-induced stimulation of adipose tissue lipid uptake and fat accretion. J. Lipid Res. 2012. 53: 1117-1125.

Acute hypoxia induces hypertriglyceridemia by decreasing plasma triglyceride clearance in mice

Jun JC, Shin MK, Yao Q, Bevans-Fonti S, Poole J, Drager LF, Polotsky VY. Acute hypoxia induces hypertriglyceridemia by decreasing plasma triglyceride clearance in mice. Am J Physiol Endocrinol Metab 303: E377-E388, 2012. First published May 22, 2012; doi:10.1152/ajpendo.00641.2011.-Obstructive sleep apnea (OSA) induces intermittent hypoxia (IH) during sleep and is associated with elevated triglycerides (TG). We previously demonstrated that mice exposed to chronic IH develop elevated TG. We now hypothesize that a single exposure to acute hypoxia also increases TG due to the stimulation of free fatty acid (FFA) mobilization from white adipose tissue (WAT), resulting in increased hepatic TG synthesis and secretion. Male C57BL6/J mice were exposed to FiO(2) = 0.21, 0.17, 0.14, 0.10, or 0.07 for 6 h followed by assessment of plasma and liver TG, glucose, FFA, ketones, glycerol, and catecholamines. Hypoxia dose-dependently increased plasma TG, with levels peaking at FiO(2) = 0.07. Hepatic TG levels also increased with hypoxia, peaking at FiO(2) = 0.10. Plasma catecholamines also increased inversely with FiO(2). Plasma ketones, glycerol, and FFA levels were more variable, with different degrees of hypoxia inducing WAT lipolysis and ketosis. FiO(2) = 0.10 exposure stimulated WAT lipolysis but decreased the rate of hepatic TG secretion. This degree of hypoxia rapidly and reversibly delayed TG clearance while decreasing [H-3]triolein-labeled Intralipid uptake in brown adipose tissue and WAT. Hypoxia decreased adipose tissue lipoprotein lipase (LPL) activity in brown adipose tissue and WAT. In addition, hypoxia decreased the transcription of LPL, peroxisome proliferator-activated receptor-gamma, and fatty acid transporter CD36. We conclude that acute hypoxia increases plasma TG due to decreased tissue uptake, not increased hepatic TG secretion.

Toll-like receptor 9 activation in neutrophils impairs chemotaxis and reduces sepsis outcome

Objectives: To investigate the role of toll-like receptor 9 on sepsis-induced failure of neutrophil recruitment to the site of infection. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Model of polymicrobial sepsis induced by cecal ligation and puncture in wild-type and toll-like receptor 9-deficient mice. Measurements and Main Results: Toll-like receptor 9-deficient mice with cecal ligation and puncture-induced severe sepsis did not demonstrate failure of neutrophil migration and consequently had a low systemic inflammatory response and a high survival rate. Upon investigating the mechanism by which toll-like receptor 9-deficiency prevents the failure of neutrophil migration, it was found that neutrophils derived from toll-like receptor 9-deficient mice with cecal ligation and puncture induced severe sepsis expressed high levels of chemokine C-X-C motif receptor 2 (CXCR2) and had reduced induction of G-protein-coupled receptor kinase 2. Conclusions: These findings suggest that the poor outcome of severe sepsis is associated with toll-like receptor 9 activation in neutrophils, which triggers G-protein-coupled receptor kinase 2 expression and CXCR2 downregulation. These events account for the reduction of neutrophil migration to the site of infection, with consequent spreading of the infection, onset of the systemic inflammatory response, and a decrease in survival. (Crit Care Med 2012; 40:2631-2637)

Ligand- and Structure-Based Drug Design Strategies and PPAR delta/alpha Selectivity

Peroxisome-proliferator-activated receptors are a class of nuclear receptors with three subtypes: a, ? and d. Their main function is regulating gene transcription related to lipid and carbohydrate metabolism. Currently, there are no peroxisome-proliferator-activated receptors d drugs being marketed. In this work, we studied a data set of 70 compounds with a and d activity. Three partial least square models were created, and molecular docking studies were performed to understand the main reasons for peroxisome-proliferator-activated receptors d selectivity. The obtained results showed that some molecular descriptors (log P, hydration energy, steric and polar properties) are related to the main interactions that can direct ligands to a particular peroxisome-proliferator-activated receptors subtype.

Identification of 2 Novel ANTXR2 Mutations in Patients With Hyaline Fibromatosis Syndrome and Proposal of a Modified Grading System

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are rare, autosomal recessive disorders of the connective tissue caused by mutations in the gene encoding the anthrax toxin receptor 2 protein (ANTXR2) located on chromosome 4q21. Characteristically, these conditions present with overlapping clinical features, such as nodules and/or pearly papules, gingival hyperplasia, flexion contractures of the joints, and osteolytic bone defects. The present report describes a pair of sibs and three other JHF/ISH patients whose diagnoses were based on typical clinical manifestations and confirmed by histopathologic analyses and/or molecular analysis. A comparison of ISH and JHF, additional thoughts about new terminology (hyaline fibromatosis syndrome) and a modified grading system are also included. (C) 2012 Wiley Periodicals, Inc.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

L-Carnitine induces recovery of liver lipid metabolism in cancer cachexia

Cancer cachexia causes metabolic alterations with a marked effect on hepatic lipid metabolism. l-Carnitine modulates lipid metabolism and its supplementation has been proposed as a therapeutic strategy in many diseases. In the present study, the effects of l-carnitine supplementation on gene expression and on liver lipid metabolism-related proteins was investigated in cachectic tumour-bearing rats. Wistar rats were assigned to receive 1 g/kg of l-carnitine or saline. After 14 days, supplemented and control animals were assigned to a control (N), control supplemented with l-carnitine (CN), tumour-bearing Walker 256 carcinosarcoma (TB) and tumour-bearing supplemented with l-carnitine (CTB) group. The mRNA expression of carnitine palmitoyltransferase I and II (CPT I and II), microsomal triglyceride transfer protein (MTP), liver fatty acid-binding protein (L-FABP), fatty acid translocase (FAT/CD36), peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and organic cation transporter 2 (OCTN2) was assessed, and the maximal activity of CPT I and II in the liver measured, along with plasma and liver triacylglycerol content. The gene expression of MTP, and CPT I catalytic activity were reduced in TB, who also showed increased liver (150%) and plasma (3.3-fold) triacylglycerol content. l-Carnitine supplementation was able to restore these parameters back to control values (p < 0.05). These data show that l-carnitine preserves hepatic lipid metabolism in tumour-bearing animals, suggesting its supplementation to be of potential interest in cachexia.

Leukotriene B4-loaded microspheres: a new therapeutic strategy to modulate cell activation

Abstract Background Leukotriene B4 (LTB4) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB4 released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB4-loaded MS. Results In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB4-loaded MS also increase peroxisome proliferator-activated receptor-α (PPARα) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS. Conclusion LTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

Effect of Echium oil compared with marine oils on lipid profile and inhibition of hepatic steatosis in LDLr knockout mice

Abstract Background In an effort to identify new alternatives for long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) supplementation, the effect of three sources of omega 3 fatty acids (algae, fish and Echium oils) on lipid profile and inflammation biomarkers was evaluated in LDL receptor knockout mice. Methods The animals received a high fat diet and were supplemented by gavage with an emulsion containing water (CON), docosahexaenoic acid (DHA, 42.89%) from algae oil (ALG), eicosapentaenoic acid (EPA, 19.97%) plus DHA (11.51%) from fish oil (FIS), and alpha-linolenic acid (ALA, 26.75%) plus stearidonic acid (SDA, 11.13%) from Echium oil (ECH) for 4 weeks. Results Animals supplemented with Echium oil presented lower cholesterol total and triacylglycerol concentrations than control group (CON) and lower VLDL than all of the other groups, constituting the best lipoprotein profile observed in our study. Moreover, the Echium oil attenuated the hepatic steatosis caused by the high fat diet. However, in contrast to the marine oils, Echium oil did not affect the levels of transcription factors involved in lipid metabolism, such as Peroxisome Proliferator Activated Receptor α (PPAR α) and Liver X Receptor α (LXR α), suggesting that it exerts its beneficial effects by a mechanism other than those observed to EPA and DHA. Echium oil also reduced N-6/N-3 FA ratio in hepatic tissue, which can have been responsible for the attenuation of steatosis hepatic observed in ECH group. None of the supplemented oils reduced the inflammation biomarkers. Conclusion Our results suggest that Echium oil represents an alternative as natural ingredient to be applied in functional foods to reduce cardiovascular disease risk factors.

Immune receptors and adhesion molecules in human pulmonary leptospirosis

Pulmonary involvement in leptospirosis has been increasingly reported in the last 20 years, being related to the severity and mortality of the disease. The pathogenesis of pulmonary hemorrhage in leptospirosis is not understood. Lung endothelial cells have been proposed as targets in the pathogenesis of lung involvement in leptospirosis through the activation of Toll-like receptor 2 or the complement system, which stimulates the release of cytokines that lead to the activation of adhesion molecules. The aim of this study was to investigate the involvement of immune pathways and of the intercellular and vascular cell adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule, respectively) in the lungs of patients with pulmonary involvement of leptospirosis. We studied the lungs of 18 patients who died of leptospirosis and compared them with 2 groups of controls: normal and noninfectious hemorrhagic lungs. Using immunohistochemistry and image analysis, we quantified the expression of the C3a anaphylatoxin receptor, intercellular adhesion molecule, vascular cell adhesion molecule, and Toll-like receptor 2 in small pulmonary vessels and in the alveolar septa. There was an increased expression of intercellular adhesion molecule (P <.03) and C3a anaphylatoxin receptor (P <.008) in alveolar septa in the leptospirosis group compared with the normal and hemorrhagic controls. In the vessels of the leptospirosis group, there was an increased expression of intercellular adhesion molecule (P=.004), vascular cell adhesion molecule (P=.030), and Toll-like receptor 2 (P=.042) compared with the normal group. Vascular cell adhesion molecule expression in vessels was higher in the leptospirosis group compared with the hemorrhagic group (P=.015). Our results indicate that immune receptors and adhesion molecules participate in the phenomena leading to pulmonary hemorrhage in leptospirosis. (C) 2012 Elsevier Inc. All rights reserved.

Tauroursodeoxycholic Acid Affects PPAR gamma and TLR4 in Steatotic Liver Transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator-activated receptor-gamma(PPAR gamma) and damage. When PPAR gamma was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPAR gamma, and the PPAR gamma antagonist treatment up-regulated toll-like receptor 4 (TLR4), specifically the TIR domain-containing adaptor inducing IFN beta (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPAR gamma antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPAR gamma and this in turn up-regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating-based strategies could reduce the inherent risk of steatotic liver failure after transplantation.

Sorafenib in Combination With Capecitabine: An Oral Regimen for Patients With HER2-Negative Locally Advanced or Metastatic Breast Cancer

Purpose Sorafenib is a multikinase inhibitor with antiangiogenic/antiproliferative activity. A randomized, double-blind, placebo-controlled phase IIB trial assessed sorafenib with capecitabine for locally advanced or metastatic human epidermal growth factor receptor 2 (HER2) -negative breast cancer. Patients and Methods Patients were randomly assigned to first-or second-line capecitabine 1,000 mg/m(2) orally twice a day for days 1 to 14 of every 21-day cycle with sorafenib 400 mg orally twice a day or placebo. The primary end point was progression-free survival (PFS). Results In total, 229 patients were enrolled. The addition of sorafenib to capecitabine resulted in a significant improvement in PFS versus placebo (median, 6.4 v 4.1 months; hazard ratio [HR], 0.58; 95% CI, 0.41 to 0.81; P = .001) with sorafenib favored across subgroups, including first-line (HR, 0.50; 95% CI, 0.30 to 0.82) and second-line (HR, 0.65; 95% CI, 0.41 to 1.04) treatment. There was no significant improvement for overall survival (median, 22.2 v 20.9 months; HR, 0.86; 95% CI, 0.61 to 1.23; P = .42) and overall response (38% v 31%; P = .25). Toxicities (sorafenib v placebo) of any grade included rash (22% v 8%), diarrhea (58% v 30%), mucosal inflammation (33% v 21%), neutropenia (13% v 4%), hypertension (18% v 12%), and hand-foot skin reaction/hand-foot syndrome (HFSR/HFS; 90% v 66%); grade 3 to 4 toxicities were comparable between treatment arms except HFSR/HFS (44% v 14%). Reasons for discontinuation in the sorafenib and placebo arms included disease progression (63% v 82%, respectively), adverse events (20% v 9%, respectively), and death (0% v 1%, respectively). Conclusion Addition of sorafenib to capecitabine improved PFS in patients with HER2-negative advanced breast cancer. The dose of sorafenib used in this trial resulted in unacceptable toxicity for many patients. A phase III confirmatory trial has been initiated with a reduced sorafenib dose.