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Background: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression.

The development of non-alcoholic steatohepatitis (NASH) has been associated with alterations in gut microbiota composition and reduced gut barrier function.

Metabolic disorders, such as obesity and type 2 diabetes have a large impact on global health, especially in industrialized countries. Tissue-specific chronic low-grade inflammation is a key contributor to complications in metabolic disorders. To support therapeutic approaches to these complications, it is crucial to gain a deeper understanding of the inflammatory dynamics and to monitor them on the individual level. To this end, blood-based biomarkers reflecting the tissue-specific inflammatory dynamics would be of great value. Here, we describe an in silico approach to select candidate biomarkers for tissue-specific inflammation by using a priori mechanistic knowledge from pathways and tissue-derived molecules. The workflow resulted in a list of candidate markers, in part consisting of literature confirmed biomarkers as well as a set of novel, more innovative biomarkers that reflect inflammation in the liver and adipose tissue. The first step of biomarker verification was on murine tissue gene-level by inducing hepatic inflammation and adipose tissue inflammation through a high-fat diet. Our data showed that in silico predicted hepatic markers had a strong correlation to hepatic inflammation in the absence of a relation to adipose tissue inflammation, while others had a strong correlation to adipose tissue inflammation in the absence of a relation to liver inflammation. Secondly, we evaluated the human translational value by performing a curation step in the literature using studies that describe the regulation of the markers in human, which identified 9 hepatic (such as Serum Amyloid A, Haptoglobin, and Interleukin 18 Binding Protein) and 2 adipose (Resistin and MMP-9) inflammatory biomarkers at the highest level of confirmation. Here, we identified and pre-clinically verified a set of in silico predicted biomarkers for liver and adipose tissue inflammation which can be of great value to study future development of therapeutic/lifestyle interventions to combat metabolic inflammatory complications.

Background Cardiovascular risk is increased in transgender persons using gender-affirming hormone therapy. To gain insight into the mechanism by which sex hormones affect cardiovascular risk in transgender persons, we investigated the effect of hormone therapy on markers of inflammation and hemostasis. Methods In this exploratory study, 48 trans women using estradiol patches plus cyproterone acetate (CPA) and 47 trans men using testosterone gel were included. They were between 18 and 50 years old and did not have a history of cardiovascular events. Measurements were performed before and after 3 and 12 months of hormone therapy. Results After 12 months, in trans women, systemic and endothelial inflammatory markers decreased (hs-CRP -66%, (95% CI -76; -53), VCAM-1–12%, (95% CI -16; -8)), while platelet activation markers increased (PF-4 +17%, (95% CI 4; 32), β-thromboglobulin +13%, (95% CI 2; 24)). The coagulation marker fibrinogen increased transiently, after 3 months (+15%, (95% CI 1; 32)). In trans men, hs-CRP increased (+71%, (95% CI 19; 145)); platelet activation and coagulation markers were not altered. In both trans women and trans men, leptin and adiponectin changed towards reference values of the experienced gender. Conclusions Platelet activation and coagulation marker concentrations increased in trans women using transdermal estradiol plus CPA, but not in trans men using testosterone. Also, concentrations of inflammatory markers decreased in trans women, while hs-CRP increased in trans men. Our results indicate that hormone therapy may affect hemostasis in transgender persons, which could be an underlying mechanism explaining the increased cardiovascular risk in this population.

The development of obesity is characterized by the metabolic overload of tissues and subsequent organ inflammation. The health effects of krill oil (KrO) on obesity-associated inflammation remain largely elusive, because long-term treatments with KrO have not been performed to date. Therefore, we examined the putative health effects of 28 weeks of 3% (w/w) KrO supplementation to an obesogenic diet (HFD) with fat derived mostly from lard. The HFD with KrO was compared to an HFD control group to evaluate the effects on fatty acid composition and associated inflammation in epididymal white adipose tissue (eWAT) and the liver during obesity development. KrO treatment increased the concentrations of EPA and DHA and associated oxylipins, including 18-HEPE, RvE2 and 14-HDHA in eWAT and the liver. Simultaneously, KrO decreased arachidonic acid concentrations and arachidonic-acid-derived oxylipins (e.g., HETEs, PGD2, PGE2, PGF2α, TXB2). In eWAT, KrO activated regulators of adipogenesis (e.g., PPARγ, CEBPα, KLF15, STAT5A), induced a shift towards smaller adipocytes and increased the total adipocyte numbers indicative for hyperplasia. KrO reduced crown-like structures in eWAT, and suppressed HFD-stimulated inflammatory pathways including TNFα and CCL2/MCP-1 signaling. The observed eWAT changes were accompanied by reduced plasma leptin and increased plasma adiponectin levels over time, and improved insulin resistance (HOMA-IR). In the liver, KrO suppressed inflammatory signaling pathways, including those controlled by IL-1β and M-CSF, without affecting liver histology. Furthermore, KrO deactivated hepatic REL-A/p65-NF-κB signaling, consistent with increased PPARα protein expression and a trend towards an increase in IkBα. In conclusion, long-term KrO treatment increased several anti-inflammatory PUFAs and oxylipins in WAT and the liver. These changes were accompanied by beneficial effects on general metabolism and inflammatory tone at the tissue level. The stimulation of adipogenesis by KrO allows for safe fat storage and may, together with more direct PPAR-mediated anti-inflammatory mechanisms, attenuate inflammation.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is characterised by dysfunctional lipid metabolism and cholesterol homeostasis, and a related chronic inflammatory response. NAFLD has become the most common cause of chronic liver disease in many countries, and its prevalence continues to rise in parallel with increasing rates of obesity. Here, we evaluated the putative NAFLD-attenuating effects of a multicomponent medicine consisting of 24 natural ingredients: Hepar compositum (HC-24).MethodsLdlr-/-.Leiden mice were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. HC-24 or vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis. Transcriptomics results were further substantiated with ELISA, immunohistochemical and liver lipid analyses.ResultsHFD feeding induced obesity and metabolic dysfunction including adipose tissue inflammation and increased gut permeability. In the liver, HFD-feeding resulted in a disturbance of cholesterol homeostasis and an associated inflammatory response. HC-24 did not affect body weight, metabolic risk factors, adipose tissue inflammation or gut permeability. While HC-24 did not alter total liver steatosis, there was a pronounced reduction in lobular inflammation in HC-24-treated animals, which was associated with modulation of genes and proteins involved in inflammation (e.g., neutrophil chemokine Cxcl1) and cholesterol homeostasis (i.e., predicted effect on ‘cholesterol’ as an upstream regulator, based on gene expression changes associated with cholesterol handling). These effects were confirmed by CXCL1 ELISA, immunohistochemical staining of neutrophils and biochemical analysis of hepatic free cholesterol content. Intrahepatic free cholesterol levels were found to correlate significantly with the number of inflammatory aggregates in the liver, thereby providing a potential rationale for the observed anti-inflammatory effects of HC-24.ConclusionsFree cholesterol accumulates in the liver of Ldlr-/-.Leiden mice under physiologically translational dietary conditions, and this is associated with the development of hepatic inflammation. The multicomponent medicine HC-24 reduces accumulation of free cholesterol and has molecular and cellular anti-inflammatory effects in the liver.

Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disorder that is associated with an increased incidence of cardiovascular disease and type 2 diabetes. Animal models adequately mimicking this condition are scarce. We herein investigate whether Ldlr&minus, /&minus, Leiden mice on different high-fat diets represent a suitable NASH model. Ldlr&minus, Leiden mice were fed a healthy chow diet or fed a high-fat diet (HFD) containing lard or a fast food diet (FFD) containing milk fat. Additionally, the response to treatment with obeticholic acid (OCA) was evaluated. Both high-fat diets induced obesity, hyperlipidemia, hyperinsulinemia, and increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Mice on both diets developed progressive macro- and microvesicular steatosis, hepatic inflammation, and fibrosis, along with atherosclerosis. HFD induced more severe hyperinsulinemia, while FFD induced more severe hepatic inflammation with advanced (F3) bridging fibrosis, as well as more severe atherosclerosis. OCA treatment significantly reduced hepatic inflammation and fibrosis, and it did not affect atherosclerosis. Hepatic transcriptome analysis was compared with human NASH and illustrated similarity. The present study defines a translational model of NASH with progressive liver fibrosis and simultaneous atherosclerosis development. By adaptation of the fat content of the diet, either insulin resistance (HFD) or hepatic inflammation and fibrosis (FFD) can be aggravated.

The obesity epidemic increases the interest to elucidate impact of short‐chain fatty acids on metabolism, obesity, and the brain. We investigated the effects of propionic acid (PA) and caproic acid (CA) on metabolic risk factors, liver and adipose tissue pathology, brain function, structure (by MRI), and gene expression, during obesity development in Ldlr−/−.Leiden mice. Ldlr−/−.Leiden mice received 16 weeks either a high‐fat diet (HFD) to induce obesity, or chow as reference group. Next, obese HFD‐fed mice were treated 12 weeks with (a) HFD + CA (CA), (b) HFD + PA (PA), or (c) a HFD‐control group. PA reduced the body weight and systolic blood pressure, lowered fasting insulin levels, and reduced HFD‐induced liver macrovesicular steatosis, hypertrophy, inflammation, and collagen content. PA increased the amount of glucose transporter type 1‐positive cerebral blood vessels, reverted cerebral vasoreactivity, and HFD‐induced effects in microstructural gray and white matter integrity of optic tract, and somatosensory and visual cortex. PA and CA also reverted HFD‐induced effects in functional connectivity between visual and auditory cortex. However, PA mice were more anxious in open field, and showed reduced activity of synaptogenesis and glutamate regulators in hippocampus. Therefore, PA treatment should be used with caution even though positive metabolic, (cerebro) vascular, and brain structural and functional effects were observed.The obesity epidemic increases the interest to elucidate impact of short‐chain fatty acids on metabolism, obesity, and the brain. We investigated the effects of propionic acid (PA) and caproic acid (CA) on metabolic risk factors, liver and adipose tissue pathology, brain function, structure (by MRI), and gene expression, during obesity development in Ldlr−/−.Leiden mice. Ldlr−/−.Leiden mice received 16 weeks either a high‐fat diet (HFD) to induce obesity, or chow as reference group. Next, obese HFD‐fed mice were treated 12 weeks with (a) HFD + CA (CA), (b) HFD + PA (PA), or (c) a HFD‐control group. PA reduced the body weight and systolic blood pressure, lowered fasting insulin levels, and reduced HFD‐induced liver macrovesicular steatosis, hypertrophy, inflammation, and collagen content. PA increased the amount of glucose transporter type 1‐positive cerebral blood vessels, reverted cerebral vasoreactivity, and HFD‐induced effects in microstructural gray and white matter integrity of optic tract, and somatosensory and visual cortex. PA and CA also reverted HFD‐induced effects in functional connectivity between visual and auditory cortex. However, PA mice were more anxious in open field, and showed reduced activity of synaptogenesis and glutamate regulators in hippocampus. Therefore, PA treatment should be used with caution even though positive metabolic, (cerebro) vascular, and brain structural and functional effects were observed.

Background and Aims: Observational studies show a peak incidence in cardiovascular events during and early after clinical conditions associated with substantial systemic inflammation, such as pneumonia. The acuteness of this increased risk suggests rapid plaque destabilization and associated intraplaque inflammation. We evaluated whether lipopolysaccharides (LPS)-evoked acute systemic inflammation would induce such detrimental vascular changes in murine aortas with manifest atherosclerotic lesions. Methods and Results: ApoE3*Leiden mice were fed a high cholesterol diet for 20 weeks to establish atherosclerosis. Thereafter, mice received a single intraperitoneal injection with LPS to induce systemic inflammation, or saline for control. Mice were sacrificed 2 or 15 days post-LPS injection (n = 17) or post-saline injection (n = 13). Serum amyloid A, a sensitive marker of systemic inflammation, increased 250-fold in LPS-treated mice. Aortic root plaques were assessed for total plaque area, plaque severity, and inflammatory cell content. No significant differences in total surface area of atherosclerotic plaque were found between control and LPS groups sacrificed after 2 days (resp. 0.409 ± 0.228 × 105 μm2 vs. 0.285 ± 0.169 × 105 μm2) (P = 0.31), and 15 days (resp. 0.950 ± 0.938 × 105 μm2 vs. 0.612 ± 0.413 × 105 μm2) (P = 0.80). Furthermore, plaque type and number of lesions were unaltered and intraplaque density of macrophages and lymphocytes were comparable in both the groups. Conclusions: Intraperitoneal LPS injection in ApoE3*Leiden mice triggers a profound systemic inflammatory response, but does not increase atherosclerotic plaque area or inflammatory cell density. This model of LPS-induced inflammation in atherosclerosis-prone mice argues against intraplaque alterations as an explanation for acute inflammation-induced cardiovascular event risk.

Item does not contain fulltext OBJECTIVE: Midlife obesity affects cognition and increases risk of developing dementia. Recent data suggest that intake of the short chain fatty acid butyrate could improve memory function, and may protect against diet-induced obesity by reducing body weight and adiposity. SUBJECTS: We examined the impact of a high-fat diet (HFD) followed by intervention with 5% (w/w) dietary butyrate, on metabolism, microbiota, brain function and structure in the low-density-lipoprotein receptor knockout (LDLr-/-) mouse model in mid and late life. RESULTS: In mid-adult mice, 15 weeks of HFD-induced adiposity, liver fibrosis and neuroinflammation, increased systolic blood pressure and decreased cerebral blood flow, functional connectivity assessed with neuroimaging. The subsequent 2 months butyrate intervention restored these detrimental effects to chow-fed control levels. Both HFD and butyrate intervention decreased variance in fecal microbiota composition. In late-adult mice, HFD showed similar detrimental effects and decreased cerebral white and gray matter integrity, whereas butyrate intervention attenuated only metabolic parameters. CONCLUSION: HFD induces detrimental effects in mid- and late-adult mice, which can be attenuated by butyrate intervention. These findings are consistent with reported associations between midlife obesity and cognitive impairment and dementia in humans. We suggest that butyrate may have potential in prevention and treatment of midlife obesity.

Obesity, type 2 diabetes, and other metabolic disorders have a large impact on global health, especially in Western countries. An important hallmark of metabolic disorders is chronic low-grade inflammation. A key player in chronic low-grade inflammation is dysmetabolism, which is defined as the inability to keep homeostasis resulting in loss of lipid control, oxidative stress, inflammation, and insulin resistance. Although often not yet detectable in the circulation, chronic low-grade inflammation can be present in one or multiple organs. The response to a metabolic challenge containing lipids may magnify dysfunctionalities at the tissue level, causing an overflow of inflammatory markers into the circulation and hence allow detection of early low-grade inflammation. Here, we summarize the evidence of successful application of metabolic challenge tests in type 2 diabetes, metabolic syndrome, obesity, and unhealthy aging. We also review how metabolic challenge tests have been successfully applied to evaluate nutritional intervention effects, including an “anti-inflammatory” mixture, dark chocolate, whole grain wheat and overfeeding. Additionally, we elaborate on future strategies to (re)gain inflammatory flexibility. Through epigenetic and metabolic regulation, the inflammatory response may be trained by regular mild and metabolic triggers, which can be understood from the perspective of trained immunity, hormesis and pro-resolution. New strategies to optimize dynamics of inflammation may become available.

Development of non-alcoholic fatty liver disease (NAFLD) is linked to obesity, adipose tissue inflammation, and gut dysfunction, all of which depend on diet. So far, studies have mainly focused on diet-related fecal microbiota changes, but other compartments may be more informative on host health. We present a first systematic analysis of microbiota changes in the ileum and colon using multiple diets and investigating both fecal and mucosal samples. Ldlr&minus, /&minus, Leiden mice received one of three different energy-dense (ED)-diets (n = 15/group) for 15 weeks. All of the ED diets induced obesity and metabolic risk factors, altered short-chain fatty acids (SCFA), and increased gut permeability and NAFLD to various extents. ED diets reduced the diversity of high-abundant bacteria and increased the diversity of low-abundant bacteria in all of the gut compartments. The ED groups showed highly variable, partially overlapping microbiota compositions that differed significantly from chow. Correlation analyses demonstrated that (1) specific groups of bacteria correlate with metabolic risk factors, organ dysfunction, and NAFLD endpoints, (2) colon mucosa had greater predictive value than other compartments, (3) correlating bacteria differed per compartment, and (4) some bacteria correlated with plasma SCFA levels. In conclusion, this comprehensive microbiota analysis demonstrates correlations between the microbiota and dysfunctions of gut, adipose tissue, and liver, independent of a specific disease-inducing diet.

Introduction: It is generally accepted that metabolic inflammation in the liver is an important driver of disease progression in NASH and associated matrix remodeling/fibrosis. However, the exact molecular inflammatory mechanisms are poorly defined in human studies. Investigation of key pathogenic mechanisms requires the use of pre-clinical models, for instance for time-resolved studies. Such models must reflect molecular disease processes of importance in patients. Herein we characterized inflammation in NASH patients on the molecular level by transcriptomics and investigated whether key human disease pathways can be recapitulated experimentally in Ldlr−/−.Leiden mice, an established pre-clinical model of NASH. Methods: Human molecular inflammatory processes were defined using a publicly available NASH gene expression profiling dataset (GSE48452) allowing the comparison of biopsy-confirmed NASH patients with normal controls. Gene profiling data from high-fat diet (HFD)-fed Ldlr−/−.Leiden mice (GSE109345) were used for assessment of the translational value of these mice. Results: In human NASH livers, we observed regulation of 65 canonical pathways of which the majority was involved in inflammation (32%), lipid metabolism (16%), and extracellular matrix/remodeling (12%). A similar distribution of pathways across these categories, inflammation (36%), lipid metabolism (24%) and extracellular matrix/remodeling (8%) was observed in HFD-fed Ldlr−/−.Leiden mice. Detailed evaluation of these pathways revealed that a substantial proportion (11 out of 13) of human NASH inflammatory pathways was recapitulated in Ldlr−/−.Leiden mice. Furthermore, the activation state of identified master regulators of inflammation (i.e., specific transcription factors, cytokines, and growth factors) in human NASH was largely reflected in Ldlr−/−.Leiden mice, further substantiating its translational value. Conclusion: Human NASH is characterized by upregulation of specific inflammatory processes (e.g., “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes,” “PI3K signaling in B Lymphocytes”) and master regulators (e.g., TNF, CSF2, TGFB1). The majority of these processes and regulators are modulated in the same direction in Ldlr−/−.Leiden mice fed HFD with a human-like macronutrient composition, thus demonstrating that specific experimental conditions recapitulate human disease on the molecular level of disease pathways and upstream/master regulators.

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high‐fat diet (HFD)‐fed Ldlr‐/‐.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome‐wide liver transcriptome from HFD‐fed Ldlr‐/‐.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24‐34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human‐based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr‐/‐.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory‐profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80‐positive cells) and reduced crown‐like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD‐fed Ldlr‐/‐.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

Background & Aims: Anthocyanins may have beneficial effects on lipid metabolism and inflammation and are demonstrated to have hepatoprotective properties in models of restraint-stressand chemically-induced liver damage. However, their potential to protect against non-alcoholic steatohepatitis (NASH) under conditions relevant for human pathogenesis remains unclear. Therefore, we studied the effects of the standardised anthocyanin-rich extract Mirtoselect on diet-induced NASH in a translational model of disease.Methods: ApoE*3Leiden mice were fed a Western-type cholesterol-containing diet without (HC) or with 0.1% (w/w) Mirtoselect (HCM) for 20 weeks to study the effects on diet-induced NASH.Results: Mirtoselect attenuated HC-induced hepatic steatosis, as observed by decreased macro-and microvesicular hepatocellular lipid accumulation and reduced hepatic cholesteryl ester content. This anti-steatotic effect was accompanied by local anti-inflammatory effects in liver, as demonstrated by reduced inflammatory cell clusters and reduced neutrophil infiltration in HCM. On a molecular level, HC diet significantly induced hepatic expression of pro-inflammatory genes Tnf, Emr1, Ccl2, Mpo, Cxcl1, and Cxcl2 while this induction was less pronounced or significantly decreased in HCM. A similar quenching effect was observed for HC-induced pro-fibrotic genes, Acta2 and Col1a1 and this anti-fibrotic effect of Mirtoselect was confirmed histologically. Many of the pro-inflammatory and pro-fibrotic parameters positively correlated with intrahepatic free cholesterol levels. Mirtoselect significantly reduced accumulation and crystallisation of intrahepatic free cholesterol, providing a possible mechanism for the observed hepatoprotective effects.Conclusions: Mirtoselect attenuates development of NASH, reducing hepatic lipid accumulation, inflammation and fibrosis, possibly mediated by local anti-inflammatory effects associated with reduced accumulation and crystallisation of intrahepatic free cholesterol. (C) 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction: It is generally accepted that metabolic inflammation in the liver is an important driver of disease progression in NASH and associated matrix remodeling/fibrosis. However, the exact molecular inflammatory mechanisms are poorly defined in human studies. Investigation of key pathogenic mechanisms requires the use of pre-clinical models, for instance for time-resolved studies. Such models must reflect molecular disease processes of importance in patients. Herein we characterized inflammation in NASH patients on the molecular level by transcriptomics and investigated whether key human disease pathways can be recapitulated experimentally in Ldlr−/−.Leiden mice, an established pre-clinical model of NASH. Methods: Human molecular inflammatory processes were defined using a publicly available NASH gene expression profiling dataset (GSE48452) allowing the comparison of biopsy-confirmed NASH patients with normal controls. Gene profiling data from high-fat diet (HFD)-fed Ldlr−/−.Leiden mice (GSE109345) were used for assessment of the translational value of these mice. Results: In human NASH livers, we observed regulation of 65 canonical pathways of which the majority was involved in inflammation (32%), lipid metabolism (16%), and extracellular matrix/remodeling (12%). A similar distribution of pathways across these categories, inflammation (36%), lipid metabolism (24%) and extracellular matrix/remodeling (8%) was observed in HFD-fed Ldlr−/−.Leiden mice. Detailed evaluation of these pathways revealed that a substantial proportion (11 out of 13) of human NASH inflammatory pathways was recapitulated in Ldlr−/−.Leiden mice. Furthermore, the activation state of identified master regulators of inflammation (i.e., specific transcription factors, cytokines, and growth factors) in human NASH was largely reflected in Ldlr−/−.Leiden mice, further substantiating its translational value. Conclusion: Human NASH is characterized by upregulation of specific inflammatory processes (e.g., “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes,” “PI3K signaling in B Lymphocytes”) and master regulators (e.g., TNF, CSF2, TGFB1). The majority of these processes and regulators are modulated in the same direction in Ldlr−/−.Leiden mice fed HFD with a human-like macronutrient composition, thus demonstrating that specific experimental conditions recapitulate human disease on the molecular level of disease pathways and upstream/master regulators.

Obesity-related albuminuria is associated with decline of kidney function and is considered a first sign of diabetic nephropathy. Suggested factors linking obesity to kidney dysfunction include low-grade inflammation, insulin resistance and adipokine dysregulation. Here, we investigated the effects of two pharmacological compounds with established anti-inflammatory properties, rosiglitazone and rosuvastatin, on kidney dysfunction during high-fat diet (HFD)-induced obesity. For this, human CRP transgenic mice were fed standard chow, a lard-based HFD, HFD+rosuvastatin or HFD+rosiglitazone for 42 weeks to study effects on insulin resistance; plasma inflammatory markers and adipokines; and renal pathology. Rosiglitazone but not rosuvastatin prevented HFD-induced albuminuria and renal fibrosis and inflammation. Also, rosiglitazone prevented HFD-induced KIM-1 expression, while levels were doubled with rosuvastatin. This was mirrored by miR-21 expression, which plays a role in fibrosis and is associated with renal dysfunction. Plasma insulin did not correlate with albuminuria. Only rosiglitazone increased circulating adiponectin concentrations. In all, HFD-induced albuminuria, and renal inflammation, injury and fibrosis is prevented by rosiglitazone but not by rosuvastatin. These beneficial effects of rosiglitazone are linked to lowered miR-21 expression but not connected with the selectively enhanced plasma adiponectin levels observed in rosiglitazone-treated animals.

Background: Obesity frequently associates with the development of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis. Chronic inflammation in white adipose tissue (WAT) seems to be an important driver of these manifestations. Objective: This study investigated a combination of an extensively hydrolyzed casein (eHC), docosahexaenoic acid (DHA), arachidonic acid (ARA), and Lactobacillus Rhamnosus GG (LGG) (together referred to as nutritional ingredients, NI) on the development of obesity, metabolic risk factors, WAT inflammation, NAFLD and atherosclerosis in high-fat diet-fed LDLr-/-.Leiden mice, a model that mimics disease development in humans. Methods: LDLr-/-.Leiden male mice (n = 15/group) received a high-fat diet (HFD, 45 Kcal%) for 21 weeks with or without the NI (23.7% eHC, 0.083% DHA, 0.166% ARA; all w/w and 1x109 CFU LGG gavage 3 times/week). HFD and HFD+NI diets were isocaloric. A low fat diet (LFD, 10 Kcal%) was used for reference. Body weight, food intake and metabolic risk factors were assessed over time. At week 21, tissues were analyzed for WAT inflammation (crown-like structures), NAFLD and atherosclerosis. Effects of the individual NI components were explored in a follow-up experiment (n = 7/group). Results: When compared to HFD control, treatment with the NI strongly reduced body weight to levels of the LFD group, and significantly lowered (P

Objective: Previous studies investigating flavanol-rich foods provide indications for potential cardioprotective effects of these foods, but the effects of individual flavanols remain unclear. We investigated whether the flavanol epicatechin can reduce diet-induced atherosclerosis, with particular emphasis on the cardiovascular risk factors dyslipidaemia and inflammation. Methods: ApoE*3-Leiden mice were fed a cholesterol-containing atherogenic diet with or without epicatechin (0.1% w/w) to study effects on early- and late-stage atherosclerosis (8w and 20w). Invivo effects of epicatechin on diet-induced inflammation were studied in human-CRP transgenic mice and NFκB-luciferase reporter mice. Results: Epicatechin attenuated atherosclerotic lesion area in ApoE*3-Leiden mice by 27%, without affecting plasma lipids. This anti-atherogenic effect of epicatechin was specific to the severe lesion types, with no effect on mild lesions. Epicatechin mitigated diet-induced increases in plasma SAA (in ApoE*3-Leiden mice) and plasma human-CRP (in human-CRP transgenic mice). Microarray analysis of aortic gene expression revealed an attenuating effect of epicatechin on several diet-induced pro-atherogenic inflammatory processes in the aorta (e.g. chemotaxis of cells, matrix remodelling), regulated by NFκB. These findings were confirmed immunohistochemically by reduced lesional neutrophil content in HCE, and by inhibition of diet-induced NFκB activity in epicatechin-treated NFκB-luciferase reporter mice. Conclusion: Epicatechin attenuates development of atherosclerosis and impairs lesion progression from mild to severe lesions in absence of an effect on dyslipidaemia. The observed reduction of circulating inflammatory risk factors by epicatechin (e.g. SAA, human-CRP), as well as its local anti-inflammatory activity in the vessel wall, provide a rationale for epicatechin's anti-atherogenic effects. © 2014 Elsevier Ireland Ltd.

BACKGROUND AND AIMS: Observational studies show a peak incidence of cardiovascular events after major surgery. For example, the risk of myocardial infarction increases 25-fold early after hip replacement. The acuteness of this increased risk suggests abrupt enhancement in plaque vulnerability, which may be related to intra-plaque inflammation, thinner fibrous cap and/or necrotic core expansion. We hypothesized that acute systemic inflammation following major orthopedic surgery induces such changes.METHODS: ApoE(-/-) mice were fed a western diet for 10 weeks. Thereafter, half the mice underwent mid-shaft femur osteotomy followed by realignment with an intramedullary K-wire, to mimic major orthopedic surgery. Mice were sacrificed 5 or 15 days post-surgery (n = 22) or post-saline injection (n = 13). Serum amyloid A (SAA) was measured as a marker of systemic inflammation. Paraffin embedded slides of the aortic root were stained to measure total plaque area and to quantify fibrosis, calcification, necrotic core, and inflammatory cells.RESULTS: Surgery mice showed a pronounced elevation of serum amyloid A (SAA) and developed increased plaque and necrotic core area already at 5 days, which reached significance at 15 days (p = 0.019; p = 0.004 for plaque and necrotic core, respectively). Macrophage and lymphocyte density significantly decreased in the surgery group compared to the control group at 15 days (p = 0.037; p = 0.024, respectively). The density of neutrophils and mast cells remained unchanged.CONCLUSIONS: Major orthopedic surgery in ApoE-/- mice triggers a systemic inflammatory response. Atherosclerotic plaque area is enlarged after surgery mainly due to an increase of the necrotic core. The role of intra-plaque inflammation in this response to surgical injury remains to be fully elucidated.

Obesity is associated with chronic low-grade inflammation that drives the development of metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). We recently showed that white adipose tissue (WAT) constitutes an important source of inflammatory factors. Hence, interventions that attenuate WAT inflammation may reduce NAFLD development. Male LDLr−/− mice were fed a high-fat diet (HFD) for 9 weeks followed by 7 weeks of HFD with or without rosiglitazone. Effects on WAT inflammation and NAFLD development were analyzed using biochemical and (immuno)histochemical techniques, combined with gene expression analyses. Nine weeks of HFD feeding induced obesity and WAT inflammation, which progressed gradually until the end of the study. Rosiglitazone fully blocked progression of WAT inflammation and activated PPARγ significantly in WAT. Rosiglitazone intervention did not activate PPARγ in liver, but improved liver histology and counteracted the expression of genes associated with severe NAFLD in humans. Rosiglitazone reduced expression of pro-inflammatory factors in WAT (TNF-α, leptin) and increased expression of adiponectin, which was reflected in plasma. Furthermore, rosiglitazone lowered circulating levels of pro-inflammatory saturated fatty acids. Together, these observations provide a rationale for the observed indirect hepatoprotective effects and suggest that WAT represents a promising therapeutic target for the treatment of obesity-associated NAFLD.

Background: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with abdominal obesity. Growing evidence suggests that inflammation in specific depots of white adipose tissue (WAT) has a key role in NAFLD progression, but experimental evidence for a causal role of WAT is lacking. Methods: A time-course study in C57BL/6J mice was performed to establish which WAT depot is most susceptible to develop inflammation during high-fat diet (HFD)-induced obesity. Crown-like structures (CLS) were quantified in epididymal (eWAT), mesenteric (mWAT) and inguinal/subcutaneous (iWAT) WAT. The contribution of inflamed WAT to NAFLD progression was investigated by surgical removal of a selected WAT depot and compared with sham surgery. Plasma markers were analyzed by enzyme-linked immunosorbent assay (cytokines/adipokines) and lipidomics (lipids). Results: In eWAT, CLS were formed already after 12 weeks of HFD, which coincided with maximal adipocyte size and fat depot mass, and preceded establishment of non-alcoholic steatohepatitis (NASH). By contrast, the number of CLS were low in mWAT and iWAT. Removal of inflamed eWAT after 12 weeks (eWATx group), followed by another 12 weeks of HFD feeding, resulted in significantly reduced NASH in eWATx. Inflammatory cell aggregates (−40% P

Background.Naturally occurring substances from the flavanol and anthocyanin family of polyphenols have been proposed to exert beneficial effects in the course of obesity. We hypothesized that their effects on attenuating obesity-induced dyslipidemia as well as the associated inflammatory sequelae especially have health-promoting potential.Methods.Male C57BL/6J mice (n=52) received a control low-fat diet (LFD; 10 kcal% fat) for 6 weeks followed by 24 weeks of either LFD (n=13) or high-fat diet (HFD; 45 kcal% fat;n=13) or HFD supplemented with 0.1% w/w of the flavanol compound epicatechin (HFD+E;n=13) or an anthocyanin-rich bilberry extract (HFD+B;n=13). Energy substrate utilization was determined by indirect calorimetry in a subset of mice following the dietary switch and at the end of the experiment. Blood samples were collected at baseline and at 3 days and 4, 12, and 20 weeks after dietary switch and analyzed for systemic lipids and proinflammatory cytokines. Adipose tissue (AT) histopathology and inflammatory gene expression as well as hepatic lipid content were analyzed after sacrifice.Results.The switch from a LFD to a HFD lowered the respiratory exchange ratio and increased plasma cholesterol and hepatic lipid content. These changes were not attenuated by HFD+E or HFD+B. Furthermore, the polyphenol compounds could not prevent HFD-induced systemic rise of TNF-αlevels. Interestingly, a significant reduction inTnfgene expression in HFD+B mice was observed in the AT. Furthermore, HFD+B, but not HFD+E, significantly prevented the early upregulation of circulating neutrophil chemoattractant mKC. However, no differences in AT histopathology were observed between the HFD types.Conclusion.Supplementation of HFD with an anthocyanin-rich bilberry extract but not with the flavanol epicatechin may exert beneficial effects on the systemic early inflammatory response associated with diet-induced obesity. These systemic effects were transient and not observed after prolongation of HFD-feeding (24 weeks). On the tissue level, long-term treatment with bilberry attenuated TNF-αexpression in adipose tissue.

Obesity is associated with insulin resistance, disturbed glucose homeostasis, low grade inflammation, and comorbidities such as type 2 diabetes and cardiovascular disease. The cytokine macrophage migration inhibitory factor (MIF) is an ubiquitously expressed protein that plays a crucial role in many inflammatory and autoimmune disorders. Increasing evidence suggests that MIF also controls metabolic and inflammatory processes underlying the development of metabolic pathologies associated with obesity. This is a comprehensive summary of our current knowledge on the role of MIF in obesity and obesity-associated comorbidities, based on human clinical data as well as animal models of disease.

Background— Doxycycline has been shown to effectively inhibit aneurysm formation in animal models of abdominal aortic aneurysm. Although this effect is ascribed to matrix metalloproteinase-9 inhibition, such an effect is unclear in human studies. We reevaluated the effect of doxycycline on aortic wall protease content in a clinical trial and found that doxycycline selectively reduces neutrophil-derived proteases. We thus hypothesized that doxycycline acts through an effect on vascular inflammation. Methods and Results— Sixty patients scheduled for elective open aneurysmal repair were randomly assigned to 2 weeks of low-, medium-, or high-dose doxycycline (50, 100, or 300 mg/d, respectively) or no medication (control group). Aortic wall samples were collected at the time of operation, and the effect of doxycycline treatment on vascular inflammation was evaluated. Independently of its dose, doxycycline treatment resulted in a profound but selective suppression of aortic wall inflammation as reflected by a selective 72% reduction of the aortic wall neutrophils and a 95% reduction of the aortic wall cytotoxic T-cell content (median values; P P P P P P Conclusions— A brief period of doxycycline treatment has a profound but selective effect on vascular inflammation and reduces aortic wall neutrophil and cytotoxic T-cell content. Results of this study are relevant for pharmaceutical stabilization of the abdominal aneurysm and possibly for other inflammatory conditions that involve neutrophils and/or cytotoxic T cells.

In the past few years, inflammation has emerged as a major driving force of atherosclerotic lesion development. It is now well-established that from early lesion to vulnerable plaque formation, numerous cellular and molecular inflammatory components participate in the disease process. The most prominent cells that invade in evolving lesions are monocyte-derived macrophages and T-lymphocytes. Both cell types produce a wide array of soluble inflammatory mediators (cytokines, chemokines) which are critically important in the initiation and perpetuation of the disease. This review summarizes the currently available information from mouse studies on the contribution of a specified group of cytokines expressed in atherosclerotic lesions, viz. interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IL-20) and macrophage-associated cytokines [tumour necrosis factor-α (TNF-α); macrophage migration inhibitory factor (MIF); interferon-γ (IFN-γ); colony stimulating factors G-CSF,-M-CSF,-GM-CSF) to atherogenesis. Emphasis is put on the consistency of the effects of these cytokines, i.e. inasmuch an effect depends on the experimental approach applied (overexpression/deletion, strain, gender, dietary conditions, and disease stage). An important outcome of this survey is (i) that only for a few cytokines there is sufficient consistent data allowing classifying them as typically proatherogenic (IL-1, IL-12, IL-18, MIF, IFN-γ, TNF-α, and M-CSF) or antiatherogenic (IL-10) and (ii) that some cytokines (IL-4, IL-6 and GM-CSF) can exert pro- or anti-atherogenic effects depending on the experimental conditions. This knowledge can be used for improved early detection, prevention and treatment of atherosclerosis. © The Author 2008. Chemicals / CAS: colony stimulating factor 1, 81627-83-0; gamma interferon, 82115-62-6; interleukin 12, 138415-13-1; interleukin 18, 189304-55-0; interleukin 2, 85898-30-2; Colony-Stimulating Factors; Cytokines; Interferon-gamma, 82115-62-6; Interleukins; Macrophage Migration-Inhibitory Factors; Tumor Necrosis Factor-alpha

The cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. We identify the chemokine receptors CXCR2 and CXCR4 as functional receptors for MIF. MIF triggered G αi- and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation and calcium influx through CXCR2 or CXCR4. MIF competed with cognate ligands for CXCR4 and CXCR2 binding, and directly bound to CXCR2. CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inflamed or atherosclerotic arteries involved both CXCR2 and CD74. In vivo, Mif deficiency impaired monocyte adhesion to the arterial wall in atherosclerosis-prone mice, and MIF-induced leukocyte recruitment required Il8rb (which encodes Cxcr2). Blockade of Mif but not of canonical ligands of Cxcr2 or Cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and T-cell content in plaques. By activating both CXCR2 and CXCR4, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis. Targeting MIF in individuals with manifest atherosclerosis can potentially be used to treat this condition. © 2007 Nature Publishing Group.

Background and aims Besides LDL-cholesterol, local vascular inflammation plays a key role in atherogenesis. Efficient therapies to treat the inflammatory component of the disease have not been established. The discovery of specialized inflammation-resolving mediators, such as resolvins may provide new opportunities for treatment. This study examines whether the ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1), can reduce atherosclerosis, when administered alone or in combination with a cholesterol-lowering statin. Methods ApoE*3Leiden mice were fed a hypercholesterolemic diet for 9 weeks and subsequently treated with RvE1-low (1 mg/kg/day), RvE1-high (5 mg/kg/day), atorvastatin (1.5 mg/kg/day) or the combination of atorvastatin and RvE1-low for the following 16 weeks. Results RvE1-low and RvE1-high reduced atherosclerotic lesion size to the same extent (−35%; p < 0.05), attenuated the formation of severe lesions, also seen as a proportional increase in the presence of mild lesions, but did not alter plasma cholesterol levels. Cholesterol-lowering atorvastatin reduced atherosclerosis (−27%, p < 0.05), and the combination of RvE1 and atorvastatin further attenuated lesion size (−51%, p < 0.01) and increased the content of mild lesions. RvE1 did not affect plasma SAA, E-selectin, VCAM-1 or MCP-1 but did reduce plasma EPHX4 and down-regulated the local expression of pro-atherogenic genes in the aortae, (e.g. Cd74, Cd44, Ccl2, Ccr5 and Adam17) and significantly inactivated IFN-γ (p < 0.001) and TNF-α (p < 0.001) signalling pathways. Conclusions RvE1 attenuates atherogenesis both alone and on top of a statin. The local effects of RvE1 are demonstrated by the modulated aortic expression of genes involved in inflammatory and immune responses, without altering plasma cholesterol or circulating SAA. © 2016 The Authors

Background and AimsAs dietary saturated fatty acids are associated with metabolic and cardiovascular disease, a potentially interesting strategy to reduce disease risk is modification of the quality of fat consumed. Vegetable oils represent an attractive target for intervention, as they largely determine the intake of dietary fats. Furthermore, besides potential health effects conferred by the type of fatty acids in a vegetable oil, other minor components (e.g. phytochemicals) may also have health benefits. Here, we investigated the potential long-term health effects of isocaloric substitution of dietary fat (i.e. partial replacement of saturated by unsaturated fats), as well as putative additional effects of phytochemicals present in unrefined (virgin) oil on development of non-alcoholic fatty liver disease (NAFLD) and associated atherosclerosis. For this, we used pumpkin seed oil, because it is high in unsaturated fatty acids and a rich source of phytochemicals.MethodsApoE*3Leiden mice were fed a Western-type diet (CON) containing cocoa butter (15% w/w) and cholesterol (1% w/w) for 20 weeks to induce risk factors and disease endpoints. In separate groups, cocoa butter was replaced by refined (REF) or virgin (VIR) pumpkin seed oil (comparable in fatty acid composition, but different in phytochemical content).ResultsBoth oils improved dyslipidaemia, with decreased (V) LDL-cholesterol and triglyceride levels in comparison with CON, and additional cholesterol-lowering effects of VIR over REF. While REF did not affect plasma inflammatory markers, VIR reduced circulating serum amyloid A and soluble vascular adhesion molecule-1. NAFLD and atherosclerosis development was modestly reduced in REF, and VIR strongly decreased liver steatosis and inflammation as well as atherosclerotic lesion area and severity.ConclusionsOverall, we show that an isocaloric switch from a diet rich in saturated fat to a diet rich in unsaturated fat can attenuate NAFLD and atherosclerosis development. Phytochemical-rich virgin pumpkin seed oil exerts additional anti-inflammatory effects resulting in more pronounced health effects.

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and lobular inflammation. It is unclear how the development of liver steatosis and the formation of inflammatory cell aggregates are related to each other. The present study investigated the longitudinal development of two forms of steatosis, micro- and macrovesicular steatosis, as well as lobular inflammation. ApoE*3Leiden.CETP (E3L.CETP) transgenic mice were fed a high-fat diet containing 1% w/w cholesterol (HFC) for 12 weeks to induce NASH. Livers were harvested in intervals of 4 weeks and analyzed by histological and biochemical techniques, as well as transcriptome and subsequent pathway analysis. Major findings were validated in independent NASH studies using other rodent models, i.e. HFD-treated C57BL/6J and LDLr ‑/- .Leiden mice. In E3L.CETP mice, microvesicular steatosis was rapidly induced and reached plateau levels after already 4 weeks of HFC treatment. By contrast, macrovesicular steatosis developed more gradually and progressed over time. Lobular inflammation increased after 4 weeks with a significant further progression towards the end of the study (12 weeks). Macrovesicular, but not microvesicular, steatosis was positively correlated with the number of inflammatory aggregates. This correlation was confirmed in a milder (C57BL/6J) and a more severe (LDLr ‑ /-.Leiden) NASH model. Furthermore, collagen staining showed onset of perihepatocellular fibrosis in E3L.CETP mice after 12 weeks of HFC treatment and transcriptome analysis substantiated the activation of pro-fibrotic pathways and genes. Macrovesicular steatosis correlated positively with liver fibrosis in LDLr-/-.Leiden mice with pronounced fibrosis. Collectively, this study shows that macrovesicular steatosis is associated with lobular inflammation and liver fibrosis in rodent models and highlights the importance of this form of steatosis in the pathogenesis of NASH.

Obesity-induced inflammation presumably accelerates the development of chronic kidney diseases. However, little is known about the sequence of these inflammatory events and their contribution to renal pathology. We investigated the effects of obesity on the evolution of age-dependent renal complications in mice in conjunction with the development of renal and systemic low-grade inflammation (LGI). C57BL/6J mice susceptible to develop age-dependent sclerotic pathologies with amyloid features in the kidney, were fed low (10% lard) or high-fat diets (45% lard) for 24, 40 and 52 weeks. HFD-feeding induced overt adiposity, altered lipid and insulin homeostasis, increased systemic LGI and adipokine release. HFD-feeding also caused renal upregulation of pro-inflammatory genes, infiltrating macrophages, collagen I protein, increased urinary albumin and NGAL levels. HFD-feeding severely aggravated age-dependent structural changes in the kidney. Remarkably, enhanced amyloid deposition rather than sclerosis was observed. The degree of amyloidosis correlated significantly with body weight. Amyloid deposits stained positive for serum amyloid A (SAA) whose plasma levels were chronically elevated in HFD mice. Our data indicate obesity-induced chronic inflammation as a risk factor for the acceleration of age-dependent renal amyloidosis and functional impairment in mice, and suggest that obesity-enhanced chronic secretion of SAA may be the driving factor behind this process. © 2015, Nature Publishing Group. All rights reserved.

Macrophage migration inhibitory factor (MIF) is a cytokine that participates in the host inflammatory response. A Cys-Xaa-Xaa-Cys (CXXC)-based thiol-protein oxidoreductase activity of MIF is associated with certain biological functions. Peptides spanning the CXXC region of thiol-protein oxidoreductases retain some biochemical properties of the full-length protein. We report on the characterization of CXXC-spanning MIF-(50-65) and its serine variant, C57S/C60S-MIF-(50-65). Following disulfide-mediated cyclization, MIF-(50-65) adapted a beta-turn conformation comparable with that of beta-turn-containing cyclo-57,60-[Asp57,Dap60]MIF-(50-65). MIF-(50-65) had a redox potential E'0 of -0.258 V and formed mixed disulfides with glutathione and cysteine. MIF-(50-65) but not C57S/C60S-MIF-(50-65) had oxidoreductase activity in vitro. Intriguingly, MIF-(50-65) exhibited MIF-like cellular activities. The peptide but not its variant had glucocorticoid overriding and proliferation-enhancing activity and stimulated ERK1/2 phosphorylation. MIF-(50-65) and its variant bound to the MIF-binding protein JAB1 and enhanced cellular levels of p27Kip1. As the peptide and its variant were endocytosed at similar efficiency, sequence 50-65 appears sufficient for the JAB1-related effects of MIF, whereas other activities require CXXC. Cyclo-57,60-[Asp57,Dap60]MIF-(50-65) activated ERK1/2, indicating that CXXC-dependent disulfide and beta-turn formation is associated with an activity-inducing conformation. We conclude that CXXC and sequence 50-65 are critical for the activities of MIF. MIF-(50-65) is a surprisingly short sequence with MIF-like functions that could be an excellent molecular template for MIF therapeutics.

BACKGROUND AND AIMS: The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models. METHODS: The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart. RESULTS: The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p

Contains fulltext : 118070.pdf (Publisher’s version ) (Open Access) Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFkappaB regulated mediators (i.e. IL-6 (P

Scope: Dietary intake of cocoa and/or chocolate has been suggested to exhibit protective cardiovascular effects although this is still controversial. The aim of this study was to investigate the effects of chocolate supplementation on metabolic and cardiovascular parameters. Methods and results: Four groups of ApoE*3Leiden mice were exposed to the following diet regimens. Group 1: cholesterol-free control diet (CO). Group 2: high-dose (1.0% w/w) control cholesterol (CC). Group 3: CC supplemented chocolate A (CCA) and Group 4: CC supplemented chocolate B (CCB). Both chocolates differed in polyphenol and fiber content, CCA had a relatively high-polyphenol and low-fiber content compared to CCB. Mice fed a high-cholesterol diet showed increased plasma-cholesterol and developed atherosclerosis. Both chocolate treatments, particularly CCA, further increased plasma-cholesterol and increased atherosclerotic plaque formation. Moreover, compared to mice fed a high-cholesterol diet, both chocolate-treated groups displayed increased liver injury. Mice on high-cholesterol diet had elevated plasma levels of sVCAM-1, sE-selectin and SAA, which was further increased in the CCB group. Similar effects were observed for renal inflammation markers. Conclusion: The two chocolate preparations showed unfavorable, but different effects on cardiometabolic health in E3L mice, which dissimilarities may be related to differences in chocolate composition. We conclude that discrepancies reported on the effects of chocolate on cardiometabolic health may at least partly be due to differences in chocolate composition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Background:Chronic metabolic overload results in lipid accumulation and subsequent inflammation in white adipose tissue (WAT), often accompanied by non-alcoholic fatty liver disease (NAFLD). In response to metabolic overload, the expression of genes involved in lipid metabolism and inflammatory processes is adapted. However, it still remains unknown how these adaptations in gene expression in expanding WAT and liver are orchestrated and whether they are interrelated.Methodology/Principal Findings:ApoE*3Leiden mice were fed HFD or chow for different periods up to 12 weeks. Gene expression in WAT and liver over time was evaluated by micro-array analysis. WAT hypertrophy and inflammation were analyzed histologically. Bayesian hierarchical cluster analysis of dynamic WAT gene expression identified groups of genes ('clusters') with comparable expression patterns over time. HFD evoked an immediate response of five clusters of 'lipid metabolism' genes in WAT, which did not further change thereafter. At a later time point (>6 weeks), inflammatory clusters were induced. Promoter analysis of clustered genes resulted in specific key regulators which may orchestrate the metabolic and inflammatory responses in WAT. Some master regulators played a dual role in control of metabolism and inflammation. When WAT inflammation developed (>6 weeks), genes of lipid metabolism and inflammation were also affected in corresponding livers. These hepatic gene expression changes and the underlying transcriptional responses in particular, were remarkably similar to those detected in WAT.Conclusion:In WAT, metabolic overload induced an immediate, stable response on clusters of lipid metabolism genes and induced inflammatory genes later in time. Both processes may be controlled and interlinked by specific transcriptional regulators. When WAT inflammation began, the hepatic response to HFD resembled that in WAT. In all, WAT and liver respond to metabolic overload by adaptations in expression of gene clusters that control lipid metabolism and inflammatory processes in an orchestrated and interrelated manner. © 2013 Liang et al.

A high-fat diet disturbs the composition and function of the gut microbiota and generates local gut-associated and also systemic responses. Intestinal mast cells, for their part, secrete mediators which play a role in the orchestration of physiological and immunological functions of the intestine. Probiotic bacteria, again, help to maintain the homeostasis of the gut microbiota by protecting the gut epithelium and regulating the local immune system. In the present study, we explored the effects of two probiotic bacteria, Lactobacillus rhamnosus GG (GG) and Propionibacterium freudenreichii spp. shermanii JS (PJS), on high fat-fed ApoE*3Leiden mice by estimating the mast cell numbers and the immunoreactivity of TNF-α and IL-10 in the intestine, as well as plasma levels of several markers of inflammation and parameters of lipid metabolism. We found that mice that received GG and PJS exhibited significantly lower numbers of intestinal mast cells compared with control mice. PJS lowered intestinal immunoreactivity of TNF-α, while GG increased intestinal IL-10. PJS was also observed to lower the plasma levels of markers of inflammation including vascular cell adhesion molecule 1, and also the amount of gonadal adipose tissue. GG lowered alanine aminotransferase, a marker of hepatocellular activation. Collectively, these data demonstrate that probiotic GG and PJS tend to down-regulate both intestinal and systemic pro-inflammatory changes induced by a high-fat diet in this humanised mouse model.

24 hours after an i.v. injection of 2 mg Sephadex G 200® particels ovalbumin sensitized Sprague Dawley rats show an antigen specific bronchial hyperreactivity and an unspecific hyperreactivity against serotonin. The aim of this study was to investigate the effects of Sephadex on blood parameters and lung pathology to find the morphological substrate of bronchial hyperreactivity in this animal model. In the blood neutrophilia (p < 0.01) but no eosinophilia was present. We conclude that a blood eosinophilia needs not to be necessarily correlated with hyperreactivity of the airways like claimed by other investigators for this animal model. Histologically we found that Sephadex particels are trapped in smaller-diameter arteries of the lung and lead to a granulomatous arteritis consisting mainly of ED1 positive and widely ED2 negative macrophages interspersed with eosinophils and neutrophils. Larger vessels not occluded by particels showed perivascular oedema with infiltration of eosinophils. We report here for the first time a significant hypertrophy of PAS positive goblet cells (p < 0.01) accompanied by a peribronchial infiltration with eosinophils (p < 0.01) and macrophages positive for ED1, ED2 and Ox-6 (p < 0.01) but not Ox-19 positive T-lymphocytes. The authors suggest that the peribronchial inflammation contributes importantly to the onset of bronchial hyperreactivity in this animal model and that the hypertrophy of goblet cells indicates the pathophysiological importance of peribronchial leukocytes.

Aims: Systemic complications after cardiac surgery are common in heart failure patients. However, the pathophysiological mechanisms, such as a different local inflammatory response of failing hearts, remain in question. This study examines whether failing hearts respond differently to cardioplegic arrest and reperfusion compared with non-failing hearts (controls). Methods and results: The inflammatory response was evaluated in samples collected simultaneously from the radial artery and coronary sinus, and myocardial tissue in 62 patients undergoing cardiac surgery. No myocardial release of inflammatory mediators was observed upon reperfusion in controls (n ¼ 19). In contrast, in patients with heart failure, reperfusion was characterized by a myocardial release of several cytokines. Myocardial interleukin-6 was 115% increased in non-is-chaemic heart failure (n ¼ 18, P ¼ 0.002) as compared with a 117% increase in patients with ischaemic heart failure (n ¼ 25, P ¼ 0.01). Furthermore, a myocardial release of monocyte chemoattractant protein-1 was observedin both patient groups: a 109% (P ¼ 0.001) and 114% (P ¼ 0.01) increase in patients with non-ischaemic heart failure and ischaemic heart failure, respectively. Post-operative myocardial damage, expression of inflammatory mediators, and p65-nuclear factor-kB activity were similar in all patient groups. Inflammatory cell content was increased in early ischaemic myocardial tissue in both heart failure groups compared with controls. Conclusion: Heart failure patients show a clear myocardial inflammatory response upon reperfusion, probably explained by degranulation of infiltrated inflammatory cells. Results in controls indicate that they better withstand cardioplegic arrest and reperfusion without an explicit myocardial inflammatory response.

AIMS: Combination-drug therapy takes advantage of the complementary action of their individual components, thereby potentiating its therapeutic effect. Potential disadvantages include side effects that are not foreseen on basis of the data available from drug monotherapy. Here, we used a systems biology approach to understand both the efficacy and the side effects of a cholesterol-lowering drug-combination therapy on the basis of the biological pathways and molecular processes affected by each drug alone or in combination. METHODS AND RESULTS: ApoE*3Leiden transgenic mice, a mouse model with human-like cholesterol-lowering drug responses, were treated with rosuvastatin and ezetimibe, alone and in combination. Analyses included functional responses, viz. effects on cardiovascular risk factors, inflammation, and atherosclerosis, and measurement of global gene expression, and identification of enriched biological pathways and molecular processes. Combination therapy reduced plasma cholesterol, plasma inflammation markers, and atherosclerosis stronger than the single drugs did. Systems biology analysis at the level of biological processes shows that the therapeutic benefit of combined therapy is largely the result of additivity of the complementary mechanisms of action of the two single drugs. Importantly, combination therapy also exerted a significant effect on 16 additional and mostly NF-κB-linked signaling processes, 11 of which tended to be regulated in a similar direction with monotherapy. CONCLUSION: This study shows that gene expression analysis together with bioinformatics pathway analysis has the potential to help predict and identify drug combination-specific complementary and side effects. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

We introduce the metabolomics and proteomics based Postprandial Challenge Test (PCT) to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The PCT comprised consumption of a standardized 500 ml dairy shake containing respectively 59, 30 and 12 energy percent lipids, carbohydrates and protein. During a 6 h time course after PCT 145 plasma metabolites, 79 proteins and 7 clinical chemistry parameters were quantified. Multiple processes related to metabolism, oxidation and inflammation reacted to the PCT, as demonstrated by changes of 106 metabolites, 31 proteins and 5 clinical chemistry parameters. The PCT was applied in a dietary intervention study to evaluate if the PCT would reveal additional metabolic changes compared to non-perturbed conditions. The study consisted of a 5-week intervention with a supplement mix of anti-inflammatory compounds in a crossover design with 36 overweight subjects. Of the 231 quantified parameters, 31 had different responses over time between treated and control groups, revealing differences in amino acid metabolism, oxidative stress, inflammation and endocrine metabolism. The results showed that the acute, short term metabolic responses to the PCT were different in subjects on the supplement mix compared to the controls. The PCT provided additional metabolic changes related to the dietary intervention not observed in non-perturbed conditions. Thus, a metabolomics based quantification of a standardized perturbation of metabolic homeostasis is more informative on metabolic status and subtle health effects induced by (dietary) interventions than quantification of the homeostatic situation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0320-5) contains supplementary material, which is available to authorized users.

Objective Obesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress–induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet–induced OA in mice. Methods Human C-reactive protein (CRP)–transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined. Results Male mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet–induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point. Conclusion High-fat diet–induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator–activated receptor γ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.

BackgroundAn Alternating high- cholesterol dietary regimen has proven to be beneficial when compared to daily high- cholesterol feeding. In the current study we explored whether the same strategy is applicable to a high- fat dietary regimen.ObjectiveTo investigate whether an alternating high- fat dietary regimen can effectively diminish insulin resistance, hepatic and renal inflammation and renal dysfunction as compared to a continuous high- fat diet.DesignFour groups of male ApoE*3Leiden mice (n=15) were exposed to different diet regimens for 20 weeks as follows: Group 1: low- fat diet (10 kcal% fat); Group 2: intermediate- fat diet (25 kcal% fat); Group 3: high- fat diet (45 kcal% fat) and Group 4: alternating- fat diet (10 kcal% fat for 4 days and 45 kcal% fat for 3 days in a week).ResultsCompared to high fat diet feeding, the alternating and intermediate- fat diet groups had reduced body weight gain and did not develop insulin resistance or albuminuria. In addition, in the alternating and intermediate- fat diet groups, parameters of tissue inflammation were markedly reduced compared to high fat diet fed mice.ConclusionBoth alternating and intermediate- fat feeding were beneficial in terms of reducing body weight gain, insulin resistance, hepatic and renal inflammation and renal dysfunction. Thus beneficial effects of alternating feeding regimens on cardiometabolic risk factors are not only applicable for cholesterol containing diets but can be extended to diets high in fat content.