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Short-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being unclear. We aimed to investigate the role of gut microbiota in the induction of metabolic benefits of dietary butyrate. We performed antibiotic-induced microbiota depletion of the gut and fecal microbiota transplantation (FMT) in APOE*3-Leiden.CETP mice, a well-established translational model for developing human-like metabolic syndrome, and revealed that dietary butyrate reduced appetite and ameliorated high-fat diet-induced (HFD-induced) weight gain dependent on the presence of gut microbiota. FMT from butyrate-treated lean donor mice, but not butyrate-treated obese donor mice, into gut microbiota-depleted recipient mice reduced food intake, attenuated HFD-induced weight gain, and improved insulin resistance. 16S rRNA and metagenomic sequencing on cecal bacterial DNA of recipient mice implied that these effects were accompanied by the selective proliferation of Lachnospiraceae bacterium 28-4 in the gut as induced by butyrate. Collectively, our findings reveal a crucial role of gut microbiota in the beneficial metabolic effects of dietary butyrate as strongly associated with the abundance of Lachnospiraceae bacterium 28-4. more...

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights. more...

The use of acellular fish skin grafts (FSG) for the treatment of burn wounds is becoming more common due to its beneficial wound healing properties. In our previous study we demonstarted that FSG is a scaffold biomaterial that is rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) conjugated to phosphatidylcholines. Here we investigated whether EPA and DHA derived lipid mediators are influenced during the healing of burn wounds treated with FSG. Deep partial and full thickness burn wounds (DPT and FT, respectively) were created on Yorkshire pigs (n = 4). DPT were treated with either FSG or fetal bovine dermis while FT were treated either with FSG or cadaver skin initially and followed by a split thickness skin graft. Punch biopsies were collected on days 7, 14, 21, 28 and 60 and analyzed in respect of changes to approximately 45 derivatives of EPA, DHA, arachidonic acid (AA), and linoleic acid (LA) employing UPLC-MS/MS methodology. Nine EPA and DHA lipid mediators, principally mono-hydroxylated derivatives such as 18-HEPE and 17-HDHA, were significantly higher on day 7 in the DPT when treated with FSG. A similar but non-significant trend was observed for the FT. The results suggest that the use of FSG in burn wound treatment can alter the formation of EPA and DHA mono hydroxylated lipid mediators in comparison to other grafts of mammalian origin. The differences observed during the first seven days after treatment indicates that FSG affects the early stages of wound healing. more...

Background&Aims: GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 PUFAs, and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Methods: Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Results: Our work for the first time highlight the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of β -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. Conclusion: these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention. more...

The use of acellular fish skin grafts (FSG) for the treatment of burn wounds is becoming more common due to its beneficial wound healing properties. In our previous study we demonstarted that FSG is a scaffold biomaterial that is rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) conjugated to phosphatidylcholines. Here we investigated whether EPA and DHA derived lipid mediators are influenced during the healing of burn wounds treated with FSG. Deep partial and full thickness burn wounds (DPT and FT respectively) were created on Yorkshire pigs (n=4). DPT were treated with either FSG or fetal bovine dermis while FT were treated either with FSG or cadaver skin initially and followed by a split thickness skin graft. Punch biopsies were collected on days 7, 14, 21, 28 and 60 and analysed in respect of changes to approximately 45 derivatives of EPA, DHA, arachidonic acid (AA), and linoleic acid (LA) employing UPLC-MS/MS methodology. Several EPA and DHA derivatives, including 18-HEPE and 17-HDHA, were significantly higher on day 7 in the DPT when treated with FSG. A similar but non-significant trend was observed for the FT. In addition, prostaglandin F2α and its 15-keto derivative (AA pathway) as well as 13-HODE and 13-HOTrE (LA pathway) were significantly lower in the FSG treated FT. The results suggest that the use of FSG in burn wound treatment can alter the formation of EPA and DHA mono hydroxylated lipid mediators in comparison to other grafts of mammalian origin. The differences observed during the first seven days after treatment indicates that FSG affects the early stages of wound healing.HighlightsThis is the first study to investigate whether omega-3 rich biomaterial such as fish skin graft can affect the lipidome of burn wound healingThe study monitors the formation of lipid mediators during 60 days of burn wound healingThis is the first study to show an increase in the formation of mono-hydroxylated derivatives of EPA and DHA after seven days of treatment with fish skin graftThe study showed significantly decreased formation of lipid mediators involved in pain modulation in wounds treated with fish skin graft more...

Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages. In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the pro-resolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid. These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus. more...

ObjectiveButyrate exerts metabolic benefits in mice and humans, the underlying mechanisms being still unclear. We aimed to investigate the effect of butyrate on appetite and energy expenditure, and to what extent these two components contribute to the beneficial metabolic effects of butyrate.DesignAcute effects of butyrate on appetite and its method of action were investigated in mice following an intragastric gavage or intravenous injection of butyrate. To study the contribution of satiety to the metabolic benefits of butyrate, mice were fed a high-fat diet with butyrate, and an additional pair-fed group was included. Mechanistic involvement of the gut-brain neural circuit was investigated in vagotomised mice.ResultsAcute oral, but not intravenous, butyrate administration decreased food intake, suppressed the activity of orexigenic neurons that express neuropeptide Y in the hypothalamus, and decreased neuronal activity within the nucleus tractus solitarius and dorsal vagal complex in the brainstem. Chronic butyrate supplementation prevented diet-induced obesity, hyperinsulinaemia, hypertriglyceridaemia and hepatic steatosis, largely attributed to a reduction in food intake. Butyrate also modestly promoted fat oxidation and activated brown adipose tissue (BAT), evident from increased utilisation of plasma triglyceride-derived fatty acids. This effect was not due to the reduced food intake, but explained by an increased sympathetic outflow to BAT. Subdiaphragmatic vagotomy abolished the effects of butyrate on food intake as well as the stimulation of metabolic activity in BAT.ConclusionButyrate acts on the gut-brain neural circuit to improve energy metabolism via reducing energy intake and enhancing fat oxidation by activating BAT. more...

Critical conditions such as sepsis following infection or traumatic injury disturb the complex state of homeostasis that may lead to uncontrolled inflammation resulting in organ failure, shock and death. They are associated with endogenous mediators that control the onset of acute inflammatory response, but the central problem remains the complete resolution of inflammation. Omega-3 enriched lipid emulsions (Ω-3+ LEs) were used in experimental studies and clinical trials to establish homeostasis, yet with little understanding about their role on the resolution of inflammation and tissue regeneration. Here, we demonstrate that Ω-3 lipid emulsions (LEs) orchestrate inflammation-resolution/regeneration mechanism during sterile peritonitis and murine polymicrobial sepsis. Ω-3+ LEs recessed neutrophil infiltration, reduced pro-inflammatory mediators, reduced the classical monocyte and enhanced the non-classical monocytes/macrophages recruitment and finally increased the efferocytosis in sepsis. The actions of Ω-3+ LE were 5-lipoxygenase (5-LOX) and 12/15-lipoxygenase (12/15-LOX) dependent. Ω-3+ LEs shortened the resolution interval by 56%, stimulated the endogenous biosynthesis of resolution mediators lipoxin A4, protectin DX and maresin 1 and contributed to tissue regeneration. Ω-3+ LEs protected against hypothermia and weight loss and enhanced survival in murine polymicrobial sepsis. We highlighted a role of Ω-3+ LEs in regulating key mechanisms within the resolution terrain during murine sepsis. This might form the basis for a rational design of sepsis specific clinical nutrition. more...

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut–brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut–brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Usingin vivowide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENTPrevious studies have shown a bidirectional communication along the gut–brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut–brain axis and as a potential therapeutic to improve recovery after stroke. more...

Introduction Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be converted into lipid mediators here collectively named oxylipins. These include classical eicosanoids, but also pro-resolving mediators. A balanced production of pro-inflammatory and pro-resolving oxylipins is of importance for adequate inflammatory responses and subsequent return to homeostasis. Objectives Here we investigated if PUFA metabolism is disturbed in COPD patients. Methods Free PUFA and oxylipin levels were measured in induced sputum samples from the Bergen COPD cohort and COPD exacerbation study using liquid chromatography-mass spectrometry. Additionally, effects of whole cigarette smoke on PUFA metabolism in air-liquid interface cultures of primary bronchial epithelial cells were assessed. Results Significantly lower levels of free alpha-linolenic acid, linoleic acid and eicosapentaenoic acid (EPA) were detected in sputum from stable COPD patients compared to controls. During acute exacerbation (AE), levels of free arachidonic acid and docosapentaenoic acid were higher than in stable COPD patients. Furthermore, levels of omega-3 EPA- and docosahexaenoic acid-derived oxylipins were lower in sputum from stable COPD patients compared to controls. Cyclooxygenase-2-converted mediators were mostly increased during AE. In vitro studies additionally showed that cigarette smoke exposure may also directly contribute to altered epithelial PUFA metabolism, and indirectly by causing airway epithelial remodelling. Conclusions Our findings show significant differences in PUFA metabolism in COPD patients compared to controls, further changed during AE. Airway epithelial remodelling may contribute to these changes. These findings provide new insight in impaired inflammatory resolution in COPD. more...

Gut microbiota have been implicated in the development of atherosclerosis and cardiovascular disease. Since the prebiotic inulin is thought to beneficially affect gut microbiota, we aimed to determine the effect of inulin supplementation on atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice. Female E3L.CETP mice were fed a western-type diet containing 0.1% or 0.5% cholesterol with or without 10% inulin. The effects of inulin were determined on: microbiota composition, cecal short-chain fatty acid (SCFA) levels, plasma lipid levels, atherosclerosis development, hepatic morphology and hepatic inflammation. Inulin with 0.5% dietary cholesterol increased specific bacterial genera and elevated levels of cecal SCFAs, but did not affect plasma cholesterol levels or atherosclerosis development. Surprisingly, inulin resulted in mild hepatic inflammation as shown by increased expression of inflammation markers. However, these effects were not accompanied by increased hepatic macrophage number. Analogously, inulin induced mild steatosis and increased hepatocyte size, but did not affect hepatic triglyceride content. Inulin with 0.1% dietary cholesterol did not affect hepatic morphology, nor hepatic expression of inflammation markers. Overall, inulin did not reduce hypercholesterolemia or atherosclerosis development in E3L.CETP mice despite showing clear prebiotic activity, but resulted in manifestations of hepatic inflammation when combined with a high percentage of dietary cholesterol. more...

Our body contains a wide variety of fatty acids that differ in chain length, the degree of unsaturation, and location of the double bonds. As the various fatty acids play distinct roles in health and disease, methods that can specifically determine the fatty acid profile are needed for fundamental and clinical studies. Here we describe a method for the separation and quantification of fatty acids ranging from 8 to 24 carbon chain lengths in blood samples using gas chromatography-mass spectrometry following derivatization using pentafluorobenzyl bromide. This method quantitatively monitors fatty acid composition in a manner that satisfies the requirements for comprehensiveness, sensitivity, and accuracy. more...

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid. more...

Our body contains a wide variety of fatty acids that differ in chain length, the degree of unsaturation, and location of the double bonds. As the various fatty acids play distinct roles in health and disease, methods that can specifically determine the fatty acid profile are needed for fundamental and clinical studies. Here we describe a method for the separation and quantification of fatty acids ranging from 8 to 24 carbon chain lengths in blood samples using gas chromatography-mass spectrometry following derivatization using pentafluorobenzyl bromide. This method quantitatively monitors fatty acid composition in a manner that satisfies the requirements for comprehensiveness, sensitivity, and accuracy. more...

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid. more...

Tobacco smoke is the main risk factor for development of Chronic Obstructive Pulmonary Disease (COPD), a disease characterized by chronic airway inflammation and tissue remodelling resulting in loss of lung function. Airway inflammation in COPD persists even after smoking cessation, suggesting a problem with inflammatory resolution. The resolution phase of inflammation is initiated by pro-resolving compounds, derived from e.g. polyunsaturated fatty acids (PUFAs) that are stored in cell membranes and uncoupled upon inflammatory triggering. PUFAs are subsequently converted into lipid mediators such as pro-inflammatory leukotrienes and prostaglandins, or into pro-resolving mediators such as protectins and resolvins. To investigate if fatty acid metabolism was affected in patients with stable COPD, and during or after an acute exacerbation, we analysed induced sputum samples from the Bergen COPD Cohort and COPD Exacerbation study. Induced sputum from 27 controls without COPD, 29 stable COPD patients, 37 COPD patients during exacerbation and 27 COPD patients post exacerbation underwent LC-MS/MS analysis to determine free fatty acid (FFA) and oxylipin levels. Analysis revealed lower FFA levels in sputum from stable COPD patients compared to controls. Furthermore, 5- and 12-lipoxygenase (LOX)-mediated oxylipin levels were generally reduced in COPD patients compared to controls. 15-LOX and cyclooxygenase-2-mediated oxylipin levels (e.g. prostaglandins and TXB2) were comparable between stable COPD patients and control, however increased during exacerbation, some of which remained elevated thereafter. Our findings show marked differences in fatty acid metabolism in COPD patients that may contribute to future strategies to promote inflammatory resolution in the lungs of COPD patients. more...

Background Traditionally, immunological responses have been studied in-vitro whereby immune cells are subject to certain stimuli following which alterations in RNA and/or protein expression are measured. Recently, we have challenged this model by showing that mast cells (cells specialised in allergic reactions) triggered repeatedly through their Ig Fc epsilon receptor undergo a reprogramming of their responses (Suurmond et al. JACI, 2016). The differential expression of genes upon a single trigger were dampened and de-novo transcribed genes were observed. The latter belonged to the antigen presentation and pathogen defence response pathways implying a dramatic change in the cellular response mode. We have now extended this work by challenging mast cells with plate-bound IgG in a repeated mode, a model relevant for arthritic diseases where mast cells and IgG immune complexes are likely to interact. Materials and methods Human cord blood-derived mast cells were treated for 2 weeks with plate-bound IgG. The expression profile of naive or treated mast cells was measured through RNA sequencing, quantitative RT-PCR, flow cytometry. Protein secretion was measured with ELISA and Luminex assays. Metabolic changes were measured using HPLC mass-spectrometry. Results Similar to our previous work on Fc Epsilon receptor, we observe a dampening of the normal IgG responses with a set of novel genes upregulated. Interestingly, de-novo expressed genes consisted of DHCR7 and DHCR24, key enzymes in the cholesterol pathway. Pathway analysis confirms an enrichment of genes in this pathway following repeated IgG triggering. Preliminary data on metabolic profiling reveals a decrease in phospholipid levels in repeatedly activated mast cells. Conclusions Our study provides evidence that mast cells are also reprogrammed upon repeated IgG triggering. In contrast to repeated Fc Epsilon Receptor triggering, different pathways are affected, implying stimulus-specific effects. Our work has important implications for revisiting the traditional immunological models in the (in-vitro) modelling of chronic (arthritic) diseases. more...