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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.

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.

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

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.

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.

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.

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.

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.