The liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol,... Show moreThe liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol, a final intermediate in cholesterol biosynthesis, has been identified as a selective LXR ligand that suppresses inflammation without inducing lipogenesis. Δ24-Dehydrocholesterol reductase (DHCR24) converts desmosterol into cholesterol, and we previously showed that the DHCR24 inhibitor SH42 increases desmosterol to activate LXR and attenuate experimental peritonitis and metabolic dysfunction-associated steatotic liver disease. Here, we aimed to evaluate the effect of SH42 on atherosclerosis development in APOE∗3-Leiden.CETP mice and low-density lipoproteins (LDL) receptor knockout mice, models for lipid- and inflammation-driven atherosclerosis, respectively. In both models, SH42 increased desmosterol without affecting plasma lipids. While reducing liver lipids in APOE∗3-Leiden.CETP mice, and regulating populations of circulating monocytes in LDL receptor knockout mice, SH42 did not attenuate atherosclerosis in either model. Show less
Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA... Show moreUnsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function. Show less
Broos, J.Y.; Loonstra, F.C.; Ruiter, L.R.J. de; Gouda, M.; Fung, W.H.; Schoonheim, M.M.; ... ; Kooij, G. 2023
Background and ObjectivesExcessive activation of certain lipid mediator (LM) pathways plays a role in the complexpathogenesis of multiple sclerosis (MS). However, the relationship between bioactive... Show moreBackground and ObjectivesExcessive activation of certain lipid mediator (LM) pathways plays a role in the complexpathogenesis of multiple sclerosis (MS). However, the relationship between bioactive LMs anddifferent aspects of CNS-related pathophysiologic processes remains largely unknown.Therefore, in this study, we assessed the association of bioactive LMs belonging to the ω-3/ω-6lipid classes with clinical and biochemical (serum neurofilament light [sNfL] and serum glialfibrillary acidic protein [sGFAP]) parameters and MRI-based brain volumes in patients withMS (PwMS) and healthy controls (HCs).MethodsA targeted high-performance liquid chromatography-tandem mass spectrometry approach was usedon plasma samples of PwMS and HCs of the Project Y cohort, a cross-sectional population-basedcohort that contains PwMS all born in 1966 in the Netherlands and age-matched HCs. LMs werecompared between PwMS and HCs and were correlated with levels of sNfL, sGFAP, disability(Expanded Disability Status Scale [EDSS]), and brain volumes. Finally, significant correlates wereincluded in a backward multivariate regression model to identify which LMs best related to disability.ResultsThe study sample consisted of 170 patients with relapsing remitting MS (RRMS), 115 patientswith progressive MS (PMS), and 125 HCs. LM profiles of patients with PMS significantly differedfrom those of patients with RRMS and HCs, particularly patients with PMS showed elevatedlevels of several arachidonic acid (AA) derivatives. In particular, 15-hydroxyeicosatetraenoic acid(HETE) (r = 0.24, p < 0.001) correlated (average r = 0.2, p < 0.05) with clinical and biochemicalparameters such as EDSS and sNfL. In addition, higher 15-HETE levels were related to lowertotal brain (r = −0.24, p = 0.04) and deep gray matter volumes (r = −0.27, p = 0.02) in patientswith PMS and higher lesion volume (r = 0.15, p = 0.03) in all PwMS.DiscussionIn PwMS of the same birth year, we show that ω-3 and ω-6 LMs are associated with disability,biochemical parameters (sNfL, GFAP), and MRI measures. Furthermore, our findings indicatethat, particularly, in patients with PMS, elevated levels of specific products of the AA pathway,such as 15-HETE, associate with neurodegenerative processes. Our findings highlight thepotential relevance of ω-6 LMs in the pathogenesis of MS. Show less
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... Show moreShort-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. Show less
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... Show moreShort-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. Show less
Gagestein, B.; von Hegedus, J.H.; Kwekkeboom, J.C.; Heijink, M.; Blomberg, N.; Wel, T. van der; ... ; Stelt, M. van der 2022
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... Show moreThe 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. Show less
Gagestein, B.; Hegedus, J.H. von; Kwekkeboom, J.C.; Heijink, M.; Blomberg, N.; Wel, T. van der; ... ; Stelt, M. van der 2022
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... Show moreThe 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. Show less
GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by omega-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of... Show moreGPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by omega-3 Polyunsaturated Fatty Acids (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. 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. Our work for the first time highlights 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 beta -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. 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. Show less
Jensen, K.N.; Heijink, M.; Giera, M.; Freysdottir, J.; Hardardottir, I. 2022
Purpose: To determine the effects of dietary omega-3 polyunsaturated fatty acids (PUFAs) on recruitment of natural killer (NK) cells and resolution responses in antigen-induced peritonitis in mice.... Show morePurpose: To determine the effects of dietary omega-3 polyunsaturated fatty acids (PUFAs) on recruitment of natural killer (NK) cells and resolution responses in antigen-induced peritonitis in mice. Methods: Mice were fed fish oil-enriched or control diets, immunized twice and challenged intraperitoneally with methylated bovine serum albumin. Prior to and at different time-points following inflammation induction, expression of surface molecules on peritoneal cells was determined by flow cytometry, concentration of soluble mediators in peritoneal fluid by ELISA or Luminex, and of lipid mediators by LC-MS/MS, and number of apoptotic cells in mesenteric lymph nodes by TUNEL staining. Results: Mice fed the fish oil diet had higher number of CD11b(+)CD27(-) NK cells as well as a higher proportion of CD107a(+) NK cells in their peritoneum 6 h after inflammation induction than mice fed the control diet. They also had higher numbers of CCR5(+) NK cells and higher concentrations of CCL5 and CXCL12. Additionally, a higher fraction of apoptotic neutrophils but lower fraction of CD47(+) neutrophils were present in the peritoneum of mice fed the fish oil diet 6 h after inflammation induction and the fish oil fed mice had a shorter resolution interval. They also had lower concentrations of pro-inflammatory mediators but higher concentrations of the anti-inflammatory/pro-resolution mediators TGF-beta, IGF-1, and soluble TNF RII, as well as higher ratios of hydroxyeicosapentaenoic acid (HEPE) to hydroxyeicosatetraenoic acid (HETE) than mice fed the control diet. Conclusion: The results demonstrate that dietary fish oil increases the number of mature NK cells at the inflamed site in antigen-induced peritonitis and enhances several key hallmarks of resolution of inflammation, casting light on the potential mechanisms involved. Show less
Manders, M.; Dissel, A.M.C. van; Brouwers, W.; Fink, M.; Spoelstra, J.; Hoop, R. de; ... ; Vroomen, O. de 2022
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... Show moreThe 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. Show less
Kokova, D.; Verhoeven, A.; Perina, E.A.; Ivanov, V.V.; Heijink, M.; Yazdanbakhsh, M.; Mayboroda, O.A. 2021
Opisthorchiasis, is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. A chronic form of the disease implies a prolonged coexistence of a host and the parasite. The... Show moreOpisthorchiasis, is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. A chronic form of the disease implies a prolonged coexistence of a host and the parasite. The pathological changes inflicted by the worm to the host's hepatobiliary system are well documented. Yet, the response to the infection also triggers a deep remodeling of the host systemic metabolism reaching a new homeostasis and affecting the organs beyond the worm location. Understanding the metabolic alternation in chronic opisthorchiasis, could help us to pinpoint pathways that underlie infection opening possibilities for the development of more selective treatment strategies. Here, with this report we apply an integrative, multicompartment metabolomics analysis, using multiple biofluids, stool samples and tissue extracts to describe metabolic changes in Opisthorchis felineus infected animals at the chronic stage. We show that the shift in lipid metabolism in the serum, a depletion of the amino acids pool, an alteration of the ketogenic pathways in the jejunum and a suppressed metabolic activity of the spleen are the key features of the metabolic host adaptation at the chronic stage of O. felineus infection. We describe this combination of the metabolic changes as a "metabolically mediated immunosuppressive status of organism" which develops during a chronic infection. This status in combination with other factors (e.g., parasite-derived immunomodulators) might increase risk of infection-related malignancy. Show less
Hegedus, J.H. von; Kahnt, A.S.; Ebert, R.; Heijink, M.; Toes, R.E.M.; Giera, M.; Ioan-Facsinay, A. 2020
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... Show moreInflammation 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 proresolving 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. Show less
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... Show moreRecovery 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. Using in vivo wide-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. Show less
Does, A.M. van der; Heijink, M.; Mayboroda, O.A.; Persson, L.J.; Aanerud, M.; Bakke, P.; ... ; Giera, M. 2019
Introduction: Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be... Show moreIntroduction: 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. Show less
Chakraborty, T.; Thuer, E.; Heijink, M.; Toth, R.; Bodai, L.; Vagvolgyi, C.; ... ; Gacser, A. 2018
