Full thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin... Show moreFull thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin barrier is located in the stratum corneum (SC) and consists of corneocytes embedded in a lipid matrix. In this matrix, deviations in the composition of the FTMs lipid matrix may contribute to the impaired skin barrier when compared to NHS. One of the most abundant changes in lipid composition is an increase in monounsaturated lipids for which stearoyl-CoA desaturase-1 (SCD-1) is responsible. To improve the SC lipid composition, we reduced SCD-1 activity during the generation of the FTMs. These FTMs were subsequently assessed on all major aspects, including epidermal homeostasis, lipid composition, lipid organization, and barrier functionality. We demonstrate that SCD-1 inhibition was successful and resulted in FTMs that better mimic the lipid composition of FTMs to NHS by a significant reduction in monounsaturated lipids. In conclusion, this study demonstrates an effective approach to normalize SC monounsaturated lipid concentration and may be a valuable tool in further optimizing the FTMs in future studies. Show less
Sluis, R.J. van der; Depuydt, M.A.C.; Eck, M. van; Hoekstra, M. 2020
The contribution of individual lipoprotein species to the generation of the adrenal cholesterol pool used for the synthesis of anti-inflammatory glucocorticoid species remains unknown. Here we... Show moreThe contribution of individual lipoprotein species to the generation of the adrenal cholesterol pool used for the synthesis of anti-inflammatory glucocorticoid species remains unknown. Here we examined the impact of specific lowering of very low-density lipoprotein (VLDL) and low-density (LDL) levels on adrenal cholesterol and glucocorticoid homeostasis. Hereto, lethally-irradiated hypercholesterolemic apolipoprotein E (APOE) knockout mice received APOE-containing bone marrow from wild-type mice (n = 6) or APOE knockout control bone marrow (n = 10) and were subsequently fed a regular chow diet. Transplantation with wild-type bone marrow was associated with a 10-fold decrease in VLDL/LDL-cholesterol levels. No changes were observed in adrenal weights, adrenal cholesterol content, or basal plasma corticosterone levels. However, food deprivation-induced corticosterone secretion was 64% lower (P < 0.05) in wild-type bone marrow recipients as compared to APOE knockout bone marrow recipients, in the context of similar plasma adrenocorticotropic hormone (ACTH) levels. A parallel 19-29% decrease in adrenal relative mRNA expression levels of ACTH-responsive genes SR-BI (P < 0.01), STAR (P < 0.05), and CYP11A1 (P < 0.05) was detected. In support of relative glucocorticoid insufficiency, blood lymphocyte and eosinophil concentrations were respectively 2.4-fold (P < 0.01) and 8-fold (P < 0.001) higher in wild-type bone marrow recipients under food deprivation stress conditions. In conclusion, we have shown that a selective lowering of VLDL/LDL levels in APOE knockout mice through a transplantation with APOE-containing wild-type bone marrow is associated with a decreased maximal adrenal glucocorticoid output. Our studies provide experimental support for the hypothesis that, in vivo, VLDL/LDL serves as the primary source of cholesterol used for glucocorticoid synthesis during food deprivation stress. Show less
Boer, D.E.C.; Smeden, J. van; Al-Khakany, H.; Melnik, E.; Dijk, R. van; Absalah, S.; ... ; Bouwstra, J.A. 2020
Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin... Show morePatients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs). We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier. Show less
Martins Cardoso, R.; Creemers, E.; Absalah, S.; Hoekstra, M.; Gooris, G.S.; Bouwstra, J.A.; Eck, M. van 2019
Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to... Show moreScavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI−/−) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI−/− epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI−/− mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins. Show less
Liu, J.; Kuipers, E.N.; Sips, H.C.M.; Dorleijn, J.C.; Dam, A.D. van; Christodoulides, C.; ... ; Kooijman, S. 2019
Brown adipose tissue (BAT) is a potential target to treat cardiometabolic disorders because of its capacity to combust glucose and fatty acids for thermoregulation. Its cellular and molecular... Show moreBrown adipose tissue (BAT) is a potential target to treat cardiometabolic disorders because of its capacity to combust glucose and fatty acids for thermoregulation. Its cellular and molecular investigation in humans is hampered by the limited availability of cell material and the heterogeneity of BAT between and within individuals. In this study, monoclonal lines of conditionally immortalized brown preadipocytes (iBPAs) of mouse and human origin were generated. Conditional immortalization was achieved by doxycycline-controlled expression of simian virus 40 large tumor antigen (LT) with a repressor-based Tet-On system. In the presence of doxycycline, both the murine and human cell lines showed long-term proliferation capacity with a population doubling time of similar to 28 h. After switching off LT expression by doxycycline removal and exposure to adipogenic differentiation medium, cells from both species acquired brown adipocyte properties. This was evidenced by the accumulation of multilocular lipid droplets, the upregulation of brown adipocyte markers including uncoupling protein 1 and an increase in lipolysis and oxygen consumption following adrenergic stimulation. Switching off LT expression before the onset of adipogenic differentiation was only critical for inducing adipogenesis in the human iBPAs, while their murine counterparts showed adipogenesis upon exposure to the adipogenic differentiation cocktail regardless of LT expression. When switched to proliferation medium, cultures of adipogenically differentiated human iBPAs de-differentiated and resumed cell division without losing their adipogenic capacity. We suggest that iBPAs represent an easy-to-use model for fundamental and applied research into BAT offering unique experimental opportunities due to their capacity to switch between proliferative and differentiated states. Show less
Helder, R.W.J.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; El Ghalbzouri, A.; Bouwstra, J.A. 2019
Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a... Show moreFull thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients. Show less
Berkers, M.A.J.; Boiten, W.A.; Absalah, S.; Smeden, J. van; Lavrijsen, A.P.M.; Bouwstra, J.A. 2019
Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics... Show moreEx vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases. Show less
Rakhshandehroo, M.; Eijkeren, R.J. van; Gabriel, T.L.; Haar, C. de; Gijzel, S.M.W.; Hamers, N.; ... ; Kalkhoven, E. 2019
Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell... Show moreBackground: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. beta-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFN gamma and 114 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes. Show less
Boiten, W.A.; Helder, R.W.J.; Smeden, J. van; Bouwstra, J.A. 2019
The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the... Show moreThe cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized. Show less
Does, A.M. van der; Heijink, M.; Mayboroda, O.A.; Persson, L.J.; Aanerud, M.; Bakke, P.; ... ; Giera, M. 2019
Long-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in... Show moreLong-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in response to hypercholesterolemia are however unknown. In this study, we investigated the skin lipid composition and associated barrier function in young adult low-density lipoprotein receptor knockout (LDLR−/−) and apolipoprotein E knockout (APOE−/−) mice, two commonly used hypercholesterolemic mouse models characterized by the accumulation of apolipoprotein B containing lipoproteins. No effects were observed on cholesterol content in the epidermis in LDLR−/− mice nor in the more extremely hypercholesterolemic APOE−/− mice. Interestingly, the free fatty acids in the APOE−/− epidermis shifted towards shorter and unsaturated chains. Genes involved in the synthesis of cholesterol and fatty acids were downregulated in APOE−/− skin suggesting a compensation for the higher influx of plasma lipids, most probably as cholesteryl esters. Importantly, in vivo transepidermal water loss and permeability studies with murine lipid model membranes revealed that the lipid composition of the APOE−/− skin resulted in a reduced skin barrier function. In conclusion, severe hypercholesterolemia associated with increased apolipoprotein B containing lipoproteins affects the epidermal lipid composition and its protective barrier. 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
Rakhshandehroo, M.; Eijkeren, R.J. van; Gabriel, T.L.; Haar, C. de; Gijzel, S.M.W.; Hamers, N.; ... ; Kalkhoven, E. 2019
Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell... Show moreBackground: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. beta-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFN gamma and 114 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes. Show less
Rakhshandehroo, M.; Eijkeren, R.J. van; Gabriel, T.L.; Haar, C. de; Gijzel, S.M.W.; Hamers, N.; ... ; Kalkhoven, E. 2019
Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell... Show moreBackground: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. beta-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFN gamma and 114 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes. Show less
