Multiple sclerosis (MS) is a chronic neuroinflammatory disease in which demyelinating white matter lesions accumulate and expand, driving irreversible disability. Here we identify a distinct population of foamy GPNMB+ microglia/macrophages associated with lesion expansion in secondary progressive MS. Using integrated lipidomic, transcriptomic, proteomic, chemical proteomic and histological analyses of human postmortem MS lesions, we show that lesions containing foamy microglia/ macrophages exhibit disrupted lipid metabolism, lysosomal stress and markers associated with heightened phagocytosis and antigen presentation without classical pro-inflammatory signatures. These lesions are enriched for oxylipins, bismonoacylglycerolphosphates and cholesterol esters, and are associated with increased B cell infiltration and IgG1. Monoacylglycerol lipase (MAGL), a lipid-metabolizing enzyme enriched in lesions with foamy microglia/macrophages, emerged as a potential therapeutic target. Inhibition of MAGL promoted lesion recovery and reduced microgliosis in a mouse model of demyelination. Finally, oxylipins in cerebrospinal fluid correlate with the proportion of foamy lesions, suggesting potential biomarkers for progression. Our findings implicate disturbed lipid metabolism in chronic MS pathology and suggest that foamy microglia/ macrophages are an interesting cell type to target for progressive disease.

Macrophages are specialised cells that degrade a range of substrates during their lifetime. In inherited lysosomal storage disorders, particularly the sphingolipidoses, macrophages transform into storage cells and contribute to pathology. An appropriate cultured macrophage model is desired for fundamental research and the assessment of considered therapeutic interventions. We compared commonly used macrophage cell lines, RAW264.7, J774A.1, and THP-1 cells, with human monocyte-derived macrophages (HMDMs) isolated from peripheral blood. Specific lysosomal glucosidases were analysed by enzymatic activity measurements and visualised with fluorescent activity-based probes. Special attention was given to lysosomal glucocerebrosidase (GBA1), the enzyme deficient in Gaucher disease in which lipid-laden macrophages are a hallmark. In macrophage cell lines and HMDMs, various (glyco)sphingolipids relevant to GBA1 activity were determined. Finally, the feasibility of inactivation of GBA1 with a cell-permeable suicide inhibitor was established, as well as the monitoring of uptake of therapeutic recombinant human GBA1. Major differences among various cell lines were noted in terms of morphology, lysosomal enzyme expression, and glycosphingolipid content. HMDMs appear to be the most suitable model for investigations into GBA1 and Gaucher disease. Moreover, they serve as a valuable model for mannose-receptor mediated uptake of therapeutic human GBA1, effectively mimicking enzyme replacement therapy for Gaucher disease.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by expanding demyelinating lesions, leading to severe and irreversible disability. The mechanisms driving lesion expansion, however, remain poorly understood. Here, using a multi-omics approach, we identified foamy microglia as primary contributors to the molecular profile of lesions and disease progression in secondary progressive MS. Lesions with foamy microglia are marked by the accumulation of cholesterol esters, bismonoacylglycerolphosphates (BMP), and oxylipins, along with high B-cell infiltration, increased levels of immunoglobulin G1, and elevated expression of Fcgamma- and complement receptors. Lesions with foamy GPNMB+-microglia display markers of enhanced phagocytosis, lipid metabolism, lysosomal dysfunction, and antigen presentation, but lack classical pro-inflammatory markers. Our data suggest that sustained phagocytosis of myelin overwhelms microglial endo-lysosomal capacity, leading to lipid droplet and oxylipin formation. This microglial phenotype may induce further recruitment of adaptive immune cells, axonal damage, drive lesion expansion and prevent remyelination. Monoacylglycerol lipase, involved in producing oxylipin precursors, was identified as a potential therapeutic target to disrupt this cycle and prevent chronic lesion expansion. ### Competing Interest Statement AP, WM, CCB, UG and LC are employees of F. Hoffmann-La Roche Ltd, which holds patents on inhibitors of MGLL. MvdS is an author on patents describing inhibitors of MGLL. All other authors have no competing interests to declare.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

To describe the care provided in the Netherlands in geriatric rehabilitation (GR) after a hip fracture, using care pathways and diagnosis treatment combinations from various geriatric rehabilitation facilities. Care provided in GR after hip fracture is difficult to define due to the diversity in care pathways and large practice variation. Further research is needed to investigate whether a standardized care pathway is effective for GR. After acute hospital admission, patients with a hip fracture are frequently discharged to skilled nursing homes providing geriatric rehabilitation (GR). There are few evidence-based studies regarding specific treatment times and assessments during GR. This study aims to provide a description of care for hip fracture patients during GR in the Netherlands. Descriptive study analyzing the care pathways from GR facilities, regarding healthcare professionals involved, allocated treatment time per profession, total length of rehabilitation stay, and assessment instruments. Based on the reimbursement algorithm (diagnostic treatment combination = DBCs), of 25 patients, the registered actual treatment time per profession was calculated. The care pathways pivoted on three groups of health care professionals: medical team (MT), physiotherapy (PT), and occupational therapy (OT). There was some discrepancy between the allocated time in the care pathways and the calculated mean actual treatment time from the DBCs. First week: MT 120–180 min, DBC 120 (SD: 59) minutes; PT 120–230 min, DBC 129 (SD: 58) minutes; and OT 65–165 min, DBC 93 (SD: 61) minutes. From week two onwards, MT 15–36 min, DBC 49 (SD: 29) minutes; PT 74–179 min, DBC 125 (SD: 50) minutes; and OT 25–60 min, DBC 47 (SD: 44) minutes. Dieticians, psychologists, and social workers were sporadically mentioned. There was heterogeneity in the assessment and screening tools. It is difficult to define current standard care in GR after hip fracture in the Netherlands due to the diversity in care pathways and large practice variation. This is a problem in conducting randomized effectiveness research with care provided as control. NL7491 04-02-2019.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N ‐glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Fluorescent activity‐based probes (ABPs) that covalently label GCase in reaction‐based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage‐like RAW264.7 cells. Specific attention was paid to the impact of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate‐buffered medium. Here, we report how HEPES‐buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES‐containing medium was also found to reduce maturation of other lysosomal enzymes (α‐glucosidase and β‐glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD‐patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.

Macrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling systems. In addition, macrophages have various systems that are involved in the uptake of extracellular and intracellular cargo. The lysosomes in macrophages play a central role in the digestion of all sorts of macromolecules and the entry of nutrients to the cytosol, and, thus, the regulation of endocytic processes and autophagy. Simplistically viewed, two macrophage phenotype extremes exist. On one end of the spectrum, the classically activated pro-inflammatory M1 cells are present, and, on the other end, alternatively activated anti-inflammatory M2 cells. A unique macrophage population arises when lipid accumulation occurs, either caused by flaws in the catabolic machinery, which is observed in lysosomal storage disorders, or as a result of an acquired condition, which is found in multiple sclerosis, obesity, and cardiovascular disease. The accompanying overload causes a unique metabolic activation phenotype, which is discussed here, and, consequently, a unifying phenotype is proposed.

Lyso-glycosphingolipids are generated in excess in glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating glycosphingolipids drives the formation of lyso-glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-glycosphingolipid species. This review discusses how the various lyso-glycosphingolipids are synthesized, how they may contribute to abnormal immunity in glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.

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 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.

Several diseases are caused by inherited defects in lysosomes, the so-called lysosomal storage disorders (LSDs). In some of these LSDs, tissue macrophages transform into prominent storage cells, as is the case in Gaucher disease. Here, macrophages become the characteristic Gaucher cells filled with lysosomes laden with glucosylceramide, because of their impaired enzymatic degradation. Biomarkers of Gaucher cells were actively searched, particularly after the development of costly therapies based on enzyme supplementation and substrate reduction. Proteins selectively expressed by storage macrophages and secreted into the circulation were identified, among which glycoprotein non-metastatic protein B (GPNMB). This review focusses on the emerging potential of GPNMB as a biomarker of stressed macrophages in LSDs as well as in acquired pathologies accompanied by an excessive lysosomal substrate load in macrophages.

Glycosphingolipids (GSLs), the main topic of this review, are a subclass of sphingolipids. With their glycans exposed to the extracellular space, glycosphingolipids are ubiquitous components of the plasma membrane of cells. GSLs are implicated in a variety of biological processes including specific infections. Several pathogens use GSLs at the surface of host cells as binding receptors. In addition, lipid-rafts in the plasma membrane of host cells may act as platform for signaling the presence of pathogens. Relatively common in man are inherited deficiencies in lysosomal glycosidases involved in the turnover of GSLs. The associated storage disorders (glycosphingolipidoses) show lysosomal accumulation of substrate(s) of the deficient enzyme. In recent years compounds have been identified that allow modulation of GSLs levels in cells. Some of these agents are well tolerated and already used to treat lysosomal glycosphingolipidoses. This review summarizes present knowledge on the role of GSLs in infection and subsequent immune response. It concludes with the thought to apply glycosphingolipid-lowering agents to prevent and/or combat infections.

Obesity is taking on worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar N-adamantine-methyloxypentyl-deoxynojirimycin (AMP-DNM), which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents. Moreover, we demonstrate that the mechanism mediating these favorable actions depends on oral, but not central, administration of AMP-DNM, which ultimately stimulates systemic glucagon-like peptide 1 (GLP1) secretion. We evidence an essential role of brain GLP1 receptors (GLP1r), as AMP-DNM fails to promote satiety and activate BAT in mice lacking the brain GLP1r as well as in mice treated intracerebroventricularly with GLP1r antagonist exendin-9. In conclusion, AMP-DNM markedly ameliorates metabolic abnormalities in obese rodents by restoring satiety and activating BAT through central GLP1r, while improving glucose homeostasis by mechanisms independent of central GLP1r.

Wu, Liang, Jiang, Jianbing, Jin, Yi, Kallemeijn, Wouter W, Kuo, Chi-Lin, Artola, Marta, Dai, Wei, van Elk, Cas, van Eijk, Marco, van der Marel, Gijsbert A, Codée, Jeroen D C, Florea, Bogdan I, Aerts, Johannes M F G, Overkleeft, Herman S and Davies, Gideon J 2017. Activity-based probes for functional interrogation of retaining -glucuronidases. Nature Chemical Biology 13 , pp. 867-873. β 10.1038/nchembio.2395 file

Medical care in Dutch nursing homes is provided by elderly care physicians (ECPs). The three-year specialist training program for ECPs consists of three training periods in an educational nursing home, three internships, and a theoretical course lasting 100-130 days. Evidence-based medicine (EBM) is taught during the theoretical course. Currently, the EBM training in the ECP program at Leiden University Medical Centre comprises: 1) several lectures on the basics of research and critical reading, 2) the writing of three critically appraised topics (CATs) with questions initiated by the trainees themselves, and 3) participation in a group of ECP trainees to analyse an existing dataset and present the results to their peers. Teaching EBM is essential to create lifelong learners who can critically appraise information and assess the applicability of this information for the individual patient. This applies, in particular, to elderly care medicine, due to the underrepresentation of frail elderly persons and nursing home residents in medical research. Although classroom teaching of EBM improves knowledge, clinically integrated teaching improves not only knowledge but also related skills, attitude and behaviour. Compared with traditional teaching, a blended learning approach is more effective in improving the attitude towards EBM and results in a higher self-reported use of EBM in clinical practice. The EBM training is regularly evaluated and updated to maintain a state-of-the-art program. Our latest innovation is the integration of a prospective cohort study assessing the relation between caffeine and behavioural symptoms, and the EBM training program. Of all patients admitted to Dutch nursing homes, ≥ 50% are diagnosed with cognitive disorders or dementia. In patients with dementia behavioural symptoms are often the main reason for nursing home admission, often due to the heavy burden placed on the caregivers, resulting in a high demand for care. In addition, behavioural symptoms lower the patient’s quality of life. Behavioural symptoms are present in ≥ 80% of patients with dementia in a nursing home. In patients with dementia the aetiology of behavioural symptoms is complex and thought to be multifactorial. To manage these symptoms, national guidelines recommend a detailed analysis of the patient, including contributing physical, psychological, social and environmental factors. Despite the fact that many pharmacological and psychosocial interventions have been studied, no standardised solution is available and all interventions targeting behavioural symptoms must be tailored.

Background: In geriatric rehabilitation, functional prognosis informs expectations regarding recovery and discharge planning.Assessing pre-existent comorbidity may aid functional prognosis but existing indices have limitations in older populations.We investigated the predictive validity of the weighted Functional Comorbidity Index (wFCI) for recovery of mobility in an older cohort undergoing rehabilitation.Methods: Patients admitted to a community hospital for rehabilitation between January and September 2017 were recruited to an observational cohort study.Those admitted for end-of-life care were excluded.Outcome measures were: mobility at discharge assessed with the elderly mobility scale (EMS; range 0-20) and change in mobility (EMS gain/ day).Comorbidity indices were collected at baseline: Charlson index (range 0-33), FCI (range 0-18) and wFCI (range 0-54).Univariate (Mann-Whitney U) and binary logistic regression analyses were performed.Variables were dichotomized around their median and 3 models were created to compare comorbidity indices.The wFCI classified comorbidity as: absent (0), and having no (1), partial (2) or severe (3) impact on function.Age, gender, basic activities of daily living (Barthel range 0-20) and cognitive function (MoCA range 0-30) at baseline were added to the models to control for confounding.Results: 75 patients (mean age 82 years; 61% female) were included.Median (IQR) Barthel index and MoCA were 9 (5) and 20 (10) respectively.Median EMS was 5 (4) and 11 (6) at admission and discharge respectively.Median (IQR) gain/day was 0.19 (0.25).Charlson and FCI did not independently contribute to the prediction models, but wFCI was associated with lower EMS at discharge (OR [95% CI]: 0.72 [0.54-0.93])and lower EMS gain/day (OR: 0.80 [0.64-1.00]). Conclusion:The wFCI was independently predictive of mobility at discharge and mobility gain per day.Further research is required to understand how the wFCI can be incorporated into routine decision making in rehabilitation practice.

OBJECTIVE:This article describes and discusses the viewpoints of elderly care physicians and municipal coroners regarding the qualification of natural or unnatural deaths of nursing home residents with advanced dementia who fall.DESIGN:A digital questionnaire was sent to all elderly care physicians-in-training and their trainers in the Netherlands, and to all 23 municipal coroners in the Mid-Holland region.METHOD:As well as questions on knowledge of the Dutch law and prognostic figures concerning dementia-related death, the questionnaire described two cases with minimal differences. The respondents were asked if they would issue a certificate of natural death in each case, and if not, would they have preferred to do so. They were also asked to explain their answers.RESULTS:Of the 405 elderly care physicians who responded, 68% and 49%, respectively, would have issued a certificate stating the cause to be natural death in each case, or if they did not they would have preferred to do so. All 15 coroners who filled in the questionnaire came to the conclusion of unnatural death.CONCLUSION:The majority of the elderly care physicians feel that a certificate of natural death can be issued if a fall in a nursing home results in a fracture, as this can be considered as part of the disease process of dementia.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.