Dr Johanna von Gerichten

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)–dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides—namely monosialoganglioside GM3 and disialoganglioside GD3—as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S−/−) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S−/− mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.

Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal β-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa−/− mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by β-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa−/− Neu3−/− mice were healthy at birth, but died at 1.5 to 4.5 months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa−/− Neu3−/− mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa−/− Neu3−/− mice exhibited progressive neurodegeneration with neuronal loss, Purkinje celldepletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa−/− Neu3−/− mice. Thus, the Hexa−/− Neu3−/− mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, -galactosylceramide (GalCer) and -glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce -GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous -GlcCer, -GalCer, and -GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of -GlcCers versus -GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific -GlcCers due to loss of -glucoceramidase 2 activity. Vice versa, -GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm -GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic -GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations

Phospholipids have excellent biocompatibility and are therefore often used as main components of liposomal drug carriers. In traditional bioanalytics, the in-vivo distribution of liposomal drug carriers is assessed using radiolabeled liposomal constituents. This study presents matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) as an alternative, label-free method for ex-vivo molecular imaging of liposomal drug carriers in mouse tissue. To this end, indocyanine green as cargo and two liposomal markers, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with monodisperse polyethylene glycol (PEG36-DSPE) were incorporated into liposomal carriers and administered to mice. We used MALDI MSI of the two lipid markers in both positive and negative ion mode for visualization of liposome integrity and distribution in mouse organs. Additional MSI of hemoglobin in the same tissue slice and pixel-by-pixel computational analysis of co-occurrence of lipid markers and hemoglobin served as indicator of liposome localization either in parenchyma or in blood vessels. Our proof-of-concept study suggests that liposomal components and indocyanine green distributed into all investigated organs.

Impaired regulation of immune function characterised by chronic inflammation together with a declining protective immune response is a major challenge to healthy ageing. It is therefore important to understand the mechanisms that regulate immune function and the impact of ageing upon such processes. Appropriate induction and resolution of the immune response require adequate availability of polyunsaturated fatty acids (PUFAs) for incorporation into cell membranes. However, humans are unable to synthesise PUFAs de novo and are dependent upon dietary intake for pre-formed PUFAs or synthesis by the liver from the essential fatty acids, linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (aLNA, 18:3n-3). We have shown that activation of peripheral blood mononuclear cells increases PUFA biosynthesis from essential fatty acids via a mechanism that involves altered epigenetic regulation of a key gene in the pathway. Moreover, induction of PUFA synthesis is directly involved in the regulation of lymphocyte activation and proliferation. The aim of the Biotechnology and Biological Sciences Research Council responsive mode award described in this paper, ‘How does polyunsaturated fatty acid biosynthesis regulate T-lymphocyte function?’, is to determine how PUFA biosynthesis regulates T-cell function and the effect of ageing on this process. The project will identify points of regulation in the biosynthetic pathway and how these might influence the capacity for up-regulation of PUFA synthesis in older individuals. We will use stable isotope tracers of LA and aLNA to determine whether newly synthesised PUFAs are preferential substrates for synthesis of lipid mediators and whether they are involved in formation of membrane microdomains that mediate cell signalling.

Longer-chain polyunsaturated fatty acids (LCPUFAs) ≥20 carbons long are required for leukocyte function. These can be obtained from the diet, but there is some evidence that leukocytes can convert essential fatty acids (EFAs) into LCPUFAs. We used stable isotope tracers to investigate LCPUFA biosynthesis and the effect of different EFA substrate ratios in human T lymphocytes. CD3+ T cells were incubated for up to 48 h with or without concanavalin A in media containing a 18:2n-6:18:3n-3 (EFA) ratio of either 5:1 or 8:1 and [13C]18:3n-3 plus [d5]18:2n-6. Mitogen stimulation increased the amounts of 16:1n-7, 18:1n-9, 18:2n-6, 20:3n-6, 20:4n-6, 18:3n-3, and 20:5n-3 in T cells. Expression of the activation marker CD69 preceded increased FADS2 and FADS1 mRNA expression and increased amounts of [d5]20:2n-6 and [13C]20:3n-3 at 48 h. In addition, 22-carbon n-6 or n-3 LCPUFA synthesis was not detected, consistent with the absence of ELOVL2 expression. An EFA ratio of 8:1 reduced 18:3n-3 conversion and enhanced 20:2n-6 synthesis compared to a 5:1 ratio. Here, [d5]9- and [d5]-13-hydroxyoctadecadienoic (HODE) and [13C]9- and [13C]13-hydroxyoctadecatrienoic acids (HOTrE) were the major labelled oxylipins in culture supernatants; labelled oxylipins ≥20 carbons were not detected. An EFA ratio of 8:1 suppressed 9- and 13-HOTrE synthesis, but there was no significant effect on 9- and 13-HODE synthesis. These findings suggest that partitioning of newly assimilated EFA between LCPUFA synthesis and hydroxyoctadecaenoic acid may be a metabolic branch point in T-cell EFA metabolism that has implications for understanding the effects of dietary fats on T lymphocyte function.