Supplementary key words glycolipids • sphingolipids • mass spectrometry • immunology • bacteria • invariant natural killer T cells • Western diet • experimental colitis • influenza A virus • cluster of differentiation 1d Besides recognition of classical major histocompatibility complex class I or II molecules presenting oligopeptides by T lymphocytes, a wide variety of nonclassical nearly monomorphic major histocompatibility complex molecules, including CD1, may be recognized by  and  T cells as Abstract The glycosphingolipid, -galactosylceramide (GalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of GalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS 2 , we screened murine intestinal tracts to identify and quantify GalCers, and we investigated the GalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of GalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor GalCer. The identified GalCer contained a (R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis. Unlike -anomeric structures, but similar to GalCers from B. fragilis, the synthetic form of the murine GalCer induced iNKT cell activation in vitro. Last, we observed a decrease in GalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that GalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future

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Functional skin barrier requires sphingolipid homeostasis. 3-ketodihydrosphingosine reductase or KDSR is a key enzyme of sphingolipid anabolism catalyzing the reduction of 3-ketodihydrosphingosine to sphinganine. Biallelic mutations in the KDSR gene may cause erythrokeratoderma variabilis et progressive-4, later specified as PERIOPTER syndrome, emphasizing a characteristic periorifical and ptychotropic erythrokeratoderma. We report another patient with compound heterozygous mutations in KDSR, born with generalized harlequin ichthyosis, which progressed into palmoplantar keratoderma. To determine whether patient-associated KDSR mutations lead to KDSR substrate accumulation and/or unrecognized sphingolipid downstream products in stratum corneum we analyzed lipids of this and previously published patients with non-identical biallelic mutations in KDSR. In stratum corneum of both patients we identified hitherto unobserved skin ceramides with an unusual keto-type sphingoid base in lesional and non-lesional areas, which accounted for up to 10% of the measured ceramide species. Furthermore, an overall shorter mean chain length of free and bound sphingoid bases was observed—shorter mean chain length of free sphingoid bases was also observed in lesional psoriasis vulgaris SC, but not generally in lesional atopic dermatitis SC. Formation of keto-type ceramides is probably due to a bottle neck in metabolic flux through KDSR and a bypass by ceramide synthases, which highlights the importance of tight intermediate regulation during sphingolipid anabolism and reveals substrate deprivation as potential therapy.