Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation

The glycosphingolipid, alpha-galactosylceramide (alpha 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 alpha GalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify alpha GalCers, and we investigated the alpha GalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of alpha GalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor alpha GalCer. The identified alpha GalCer contained a beta(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis. Unlike beta-anomeric structures, but similar to alpha GalCers from B. fragilis, the synthetic form of the murine alpha GalCer induced iNKT cell activation in vitro. Last, we observed a decrease in alpha 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 alpha 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 studies.