Ontogeny-independent expression of LPCAT2 in granuloma macrophages during experimental visceral leishmaniasis [scRNA-seq]
Granulomas are organized inflammatory lesions that form in response to persistent stimuli such as infections. Murine infection with Leishmania donovani results in the formation of granulomas around infected Kupffer cells in the liver and serves as a well-defined model of immune granuloma formation. The formation and resolution of granulomatous inflammation requires dynamic shifts in immune cell activation states, imposing significant metabolic demands. As mediators of energy homeostasis and cell signaling, lipids and lipid metabolism play a key role in regulating immune cell function during inflammation and the response to infection. However, the extent to which alterations in lipids are spatially linked to altered immune cell transcription has yet to be resolved. In this study, we performed a multimodal imaging analysis combining MALDI mass spectrometry, spatial and single cell transcriptomics, proteomics of flow-sorted macrophages and histopathology of L. donovani induced hepatic granulomas. Using this spatially-integrated approach, we identified LPCAT2-mediated membrane re-modelling of myeloid cells as a novel feature of these granulomas. Our study provides new insights into local immunometabolic changes associated with granuloma formation and macrophage activation. Overall design: Adult female C57BL/6J mice were maintained and bred at the University of York, UK. The mice were housed in ventilated cages under specified pathogen-free conditions with access to food and water ad libitum. Identification of the mouse colony was confirmed through genetic profiling of microsatellite markers, verifying C57BL/6J genetic background with minor variations at 3 markers. Healthy (6-8 week old females) were infected intravenously with 3x107 amastigotes of the Ethiopian LV9 strain of Leishmania donovani to establish visceral infection. At 28 days post-infection, mice were euthanized by CO2 inhalation followed by cervical dislocation according to Home Office regulations. Livers were harvested after euthanization and the major lobe cut and snap-frozen in liquid nitrogen while bathed in isopenane and then kept in -80 degrees until used. The remaining liver was harvested in 2% fetal calf serum (FCS) in 1x PBS and immediately processed into a single cell-suspension. Age-matched uninfected female mice from the same colony were used as baseline controls for liver tissue. For single-cell RNA seq 6 infected mice were run as individual samples selected from 2 independent experiments and the 2 naive samples were each pooled from 3x uninfected mice.