Decoding acute neuroinflammatory states from the 3D architecture of in vitro microglia

Abstract

Microglia, core brain immune defenders, rapidly polarize into pro- or anti-inflammatory states that shape neuronal survival during acute brain inflammation. Yet, how these inflammatory states are encoded at the native cellular level remains unclear. While microglial states associate with specific molecular and organelle markers, it is unknown whether their cellular architecture integrates robust metabolic and structural features. Here, we quantitatively decode inflammatory states from the 3D architecture of individual in vitro mouse microglia (BV-2). Using soft X-ray tomography on established BV-2 polarization, we identify coordinated intracellular organization distinguishing homeostatic, pro-inflammatory, and anti-inflammatory cells, including characteristic lipid droplet-endosome architectures. We further link lipid-endosome reorganization with mTORC1 pathway, by functionally implicating sestrin-2 in promoting anti-inflammation. Finally, we resolve the time-dependent remodeling of lipid droplet 3D profiles by their distinct lipidomic composition across inflammatory states. Overall, our work enables deciphering microglia states in disease-relevant models, with potential to ultimately understand brain immunity.