RESUMO
The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.
Assuntos
Autofagia/fisiologia , Células Endoteliais/fisiologia , Infiltração de Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Junções Intercelulares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologiaRESUMO
Correlative light and electron microscopy (CLEM) greatly facilitate capturing the ultrastructure of spatially and/or temporally rare events. Here, we present a protocol for targeting regions of interests (ROIs) in tissue endothelial cells (ECs) using X-ray micro-computed tomography (µCT). We describe steps for ROI targeting guided by vasculature patterns and positions of EC nuclei visualized by light and X-ray microscopy. The protocol is applicable to thin or translucent tissues that contain defined landmarks visible in both light and X-ray microscopy. For complete details on the use and execution of this protocol, please refer to Reglero-Real et al.1.