Profiling the proteomic inflammatory state of human astrocytes using DIA mass spectrometry

BACKGROUND: Astrocytes are the most abundant cells in the central nervous system and are responsible for a wide range of functions critical to normal neuronal development, synapse formation, blood-brain barrier regulation, and brain homeostasis. They are also actively involved in initiating and perpetuating neuroinflammatory responses. However, information about their proteomic phenotypes under inflammation is currently limited. METHOD: Data-independent acquisition mass spectrometry was applied to extensively characterize the profile of more than 4000 proteins in immortalized human fetal astrocytes under distinct inflammatory conditions induced by TNF, IL-1β, and LPS, while multiplex immunoassay-based screening was used to quantify a wide range of cytokines released under these inflammatory conditions. Then, immunocytochemistry and western blotting were used to verify the activation of canonical and non-canonical NF-κB upon exposure to the different stimuli. Finally, an in vitro model of the blood-brain barrier consisting of a co-culture of primary human brain microvascular endothelial cells and primary human astrocytes was used to verify the inflammatory response of astrocytes upon LPS exposure in a more complex in vitro system. RESULTS: We reported on a set of 186 proteins whose levels were significantly modulated by TNF, IL-1β, and LPS. These three stimuli induced proteome perturbations, which led to an increased abundance of key inflammatory proteins involved in antigen presentation and non-canonical NF-κB pathways. TNF and IL-1β, but not LPS, also activated the canonical NF-κB pathway, which in turn led to an extensive inflammatory response and dysregulation of cytoskeletal and adhesion proteins. In addition, TNF and LPS, but not IL-1β, increased the abundance of several interferon-stimulated gene products. Finally, TNF and IL-1β similarly upregulated the secretion of several cytokines and chemokines, whereas LPS only induced a moderate increase in IL-8, IFN-γ, and IL-1β secretion. Upregulation of proteins associated with type I IFN and non-canonical NF-κB signaling was also observed in primary astrocytes co-cultured with primary brain microvascular endothelial cells exposed to LPS. CONCLUSIONS: The present study provides comprehensive information about the proteomic phenotypes of human astrocytes upon exposure to inflammatory stimuli both in monoculture and in co-culture with human brain microvascular endothelial cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12974-018-1371-6) contains supplementary material, which is available to authorized users.