Profiling the proteomic inflammatory state of human astrocytes using DIA-mass spectrometry Vito Dozio1,2, Jean-Charles Sanchez1,2 1Department of Internal Medicine Specialties, University of Geneva, Geneva, Switzerland 2SCAHT, Geneva, Switzerland
A successful inflammatory response is fundamental for eliminating invading pathogens, promoting angiogenesis and wound healing inside the brain. Prolonged inflammation of the central nervous system can, however, lead to detrimental consequences and is linked to several classical neurodegenerative diseases. In the last decades, astrocytes gained increasing interest as active players in mediating the neuroinflammatory response. However, little is known about their molecular inflammatory phenotype as well as their ability to respond directly to lipopolysaccharide (LPS) during infection. In the present study, human astrocytes were exposed to different inflammatory stimuli (TNF, IL-1β and LPS) and data-independent acquisition (DIA) mass spectrometry was applied to extensively quantify more than 4,000 astrocytic proteins. In addition, multiplex immunoassay was used to screen astrocyte-secreted cytokines and chemokines. Cluster and pathway analyses on proteomic data were used to highlight both specific as well as common downstream biological pathways activated by TNF, IL-1β and LPS. We reported 149 proteins differentially changing after TNF exposure, 108 after IL-1β and 27 following LPS exposure. All three stimuli strongly led to upregulation of proteins involved in the antigen presentation and the non-canonical NF-κB pathways. The canonical NF-κB pathway, together with pathways related to cell adhesion and cytoskeletal remodeling, were instead only modulated by TNF and IL-1β. TNF and LPS but not IL-1β induced activation of the Type I Interferon (IFN) signaling pathway. Analysis of supernatant revealed that TNF and IL-1β upregulated very similarly the secretion of several cytokines and chemokines, with the only exception of IL-6 which was significantly more abundant in supernatant of IL-1β-stimulated cells. LPS significantly upregulated secretion of IL-8, IFN-γ and IL-1β secretion, although to a much lesser extent compared to TNF and IL-1β. Altogether these results bring comprehensive information about the inflammatory phenotype of human astrocytes at protein level together with evidences about their ability to directly and specifically respond to LPS.
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