Delineating astrocytic cytokine responses in a human stem cell model of neural trauma

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Abstract

Neuroinflammation has been shown to mediate the pathophysiological response following traumatic brain injury (TBI). Accumulating evidence implicates astrocytes as key immune cells within the central nervous system (CNS), displaying both pro- and anti-inflammatory properties. The aim of this study was to investigate how in vitro human astrocyte cultures respond to cytokines across a concentration range that approximates the aftermath of human TBI. To this end, enriched cultures of human induced pluripotent stem cells (iPSC)-derived astrocytes were exposed to interleukin-1β (IL-1β) (1-10,000pg/ml), IL-4 (1-10,000pg/ml), IL-6 (100-1,000,000pg/ml), IL-10 (1-10,000pg/ml) and tumor necrosis factor-alpha (TNF) (1-10,000 pg/ml). Following 1, 24, 48 and 72hours, cultures were fixed and immunolabeled, and the secretome/supernatant was analyzed at 24, 48 and 72h using a human cytokine/chemokine 39-plex Luminex assay. Data was compared to previous in vitro studies of neuronal cultures and clinical TBI studies. The secretome revealed concentration-, time- and/or both concentration and time-dependent production of downstream cytokines (29, 21 and 17 cytokines, respectively p<0.05). IL-1β exposure generated the most profound downstream response (27 cytokines), IL-6 and TNF had intermediate responses (13 and 11 cytokines, respectively), while IL-4 and IL-10 only led to weak responses over time or in escalating concentration (8 and 8 cytokines, respectively). Notably, expression of IL-1β, IL-6 and TNF cytokine receptor mRNA was higher in astrocyte cultures in comparison to neuronal cultures. Several secreted cytokines had temporal trajectories, which corresponded to those seen in the aftermath of human TBI. In summary, iPSC-derived astrocyte cultures exposed to cytokine concentrations reflecting those in TBI generate an increased downstream cytokine production, particularly IL-1β. Although more work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI, our data shows that iPSC-derived astrocytes represent a tractable model to study cytokine stimulation in a cell type-specific manner.