CD4+ T cell-associated cytokines induce a chronic pro-inflammatory phenotype in induced pluripotent stem cell-derived midbrain astrocytes

Astrocytes are mediators of homeostasis but contribute to neuroinflammation in Parkinson's disease (PD). Mounting evidence suggests involvement of peripheral immune cells in PD pathogenesis. Therefore, this study aimed to determine the potential role of peripheral immune secreted cytokines in modulating midbrain astrocyte reactivity. Human iPSC-derived midbrain astrocytes were exposed to 5% and 10% CD4⁺ T cell conditioned media (CD4CM) for 24 h, 72 h, and 7 days to assess chronic exposure. Additionally, astrocytes were exposed to the Th17 cell cytokine, IL-17A (10 ng/mL), alone and in combination with TNF-α (0.3 ng/mL) to assess potential synergistic effects of both cytokines at 24 h, 72 h, and 7 days. CD4CM induced acute and chronic alterations in midbrain astrocytes. Increased NFκB translocation to the nucleus, increased expression of the pro-inflammatory genes, IL-1β, CXCL10 at 24 h, C3, LCN2, IL-6 at 24 and 48 h, as well as an increase in their release of pro-inflammatory cytokines IL-6 and CXCL10 at both these time points were observed. A synergistic response to the combination of IL-17A and TNF-α on increasing inflammatory gene expression and cytokine release occurred. IL-17A and TNF-α increased intensity of S100β at 24 h, decreased nuclear area and increased circularity of astrocytes at 72 h. A synergistic effect on γH2AX intensity at 72 h and an increase in LDH release at 7 days was observed. Our results demonstrate that IL-17A and TNF-α act synergistically, enhancing midbrain astrocyte reactivity to a similar degree as CD4CM. This highlights the importance of the peripheral immune secreted cytokines in increasing the reactivity status of midbrain astrocytes, implicating their role in PD.