Direct effects of prolonged TNF-α and IL-6 exposure on neural activity in human iPSC-derived neuron-astrocyte co-cultures

Noah Goshi ¹ Doris Lam ¹ Chandrakumar Bogguri ¹ Vivek Kurien George ² Aimy Sebastian ¹ Jose Cadena ² Nicole Leon ¹ Nicholas Hum ¹ Dina Weilhammer ¹ Nicholas O Fischer ¹ Heather Ann Enright ^1*

• ¹ Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory (DOE), Livermore, United States
• ² Engineering Directorate, Lawrence Livermore National Laboratory, Livermore, CA, United States

Cognitive impairment is one of the many symptoms reported by individuals suffering from long-COVID and other post-viral infection disorders. A common factor among these conditions is a sustained level of inflammatory cytokines. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are two such cytokines that are elevated in patients diagnosed with long-COVID and ME/CFS. In this study, we characterized the changes in neural functionality, secreted cytokine profiles, and gene expression in co-cultures of human iPSC-derived neurons and primary astrocytes in response to prolonged exposure to TNF-α and IL-6. Exposure to TNF-α produced both a concentration-independent and concentration-dependent response in neural activity. Burst duration was significantly reduced within a few days of exposure regardless of concentration (1 pg/mL – 100 ng/mL) but returned to baseline after 7 days. Treatment with low concentrations of TNF-α (e.g. 1 pg/mL and 25 pg/mL) did not lead to changes in the secreted cytokine profile or gene expression but still resulted in significant changes to electrophysiological features such as interspike interval and burst duration. Conversely, treatment with high concentrations of TNF-α (e.g. 10 ng/mL and 100 ng/mL) led to reduced spiking activity, which may be correlated to changes in neural health, gene expression, and increases in secreted cytokines (e.g. IL-1β, IL-4, and CXCL-10) that were observed at higher TNF-α concentrations. Prolonged exposure to IL-6 led to changes in bursting features, with significant reduction in the number of spikes in bursts across a wide range of treatment concentrations (i.e. 1 pg/mL-10 ng/mL). In combination, the addition of IL-6 appears to counteract the changes to neural function induced by low concentrations of TNF-α, while at high concentrations of TNF-α the addition of IL-6 had little to no effect. Conversely, the changes to electrophysiological features induced by IL-6 were lost when the cultures were co-stimulated with TNF-α regardless of the concentration, suggesting that TNF-α may play a more pronounced role in altering neural function. These results indicate that increased concentrations of key inflammatory cytokines associated with long-COVID can directly impact neural function and contribute to the cognitive impairment associated with long-COVID and other post-viral infection disorders.