Poly(I:C) promotes TNFa/TNFR1-dependent oligodendrocyte death in mixed glial cultures

Steelman, Andrew J; Li, Jianrong

Microglia are the sole source of TNFa secretion after poly (I:C) stimulation in culture Both microglia and astrocytes are capable of cytokine and chemokine secretion following recognition of various pathogen associated molecular patterns including poly(I:C). To determine the cellular source of TNFα secretion in our cultures, purified microglia, astrocytes or oligodendrocytes were treated with PBS or poly(I:C) for 24 h and TNFα levels were determined by ELISA. We found that microglia, but neither astrocytes nor oligodendrocytes, produced TNFα following poly(I:C) stimulation ( Figure 5A ). To confirm this finding, we then conducted RT-PCR analysis on RNA isolated from microglia and astrocyte monocultures stimulated with or without poly(I:C) for 24 h. Both microglia and astrocytes expressed TLR3. TNFα and IL-1β transcripts were induced by poly(I:C) in microglia, but undetectable in astrocytes. Moreover, transcription of IL-6 was evident in stimulated microglia and in poly(I:C) stimulated astrocytes ( Figure 5B ). The expression of the macrophage/microglia marker Iba-1 and astrocyte marker GFAP from the same samples indicated that there was virtually no microglia contamination of astrocyte cultures, but some astrocyte contamination in microglia cultures ( Figure 5B ). Additionally, microglia but not astrocytes responded to poly(I:C) by producing TNFα in a dose-and time-dependent fashion ( Figure 5C ). These results strongly indicate that microglia are the producers of TNFα in mixed glial cultures.