Abstract
BackgroundMicroglia are the principal cells involved in the innate immune response in the CNS. Activated microglia produce a number of proinflammatory cytokines implicated in neurotoxicity but they also are a major source of anti-inflammatory cytokines, antiviral proteins and growth factors. Therefore, an immune therapy aiming at suppressing the proinflammatory phenotype while enhancing the anti-inflammatory, growth promoting phenotype would be of great benefit. In the current study, we tested the hypothesis that interferon regulatory factor 3 (IRF3), a transcription factor required for the induction of IFNβ following TLR3 or TLR4 activation, is critical to the microglial phenotype change from proinflammatory to anti-inflammatory, and that this phenotype change can be greatly facilitated by IRF3 gene transfer.MethodsCultures of primary human fetal microglia were transduced with IRF3 using recombinant adenovirus (Ad-IRF3) and subjected to microarray analysis, real-time PCR, immunoblotting and ELISA to determine inflammatory gene expression. Two different types of immune stimuli were tested, the TLR ligands, poly IC (PIC) and LPS, and the proinflammatory cytokines, IL-1/IFNγ. In addition, the role of the PI3K/Akt pathway was examined by use of a pharmacological inhibitor, LY294002.ResultsOur results show that Ad-IRF3 suppressed proinflammatory genes (IL-1α, IL-1β, TNFα, IL-6, IL-8 and CXCL1) and enhanced anti-inflammatory genes (IL-1 receptor antagonist, IL-10 and IFNβ) in microglia, regardless of the cell stimuli applied. Furthermore, Ad-IRF3 activated Akt, and LY294002 reversed the effects of Ad-IRF3 on microglial inflammatory gene expression. pAkt was critical in LPS- or PIC-induced production of IL-10 and IL-1ra. Significantly, microglial IFNβ protein production was also dependent on pAkt and required both Ad-IRF3 and immunological stimuli (PIC > IL-1/IFNγ). pAkt played much less prominent and variable roles in microglial proinflammatory gene expression. This anti-inflammatory promoting role of PI3K/Akt appeared to be specific to microglia, since astrocyte proinflammatory gene expression (as well as IFNβ expression) required PI3K/Akt.ConclusionsOur results show a novel anti-inflammatory role for the PI3K/Akt signaling pathway in microglia. They further suggest that IRF3 gene therapy could facilitate the microglial phenotype switch from proinflammatory ("M1-like") to anti-inflammatory and immunomodulatory ("M2-like"), in part, by augmenting the level of pAkt.
Highlights
Microglia are the principal cells involved in the innate immune response in the CNS
Adenovirus-mediated interferon regulatory factor 3 (IRF3) gene transfer alters the gene expression profile of cultured human microglia Our previous studies have suggested that over-expression of IRF3 by adenovirus-mediated gene transfer (AdIRF3) might suppress microglial proinflammatory cytokine expression while increasing anti-inflammatory and antiviral gene expression [21]
Cultures of primary human fetal microglia were infected with recombinant Ad-IRF3 or the control adenovirus (Ad-GFP or Ad-b-gal) for 48 h as previously described, and further treated with inflammatory stimuli (LPS, poly IC (PIC) or IL-1b/IFNg) for an additional 6 h 24 h
Summary
Microglia are the principal cells involved in the innate immune response in the CNS. Activated microglia produce a number of proinflammatory cytokines implicated in neurotoxicity but they are a major source of anti-inflammatory cytokines, antiviral proteins and growth factors. Microglia are the key cell type involved in innate immune responses in the CNS [6,7,8]. Astrocytes can participate in the CNS innate inflammatory response including antiviral immunity [14]. Studies indicate that neurons in vivo and in vitro possess pattern recognition receptors, and can respond to dsRNA by activation of the innate immune signaling pathways including the production of IFNb [15]
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