Abstract
Introduction: Brain resident macrophages, microglia activated rapidly and produce inflammatory molecules to encounter bacteria or viruses by engaging multiple signaling mediators and pathways, which includes TLRs and interferons . Although the underlying mechanism of their synergistic activities is still elusive , however the either single- TLR or interferon signaling is insufficient to explain strong synergistic activation of early immune response of efficient host defenses.
Methods: Mouse primary microglia were stimulated with either TLR7 agonist R848 (resiquimod) (1µM), IFN-α (100U/ml) or combination for 4h and gene expression profiles were measured by transcriptome qRT-PCR, RNA-seq assay and bioinformatic analyses. The underlying transcription factors (TF) and gene networks for these expression dynamics were identified by TF motif analysis and knowledge base Ingenuity Pathway Analysis (IPA). Furthermore, the associated biological pathways and TF binding affinity at the promoter of synergy induced genes were determined by DAVID and Chip-qPCR study, respectively.
Results: We demonstrated concurrent R848 and IFN-α synergistically induces microglia cells to robust production and release of immune response genes at mRNA level , of which 836 genes were upregulated (≥2, log2 fold change, P<0.0001) in R848+IFN-α-treated cells, whereas 393 in R848-treated and 394 in IFN-α-treated cells, in association of negligible down-regulatory effect. The upregulated genes from co-treated cells includes substantial number of cytokines, chemokines, interferon response genes and epigenetic regulators that belong to defense responses and innate immune system processes. While we found selective members of IRFs preferably novel IRF1 and IRF7 regulates synergistic-transcription upon co-stimulation, in contrast, NFKBs regulate R848-targeted, and STATs regulate IFN-α-targeted gene transcription in induced microglia cells.
Conclusion: Collectively, these unprecedented results identify an unexpected conclusion as in microglia cells selective TLR7-agonist R848 and IFN-α may co-elicits synergistic transcriptional events and preferably novel pathways to shape CNS defenses responses.