TLR4-mediated signalling potentiates the expression of IFNβ mRNA in human phagocytes transfected with plasmid DNA.

The detection of intracellular DNA of bacterial or viral origin is critical to induce a proper innate immune response to pathogens, which often includes the production of type I interferons. However, knowledge of how intracellular DNA is sensed, particularly in human immune cells, is still limited. This being said, we have optimized an electroporation method to efficiently transfect plasmid DNA into neutrophils and monocytes (including a plasmid carrying PKCε, since neutrophils do not express it). By doing so, we could observe a significant IFNβ mRNA expression - in the absence of LPS-stimulation - not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty plasmids: LPS, however, further upregulated IFNβ mRNA in plasmid-transfected neutrophils, regardless of PKCε-overexpression. Notably, the same findings were observed also in autologous monocytes, which however constitutively display a functional PKCε. Consistent with the previous observations, immunoblotting and coimmunoprecipitation studies not only revealed that both monocytes and neutrophils constitutively express various cytosolic DNA sensors, including IFI16, LRRFIP1 and DDX41, but also identified IFI16 as the intracellular receptor recognizing transfected DNA. Furthermore, chromatin immunoprecipitation assays, targeting the IFNβ promoter, revealed that IFNβ mRNA induction occurred through the cooperative action of IRF3 activated by transfected DNA, and NF-κB activated by LPS. Taken together, data indicate that human monocytes and neutrophils recognize and respond to microbial cytosolic DNA, in terms of IFNβ induction, via IFI16 involvement. Data also uncover that, in DNAtransfected monocytes and neutrophils, LPS further upregulates the IFNβ transcription by specifically activating NF-κB.