IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

Abstract

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

• Authors: Nicola Tamassia, Flavia Bazzoni, Vincent Le Moigne, Federica Calzetti, Caterina Masala, Giulia Grisendi, Uta Bussmeyer, Sara Scutera, Marzia De Gironcoli, Claudio Costantini, Tiziana Musso, Marco A Cassatella
• Journal: Journal of immunology (Baltimore, Md. : 1950) (J Immunol) Vol. 189 Issue 3 Pg. 1500-9 (Aug 01 2012) ISSN: 1550-6606 [Electronic] United States
• PMID: 22730532 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• RNA, Messenger
• TLR4 protein, human
• Toll-Like Receptor 4
• Interferon-beta
• DNA

Topics

• Adenoviruses, Human (genetics, immunology)
• Bartonella henselae (genetics, immunology)
• Cells, Cultured
• Cytosol (immunology)
• DNA (biosynthesis, genetics)
• HEK293 Cells
• Humans
• Interferon-beta (biosynthesis, genetics)
• Legionella pneumophila (genetics, immunology)
• Listeria monocytogenes (genetics, immunology)
• Neutrophils (immunology, metabolism, microbiology)
• Plasmids (genetics)
• RNA, Messenger (biosynthesis)
• Signal Transduction (genetics, immunology)
• Toll-Like Receptor 4 (physiology)
• Transcriptional Activation (immunology)
• Transfection (methods)
• Up-Regulation (genetics, immunology)