Journal
JOURNAL OF IMMUNOLOGY
Volume 189, Issue 3, Pages 1500-1509Publisher
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.1102985
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Funding
- Ministero dell'Istruzione
- dell'Universita' e della Ricerca
- Associazione Italiana per la Ricerca sul Cancro
- Fondazione Cassa di Risparmio
- Fondazione Italiana per la Ricerca sul Cancro
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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-beta. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-beta in response to TLR4 stimulation. Because neutrophils do not express protein kinase C epsilon, a molecule recently reported as essential for initiating the MyD88-independent/ TRIF-dependent pathway, we optimized an electroporation method to transfect PKC epsilon into neutrophils with very high efficiency. By doing so, a significant IFN-beta mRNA expression was induced, in the absence of LPS stimulation, not only in PKC epsilon--overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-beta transcript levels in plasmid-transfected neutrophils, regardless of PKC epsilon overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-b promoter, revealed that IFN-b mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-kappa B, 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-b mRNA expression. Taken together, these data raise questions about the role of PKC epsilon in driving the MyD88-independent/ TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA. The Journal of Immunology, 2012, 189: 1500-1509.
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