IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.
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.
Section of General Pathology, Department of Pathology and Diagnostics, University of Verona, 37134 Verona, Italy.
Nicola
N
Tamassia
Flavia
F
Bazzoni
Vincent
V
Le Moigne
Federica
F
Calzetti
Caterina
C
Masala
Giulia
G
Grisendi
Uta
U
Bussmeyer
Sara
S
Scutera
Marzia
M
De Gironcoli
Claudio
C
Costantini
Tiziana
T
Musso
Marco A
MA
Cassatella
D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology; D018416:Bartonella henselae / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D003600:Cytosol / Q000276:immunology; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics; D016952:Legionella pneumophila / Q000235:genetics / Q000276:immunology; D008089:Listeria monocytogenes / Q000235:genetics / Q000276:immunology; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D015533:Transcriptional Activation / Q000276:immunology*; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology*
D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D016899:Interferon-beta; D004247:DNA