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.

Zinc Finger Protein 64 Promotes Toll-like Receptor-triggered Proinflammatory and Type I Interferon Production in Macrophages by Enhancing p65 Subunit Activation*

“…4, A and B). In addition, we did not detect a deviation of the phosphorylation level of IRF3, whose activation contributes to TLR-induced transcriptional activation of the IFN-␤ gene (24,25). Hence, it is likely that the signaling components of the NF-B and IRF3 pathways in the TLR-triggered inflammatory response are not strongly affected by ZFP64.…”

A novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities

“…To further assess the in vivo functional relevance of L1 and L2, we analyzed the recruitment of the phosphorylated version of the key IFNB1-regulating transcription factor IRF3 (pIRF3) (60), RNAPII (61), Cohesin component SMC1A (16) and Mediator component Med1 (62), to L1–1 and L2 (Figure 4A) by ChIP. ChIP analysis determined that both pIRF3 and RNAPII exhibited no enrichment relative to input chromatin in the uninfected state at either L1–1 or L2, but found pIRF3 consistently recruited to both sites in the infected state (Figure 4B and C), and RNAPII only recruited to L2 in a consistent manner.…”

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

“…Circulating blood neutrophils from healthy individuals do not normally express cytokines, but can generate them in response to stimulus‐specific environmental signals . Many ligands can activate cytokine expression by human neutrophils, for instance microbial factors such as pathogen‐associated molecular patterns (PAMPs) binding to pattern recognition receptors (PRR), including TLRs, RIG‐I and DNA sensors, or host‐generated cytokines. In addition, neutrophil‐derived factors can themselves enhance/generate additional cytokine expression via autocrine feedback loops …”