Neutrophil Signaling Pathways Activated by Bacterial DNA Stimulation

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: Discussionmentioning

confidence: 99%

IFN-β Expression Is Directly Activated in Human Neutrophils Transfected with Plasmid DNA and Is Further Increased via TLR-4–Mediated Signaling

Tamassia

Bazzoni

Moigne

et al. 2012

“…Several reports indicate the presence of JNK1 in human neutrophils, although it has also been reported that JNK2 is the dominant isoform in these cells (45)(46)(47)(48)(49). More interestingly, it has been reported that inflammatory cytokine production in human PMN is independent of JNK and the downstream transcription factor complex AP-1 (50).…”

Section: Discussionmentioning

confidence: 99%

Mouse Neutrophils Require JNK2 MAPK forToxoplasma gondii-Induced IL-12p40 and CCL2/MCP-1 Release

Sukhumavasi

Egan

Denkers

2007

The MAPK family member JNK/stress-activated MAPK (SAPK) is involved in extracellular stress and proinflammatory cytokine responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are widely expressed, but JNK3 is largely restricted to tissues of the brain, testis, and heart. In this study, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been given little study. We used Western blot analysis to examine expression patterns of JNK/SAPK in wild-type and JNK2−/− polymorphonuclear leukocytes (PMN). Surprisingly, neutrophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed high levels of both JNK1 and JNK2 MAPK. JNK1 expression was steadily reduced during the neutrophil maturation in bone marrow. We used PMN infection with the protozoan parasite Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced rapid JNK2 phosphorylation and intracellular FACS staining demonstrated preferential activation in infected neutrophils. Use of JNK2−/− neutrophils revealed that this MAPK family member was required for PMN IL-12p40 and CCL2/MCP-1 production. The chemotactic response displayed a minor JNK2 dependence but phagocytosis and oxidative burst activity did not require this MAPK. These findings are important because they demonstrate 1) a previously unrecognized unusual JNK expression pattern in mouse neutrophils, 2) JNK2 in PMN is activated by Toxoplasma invasion, and 3) a requirement for JNK2 in PMN IL-12p40 and CCL2/MCP-1 production in response to a microbial pathogen.

“…In particular, Akt activation induces modulation of Bcl-2 family proteins such as Mcl-1 and phospho-Bad (39,40) and could therefore be involved in the inhibitory effect of CpG-DNA on our patient's PMN apoptosis. In addition, it has been reported that class I PI3K-catalytic subunits can lead to phosphorylation of ERK1/2 and p38MAPK (9,10,41,42); activation of these signaling pathways has been implicated in the up-regulation of CD11b expression (43,44) and L-selectin shedding (45,46) after PMN treatment with various inflammatory stimuli. In keeping with these data, we demonstrated that CpG-DNA-induced modulation of CD11b and Lselectin on the surface of our patient's PMNs is partially inhibited by pharmacological inhibitors of ERK1/2 and p38MAPK; furthermore, we found a CpG-DNA-induced increase in phosphorylation of ERK1/2 and p38MAPK in the patient's PMNs, which was reduced by PI3K inhibitors.…”

Section: Discussionmentioning

confidence: 99%

TLR9 Activation Induces Normal Neutrophil Responses in a Child with IRAK-4 Deficiency: Involvement of the Direct PI3K Pathway

Hoarau

Gérard

Lescanne

et al. 2007

Polymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient’s functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient’s PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient’s PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency.