Inhibition of neutrophil apoptosis by TLR agonists in whole blood: involvement of the phosphoinositide 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased levels of Mcl-1, A1, and phosphorylated Bad.
Using flow cytometry, we investigated the effect of TLR agonists on human polymorphonuclear neutrophil (PMN) apoptosis in whole blood. LPS (TLR4), peptidoglycan (TLR2), R-848 (TLR7/8), and CpG-DNA (TLR9) were equally effective at delaying spontaneous apoptosis of PMN, while PamCSK4 (TLR1/2), macrophage-activating lipopeptide-2 (TLR2/6), flagellin (TLR5), and loxoribine (TLR7) were less effective or inactive. TLR agonists found to delay apoptosis also extended the functional life span of PMN. Analysis of signaling pathways revealed that the antiapoptotic effect of TLR agonists required NF-kappaB and PI3K activation. Furthermore, analysis of intact cells by flow cytometry showed that TLR agonists delaying PMN apoptosis increased phosphorylation of Akt, a major target of PI3K. This effect was associated with a PI3K-dependent increase in heat shock protein 27 phosphorylation, which has been reported to play a key role in PMN survival. Finally, the TLR-induced delay in PMN apoptosis was associated with increased levels of Mcl-1 and A1, which are antiapoptotic members of the Bcl-2 family. These effects were reversed by PI3K and NF-kappaB inhibitors, respectively. TLR activation also led to PI3K-dependent phosphorylation of the proapoptotic protein Bad. Taken together, our results strongly suggest a role of NF-kappaB and PI3K in TLR-induced PMN survival, leading to modulation of Bcl-2 family molecules.
François
Stéphanie
S
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 479, Faculté Xavier Bichat, Paris, France.
El Benna
Jamel
J
Dang
Pham M C
PM
Pedruzzi
Eric
E
Gougerot-Pocidalo
Marie-Anne
MA
Elbim
Carole
C
D017209:Apoptosis / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000819:agonists*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000819:agonists*; D051818:Replication Protein C; D015398:Signal Transduction; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051199:Toll-Like Receptor 7; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D051025:bcl-Associated Death Protein
D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D011956:Receptors, Cell Surface; D051818:Replication Protein C; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051199:Toll-Like Receptor 7; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D051025:bcl-Associated Death Protein; C494819:BAD protein, human; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C495346:TLR5 protein, human; C495348:TLR7 protein, human; C495371:TLR9 protein, human; C494918:AKT1 protein, human; C505407:CASP3 protein, human