Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-κB Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad

Abstract

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-κB 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-κB 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-κB and PI3K in TLR-induced PMN survival, leading to modulation of Bcl-2 family molecules.

A Polysaccharide Virulence Factor of a Human Fungal Pathogen Induces Neutrophil Apoptosis via NK Cells

“…In contrast, Thorén et al (26) recently reported that NK cells accelerate spontaneous apoptosis of isolated PMNs. In this study, to avoid the PMN isolation procedures that have been reported to alter PMN responses (21,42,43), we analyzed PMN apoptosis by flow cytometry in whole-blood conditions. In these conditions, with whole-blood PMN activation by GG, a polysaccharide extracted from A. fumigatus, we demonstrated that NK cells are a critical trigger of PMN apoptosis.…”

“…The study was approved by the local ethics committee of Necker-Enfants Malades Hospital (Paris, France). Human PMNs were isolated as described previously (21).…”

“…Cycloheximide (CHX) (10 mg/ml; Calbiochem) and LPS from Escherichia coli 055:B5 (10 ng/ml; Sigma-Aldrich) were used as proapoptotic and antiapoptotic controls, respectively (21). In some experiments, samples were pretreated for 1 h with 30 mM diphenyleneiodonium (DPI) (Sigma-Aldrich), an inhibitor of NADPH oxidase, 100 mM benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-FMK) (BD Pharmingen), an inhibitor of caspases, 50 mM Z-IETD, a caspase-8 inhibitor (BD Pharmingen), 100 mM pepstatin-A, a cathepsin D inhibitor (Sigma-Aldrich), and 100 mm calpain inhibitor III (Calbiochem-Millipore).…”

“…FITC-Annexin V (BD Pharmingen) and the impermeant nuclear dye 7-aminoactinomycin D (7-AAD; BD Via-Probe) were used to quantify apoptosis, as described previously (21,22). After pretreatment of whole blood with PBS, GG, CHX, or LPS for 24 h, samples (100 ml) were washed twice in PBS, incubated with APC-anti-CD15 (clone HI98; BD Pharmingen) for 15 min, and then incubated with FITC-annexin V for 15 min.…”

NF-κB and the immune response

“…Unlike lymphocytes, which are relatively long-lived in the absence of activation, protection from apoptosis in neutrophils is more important during the inflammatory response than in homeostasis. Indeed, many TLR ligands increase neutrophil survival in vitro, likely due to NF-kBmediated expression of antiapoptotic genes (Francois et al, 2005). Although neutrophils are capable of activating NF-kB in response to many pro-inflammatory stimuli (McDonald, 2004), they lack p52 and RelB (McDonald et al, 1997), the very subunits that are crucial for the maintenance of long-lived lymphocytes.…”