Pre-B Cell Colony Enhancing Factor (PBEF)/Nampt Phosphorylation by PI3 Kinase is Indispensable for the Inhibition of Neutrophil (PMN) Apoptosis in Patients with Sepsis

Song Jia, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto Canada; Jean Parodo, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto Canada; John Marshall, St. Michael's Hospital

We have reported that delayed apoptosis of PMN from patients with sepsis requires expression of PBEF/Nampt, the rate-limiting enzyme in a salvage pathway of NAD biosynthesis, and the activity of PI3 kinase (PI3K).

 We hypothesize that PBEF Nampt activity is dependent on PI3-kinase-mediated serine phosphorylation.

We studied PMN from healthy controls or ICU patients with severe sepsis (N=9), and HEK293 cells stably transfected with WT or mutant PBEF-myc. Apoptosis was quantified by flow cytometry as the uptake of propidium iodide, and NAD was measured by thiazylol blue assay. Phosphodeficient PBEF was created by mutation of Ser199 to Ala (A), and phosphomimetic PBEF by Ser199 mutation to Glu (D). PI3K activity was inhibited by Ly294002 (20 μM), and PBEF Nampt activity by APO866 (10 nM).

Compared with controls,septic PMN apoptosis was delayed (4.7±3.5 vs 50.0±8.6%, p<0.001), and intracellular NAD increased (17.2±6.2 vs 4.5±2.2 pMol, p<0.001). Both Ly294002 (25.2±8.0%, p<0.01) and APO866 (20.1±5.6%, p<0.01) increased septic MPN appoptosis and reduced NAD levels (Ly 4.4±3.8, APO 5.3±3.8 pMol, p<0.01). Intracellular NAD was increased in HEK293 cells transfected with rPBEF or S199D phosphomimetic PBEF (5.4±1.3 pMol to 16.2±2.4 and 16.2±2.9 pMol; p<0.01). Phosphodeficient S199A PBEF had no effect on NAD levels, but increased spontaneous apoptosis from 1.4±1.1% in controls to 11.3±2.3% (p<0.01).   Western blots of septic PMN (N=4) revealed increased expression of the active p110 subunit of PI3K; Ly294002 induced time-dependent serine dephosphorylation of PBEF (Figure). Co-immunoprecipitation studies showed that PBEF bound the PI3K p110 subunit, but not the p85 regulatory subunit in septic PMN; this interaction was blocked by Ly294002. PBEF dimerized in HEK293 cells transfected with myc- and GFP-tagged WT or S199D, but not S199A PBEF; Ly294002 prevented the dimerization of WT PBEF.

PI3 kinase serine phosphorylates PBEF, promoting its dimerization and Nampt activity, and supporting prolonged PMN survival in septic patients. Inhibition of this process restores the normal kinetics of PMN apoptosis, and potentially supports the resolution of PMN-mediated inflammation in sepsis.