Regulation of neutrophil function by NAMPT


Roberts, Kate Regulation of neutrophil function by NAMPT. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Neutrophils have capacity to cause tissue damage in chronic inflammatory disease. In rheumatoid arthritis (RA) they infiltrate joints, secrete proteases and reactive oxygen species (ROS), and express cytokines. RA neutrophils express NAMPT which is a highly conserved, pleiotropic protein, that catalyses the rate-limiting step of the NAD salvage pathway, but also has cytokine-like activity. NAMPT is elevated in inflammation and exerts pro-inflammatory effects on neutrophils. The aim of this research was to determine the role of NAMPT, as a signalling molecule and as an enzyme, in regulating neutrophil activities of pathological importance. Neutrophils were isolated from the blood of healthy donors and either stimulated with NAMPT or else NAMPT was inhibited (with FK866), in the presence and absence of pro-inflammatory cytokines in vitro. Assays for specific neutrophil functions such as production of ROS, bacterial killing and apoptosis were performed, and expression of cytokines was also examined. Transcriptome sequencing of neutrophils treated with FK866 and TNFα in combination, was carried out to investigate the wider impact of NAMPT inhibition on neutrophil gene expression. NAMPT expression was also examined in control, cytokine treated and RA patient neutrophils. NAMPT had little capacity to prime neutrophils, although it did delay neutrophil apoptosis and stabilise the anti-apoptotic protein Mcl-1. NAMPT inhibition in neutrophils, depleted NAD(P)/H and had effects on neutrophil functions and gene expression. Notably, FK866 decreased ROS production but did not affect the ability of neutrophils to kill bacteria. NAMPT-inhibited neutrophils exhibited decreased activation of signalling pathways and a diminished response to cytokines; transcriptome analysis showed that FK866 decreased TNFα-induced expression of many genes. NAMPT expression was not dynamically regulated in control neutrophils, but in RA patient neutrophils, NAMPT mRNA correlated with TNFα expression in a cohort of patients, and NAMPT protein was elevated in synovial fluid neutrophils compared to those from paired blood. Thus, NAMPT is elevated in activated inflammatory neutrophils and correlates with other markers of inflammation in RA, suggesting that it may be a marker of inflammation in these cells. Also, as a NAD biosynthetic enzyme, NAMPT regulates neutrophil functions and gene products central to RA disease pathology, without affecting bacterial killing capacity. This suggests that inhibition of NAMPT may potentially have the capacity to decrease neutrophil mediated tissue-damage observed in chronic inflammation, without adversely compromising host defence, and thus may be a future treatment option for diseases such as RA.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2012-09 (completed)
Uncontrolled Keywords: Neutrophil, NAMPT, PBEF, Visfatin, NAD, RA
Subjects: ?? RC ??
Divisions: Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 24 Jan 2013 16:28
Last Modified: 16 Dec 2022 04:37
DOI: 10.17638/00008355
Supervisors:
URI: https://livrepository.liverpool.ac.uk/id/eprint/8355
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