Abstract
Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages.
Highlights
Important elements of neutrophil and monocyte/macrophage function in innate immunity like cell adhesion, locomotion, phagocytosis and regulation of cell shape are determined by their ability to regulate actin cytoskeleton reorganization [1]
Within 3 hours, total NAD+ levels in RAW 254.7 cells were already reduced by approximately 60% (p,0.05; Figure 1B) while NADH, NADP+ and NADPH levels remained within the range of the control (Figure 1C, E, F)
We noticed that the NADPH/NADP+ ratio fluctuated somewhat during the monitoring period, both with and without the FK866 inhibitor, while this seemed less apparent for the NAD+/NADH ratio (Figure 1D, J)
Summary
Important elements of neutrophil and monocyte/macrophage function in innate immunity like cell adhesion, locomotion, phagocytosis and regulation of cell shape are determined by their ability to regulate actin cytoskeleton reorganization [1]. Monocytes migrate into target tissue areas and differentiate into macrophages. Differentiation toward an inflammatory M1 subtype can be induced by lipopolysaccharide (LPS) stimulation, while IL-4 and IL-13 promote the alternative activation of macrophages towards a suppressive M2 subtype. In keeping with their differential roles in either the initial immune response or the resolution phase of inflammation and tissue healing, the typical morphodynamic activities, including adhesion to the extracellular matrix, or the capturing, adhesion and internalization of cells, debris, or foreign particles via phagocytosis, differ between the M1- and M2polarized macrophages. Diversity in macrophage phenotype is to a large extent the result of variable cues from the tissue microenvironment [6]
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