Latterly, nicotinamide adenine dinucleotide (NAD+) has emerged as a molecule with versatile functions and of enormous impact on the maintenance of cell integrity. Besides playing key roles in almost all major aspects of energy metabolism, there is mounting evidence that NAD+ and its degradation products affect various biological activities including calcium homeostasis, gene transcription, DNA repair, and intercellular communication. This review is aimed at giving a brief insight into the life cycle of NAD+ in the cell, referring to synthesis, action and degradation aspects. With respect to their immunological relevance, the importance and function of the major NAD+ metabolizing enzymes, namely CD38/CD157, ADP-ribosyltransferases (ARTs), poly-ADP-ribose-polymerases (PARPs), and sirtuins are summarized and roles of NAD+ and its main degradation product adenosine 5'-diphosphoribose (ADPR) in cell signaling are discussed. In addition, an outline of the variety of immunological processes depending on the activity of nicotinamide phosphoribosyltransferase (Nampt), the key enzyme of the salvage pathway of NAD+ synthesis, is presented. Taken together, an efficient supply of NAD+ seems to be a crucial need for a multitude of cell functions, underlining the yet only partly revealed potency of this small molecule to influence cell fate.

No abstract

The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features.

No abstract

No abstract

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD + ) induces a transient rise in [Ca 2+ ] i in human monocytes caused by an influx of extracellular calcium. The NAD + -induced Ca 2+ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X 1 , P2X 4 , and P2X 7 receptors being engaged in the NAD + -induced rise in [Ca 2+ ] i . Among the P2X receptor subtypes, the P2X 7 receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X 7 receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD + , we found that NAD + unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD + at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD + and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.

Macrophages (MΦ) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MΦ. We found that infection of both subsets with RV resulted in a low TNF-α and a high interferon (IFN, type I and type III) release whereby M-MΦ produced far more IFNs than GM-MΦ. Thus, TNF-α production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MΦ compared to the addition of LPS to the uninfected cells the TNF-α response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-β one putative priming stimulus induced by RV. Small amounts of IFN-β were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MΦ to RV and LPS revealed an increased phosphorylation of NFκB (M-MΦ), but different to uninfected MΦ a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-β can prime MΦ to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MΦ defense to viral infection is modulated by a following exposure to a bacterial infection.

Separation of specific blood cells is necessary for a deeper insight into their role in health and disease. To obtain such cells, efficient and robust isolation methods are needed. We compare here the Fab‐based Traceless Affinity Cell Selection (TACS®) technology and the Magnetic Activated Cell Sorting (MACS®) technology to isolate human monocytes from whole blood and buffy coats as well as the differentiation of the isolated monocytes to dendritic cells (DCs). TACS® is a positive selection technology using immune affinity chromatography based on CD‐specific low affinity Fab‐fragments for the reversible capture and release of target cells. The positive selection by MACS® is based on magnetic beads coated with specific high affinity monoclonal antibodies to catch the target cells. The target cells separated by TACS® are “label‐free” while cells positively isolated by MACS® will carry the cell specific label. Our data show that the separation methods described here are well suited to obtain functional monocytes of high quality and purity. A differentiation of the cells into DCs leads to comparable results with the exception that CD1a expression levels on immature and mature DCs are elevated when monocytes are isolated using the TACS® technology. Taken together, our results suggest that the TACS® method may be of advantage when preparing monocytes and monocyte‐derived DCs for functional analyses, while the MACS® method seems to be capable of higher monocyte recoveries. © 2018 International Society for Advancement of Cytometry