Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

Abstract

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD(+) has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD(+) homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD(+) levels and expression levels of NAD(+) homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD(+) levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD(+) synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD(+) homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD(+) levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD(+). The agonist-induced rise in NAD(+) shows striking parallels to well-known second messengers and raises the possibility that NAD(+) is acting in a similar manner in this model.

Keywords: TNF-α; immune responses; lipopolysaccharide (LPS); macrophages; pyridine nucleotides; second messenger.

Figure 1. Intracellular NAD⁺ and TNF-α levels in THP-1-derived macrophages subsets

LPS effect on NAD⁺ levels in pro-inflammatory (A; M1-like) and anti-inflammatory (B; M2-like) macrophages. TNF-α levels in M1-like (C) and M2-like (D) macrophages after LPS challenge. Data shown are mean±S.E.M. for three separate experiments (n=3).

Figure 2. Effect of FK866 (100 nM) on NAD⁺ levels in M1-like (A) and M2-like (B) macrophages

Data shown are mean±S.E.M. for three separate experiments (n=3).

Figure 3. Gene expression profiles of NAD⁺ homoeostasis enzymes in M1- and M2-like macrophages relative to THP-1 monocytes

Data shown are mean±S.E.M. of fold change for three separate experiments (n=3–5). The data were analysed by one-way ANOVA. *P<0.05.

Figure 4. Pharmacological modulation of intracellular NAD⁺ levels by DPI (A), FK866 (B) and 1-MT (C) in M1-like cells

Data shown are mean±S.E.M. for three separate experiments (n=3–4).

Figure 5. Pharmacological modulation of TNF-α release by DPI (A), FK866 (B) and 1-MT (C) in M1-like cells

Data shown are mean±S.E.M. for three separate experiments (n=4).

Figure 6. Gene expression profile of NAD⁺ homoeostasis enzymes in M1-like (A) and M2-like (B) macrophages after LPS challenge

Gene expression is expressed as fold change relative to the control. Data shown are mean±S.E.M. for three separate experiments (n=4). The data were analysed by one-way ANOVA. *P<0.05.