Intracellular NAD + Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation

Nicotinamide adenine dinucleotide (NAD ) is an important cofactor in many redox and non-redox NAD -consuming enzyme reactions. Intracellular NAD level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD -depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in -deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not -deficient mice. Collectively, our data suggest that NAD depletion provides a non-transcriptional priming signal for NLRP3 activation mitochondrial perinuclear clustering, and aging-associated NAD decline can trigger NLRP3 inflammasome activation in ATP-rich environments.