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 Nlrp3-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 in vitro 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 Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.ope

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1β secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1β rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1β-secreting cells in DAMP-rich inflammatory region.ope