Abstract
Background. The Nlrp3 inflammasome is an intracellular pattern recognition receptor (PRR) and a key component of innate immunity, located in the cytoplasm of various cell types, including hematopoietic stem/progenitor cells (HSPCs). HSPCs from Nlrp3-KO mice show a significant defect in pharmacological mobilization, homing, and engraftment after transplantation. The assembly of the Nlrp3 inflammasome complex in cells is triggered by various stimuli, leading to the activation of caspase-1, which promotes the release of active IL-1β and IL-18, and causes gasdermin pore formation in the cell membrane, facilitating the release of several alarmins. Hypothesis. To explain the pleiotropic effects of Nlrp3 inflammasome activation—effects that lack a clear signaling pathway—we focused on the role of gasdermin pores and the alarmins they release as secondary mediators responsible for trafficking and metabolism in HSPCs.Material and Methods. HSPCs purified from WT and Nlrp3-KO mice were evaluated for their redox characteristics under steady-state conditions and after exposure to modulators of mitochondrial function. These included measurements of oxygen consumption rate (OCR) during steady state and following treatment with oligomycin, a mitochondrial ATP synthase inhibitor, then maximal respiratory capacity after uncoupling with FCCP, and finally after exposure to Rotenone/Antimycin A, which inhibit mitochondrial respiratory chain complexes I and III, effectively blocking electron transport. These assessments allowed analysis of mitochondrial adaptive responses to stress in WT and mutant mice. Additionally, mitochondrial membrane integrity was assessed via MitoTracker staining, dependence on anaerobic versus aerobic glycolysis was evaluated, and the expression of genes associated with all five mitochondrial complexes (I–V) of the electron transfer chain (ETC) was analyzed, along with ETC function in specific bioassays. We also examined the expression of glucose and amino acid transporters, as well as key enzymes involved in glycolysis, lipidogenesis, and amino acid metabolism. Results. We report, for the first time, that HSPCs from Nlrp3 inflammasome knockout mice exhibit defects in OCR. Under steady-state conditions, these cells show nearly a twofold decrease in both basal and maximal respiration compared to WT cells. Similarly, Nlrp3-KO cells demonstrate reduced Spare Respiratory Capacity (SRC) and ATP production relative to WT cells. This reduction correlates with decreased expression of the mitochondria-encoded cytochrome b gene (CYTB), which encodes a critical component of complex III in the ETC. Complex III accepts electrons from complex II-associated Coenzyme Q and transfers them to cytochrome c, while pumping H+ ions from FADH2 generated in the Krebs cycle into the mitochondrial intermembrane space. The decreased CYTB expression results in a modest but significant decline in overall ATP production and is partly compensated by increased activity of cytochrome c reductase. This data underscores the role of the Nlrp3 inflammasome in regulating cell metabolism by maintaining proper “tonic activation” of the mitochondrial electron transport chain. MitoTracker analysis revealed no significant changes in mitochondrial membrane potential, indicating that the main defect in Nlrp3-KO cells relates to gasdermin-dependent alarmine feedback stimulation. We also observed significantly lower expression of enzymes critical for glycolysis (PFKFB3), lipid metabolism (SREBP2, ASMase), and amino acid uptake (LAT1). Conclusions. The Nlrp3 inflammasome mediates the “tonic activation” of OCR through a positive feedback loop involving alarmins released via gasdermin pores. When functioning within the beneficial hormetic phase, this process promotes trafficking and metabolism in HSPCs.
