Background. The intracellular complement network (complosome) has been identified to regulate lymphocyte-mediated immune responses (Adv Immunol. 2018;138:35-70). Recently, we demonstrated that the complosome, in addition to lymphocytes, is also present in hematopoietic stem/progenitor cells (HSPCs) (Leukemia 2023 Jun;37(6):1401-1405). We also reported that HSPCs from mice lacking the intracellular C3 and C5 complosome proteins exhibit different responses to extracellular stress. Specifically, HSPCs from C3-KO cells are more sensitive to oxidative challenge, whereas C5-KO cells show greater resistance (Leukemia 2025 Jul;39(7):1571-1577). This explains our previous data, indicating why C3-KO mice are effective mobilizers of HSPCs and their BM cells engraft properly after transplantation, in contrast to C5-KO mice, which mobilize poorly and whose HSPCs engraft less efficiently. However, the molecular basis for this difference has not been fully clarified. Aim of the study.To further explore the role of the complosome in regulating the trafficking and metabolism of bone marrow HSPCs, we focused on the responsiveness of HSPCs from complosome-deficient C3-KO and C5-KO mice to extracellular stress caused by reactive oxygen species (ROS) and pro-inflammatory mediators, as observed during pharmacological mobilization and conditioning for transplantation.Materials and methods. HSPCs purified from WT, C3-KO, and C5-KO mice were assessed for their redox characteristics under steady-state conditions and after exposure to modulators of mitochondrial function. This included measuring oxygen consumption rate (OCR) during steady state and following treatment with oligomycin, a mitochondrial ATP synthase inhibitor, then maximal respiratory stimulation with the uncoupler FCCP, and finally after exposure to Rotenone/Antimycin A, which inhibit mitochondrial respiratory chain complexes I and III, effectively blocking the electron transport chain. These evaluations helped analyze mitochondrial adaptive responses to stress in WT and mutant mice. We also examined mitochondrial membrane integrity via MitoTracker staining, dependence on anaerobic versus aerobic glycolysis, gene expression related to all five mitochondrial complexes (I–V) of the electron transfer chain (ETC), and the function of ETC components in specific bioassays. Additionally, we assessed the expression of glucose and amino acid transporters, along with key enzymes involved in glycolysis, lipidogenesis, and amino acid metabolism. Results. For the first time, we reveal mitochondrial functional defects in complosome-deficient HSPCs. Notably, compared to WT cells, there was a decrease in baseline OCR, particularly in C3-KO cells, and reduced accumulation of MitoTracker probes in mitochondria, indicating mitochondrial abnormalities. We also observed a significant reduction in mRNA expression of CYTB, a vital component of complex III of the ETC, in HSPCs from C3-KO and C5-KO mice. Furthermore, cells from complosome-deficient animals under steady-state conditions showed increased lactate production and elevated lactate dehydrogenase (LDH) release, indicating a reliance on anaerobic glycolysis. The uptake of the glucose analog 2-NBDG increased in C3-KO cells but decreased in C5-KO cells compared to WT mice. Meanwhile, total ATP production was reduced in C3-KO cells and remained unchanged after exposure to hydrogen peroxide (H2O2), unlike in C5 mice, suggesting a greater dependence on anaerobic glycolysis in C3-KO cells. Interestingly, C3-KO HSPCs displayed higher expression of the Nlrp3 inflammasome, a regulator of cell metabolism and a driver of HSPC trafficking. Conversely, C5-KO cells showed decreased expression of glucose and amino acid transporters, along with key enzymes involved in glycolysis and lipid metabolism. This resulted in a reduced number of HSPCs in the bone marrow of C5-KO mice and impaired trafficking. Conclusions. We demonstrate for the first time that the complosome is essential for maintaining mitochondrial function in HSPCs and influences cell trafficking, metabolism, and proliferation. Therefore, a novel regulatory mechanism involving the complosome in preserving mitochondrial integrity and function in HSPCs has been identified. Modulating complosome activity in HSPCs may be crucial for promoting their expansion and successful engraftment after transplantation.

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2025
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