Novel Observation That Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) Involves Activation of Nlrp3 Inflammasome That Provides Cholesterol and Sphingolipids for the Formation of Lipid Rafts on the Outer Cell Membrane That Are Crucial for Proper Responsiveness of HSPCs to Chemoattractant and Hematopoietic Cytokines/Growth Factors

Background. Cell outer membranes contain protein receptors, which are integrated into microdomains, known as membrane lipid rafts (MLRs), floating freely in the cell membrane bilayer. MLRs are enriched for cholesterol and sphingolipids for their functional integrity, and incorporate receptors, e.g., the CXCR4 receptor for a-chemokine stromal-derived factor 1 (SDF-1), the a1b4 integrin receptor (VLA-4) for vascular cell adhesion molecule-1 (VCAM-1), common b-subunit chain for IL-3, GM-CSF and IL-5 receptors, and the c-kit receptor for stem cell factor (SCF). Therefore, the proper assembly of MLRs regulates the migration and proliferation of hematopoietic stem/progenitor cells (HSPC). Moreover, intracellular pattern recognition receptor Nlrp3 inflammasome plays, as we reported recently, an important role in the migration and proliferation of murine and human HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Mice deficient in Nlrp3 inflammasome have a defect in migration, mobilization, homing, and engraftment of bone marrow (BM) cells. They have also reduced the number of HSPCs in BM and display a defect in the formation of MLRs. Aim of the study.Based on those mentioned above, we asked how at the molecular level, Nlrp3 inflammasome regulates MLRs formation, and based on data that Nlrp3 inflammasome is involved in cell metabolism (Dev Cell. 2020 Oct 26;55(2):133-149), we asked if this could be a result of providing cholesterol and sphingolipids elements for MLRs assembly.Materials and Methods. We evaluated as the first expression of cholesterol synthesizing enzymes SREBP2, HMGCs, and HMGCR in normal murine Sca-1⁺c-kit⁺lin^- (SKL) HSPCs and SKL HSPCs exposed to KL+IL-3+Tpo. In control experiments to inhibit Nlrp3 inflammasome, we employed the specific inhibitor MCC950. We also performed a metabolome analysis of SKL HSPCs stimulated by KL+IL-3+Tpo and evaluated changes in pathways involved in lipidogenesis. MLRs formation was assessed by Western blot analysis and confocal analysis in stimulated SKL cells and control experiments in SKL cells exposed to Nlrp3 inflammasome inhibitor MCC950. Results. We noticed that stimulation of SKL cells upregulates the expression of enzymes involved in cholesterol and fatty acids metabolism (SREBP2, HMGCs, and HMGCR), which correlated with metabolome analysis performed by MetaboAnalyst 5.0 annotation that demonstrated an increase in pentose phosphate pathway that is known NADPH source for cholesterol and sphingosine synthesis. At the metabolome level, we observed an increase in the synthesis of several cholesterol isoforms in stimulated HSPCs, including CE (16:0) or cholesteryl palmitic acid, which increases protein affinity to MLRs. We also observed a significant increase in the concentration of hexosyl ceramides lipids belonging to the sphingolipid family, which serve as crucial precursors for the biosynthesis of vital dihexosyl ceramides for spontaneous phospholipid inter-bilayer transfer. All these changes depended on Nlrp3 inflammasome activation, as confirmed by employing the specific Nlrp3 inflammasome inhibitor MCC950. Conclusions. We provide for the first-time evidence that Nlrp3 inflammasome required for proper cell migration and metabolism promotes the synthesis of cholesterol and sphingolipids that are crucial components of cell surface MLRs. This data builds a novel link between innate immunity and lipid metabolism that provides components for proper MLRs formation required for optimal mobilization and homing of transplanted HSPCs.

No relevant conflicts of interest to declare.