NLRP6 in the Bone Marrow Microenvironment Regulates HSPC Quiescence Via MGP/RUNX2

Abstract

Inflammatory response in the bone marrow microenvironment affects hematopoietic stem cell (HSC) engraftment and hematopoietic reconstruction. NLR Family Pyrin Domain Containing 6 (NLRP6) involves in multiple signaling pathways to regulate inflammatory response and maintain cell homeostasis. However, the mechanism of NLRP6 in bone marrow microenvironment regulating hematopoiesis remains unclear. To analysis of the effect of NLRP6 deficiency on bone marrow microenvironment and HSC activity, bone marrow chimeras with NLRP6 deficiency in the microenvironment was performed to analyze HSPC quiescence and differentiation in the microenvironment with Nlrp6 deficiency. Single-cell sequencing technology was utilized to analyze the cellular and molecular abnormalities of NLRP6-deficient stromal cells in the bone marrow and to furtherly explore the regulatory mechanism of the NLRP6-deficient microenvironment to resident HSPCs. The results showed that NLRP6 deficiency in bone marrow microenvironment increased proportion of LSK-HSPCs in G0 phase but not effect on the homing of HSPCs. LSK-HSPCs sorted from NLRP6-/- recipients at week 8 after 1st transplantation for 2nd transplantation had higher frequency of CD45.1+ cells in the PBMCs or in total myeloid cells compared with control. NLRP6 deficiency in bone marrow microenvironment reduced CMP and GMP proportion. CMPs sorted from NLRP6-/- recipients at week 8 after 1st transplantation had lower regeneration potential through 2nd transplantation and cell proliferation assay in vitro. Transcriptome analysis of LSK-HSPC, CMP, GMP and MEP from two groups at 8 weeks after transplantation showed that NLRP6 deficiency in the microenvironment modulated cell cycle of HSPCs and is associated with NOD-like receptor signaling pathway. Single-cell sequencing of bone marrow CD45-Ter119- cells showed that NLRP6 deficiency increased CAR cells of bone marrow stroma. The molecule expression of CAR cells (including Cxcl12 and Kitl) contributing to hematopoiesis was upregulated which was associated with MAPK and NOD-like receptor signaling pathway. NLRP6 deletion in the microenvironment upregulated MGP expression of CAR cell which were furtherly supported by western blot and immunofluorescence staining. Recombinant mouse MGP protein (rmMGP) decreased Colony-Forming Units (CFUs) of WT LSK-HSPCs. Single-cell sequencing of LSK-HSPCs at week 8 after 1st transplantation showed that HSCs differentiated into Mpps which separately kept dormant or entered cell cycling. NLRP6 deficiency in the microenvironment markedly increased the quiescent cellular proportion of LSK-HSPCs compared with control, especially Flt3+ CD48- MPPs which were dormant MPPs differentiated early from HSCs. LSK-HSPCs resident in the microenvironment with NLRP6 deletion kept lower transcription of genes about cell cycling and NOD like receptor signaling pathway. Transcription factor Runx2 and its target inhibitors of cell cycle were upregulated and RUNX2 phosphorylation increased by western blot compared with control. In a word, this study suggests that NLRP6 in the bone marrow microenvironment promotes HSPC quiescence induced by RUNX2 through upregulating MGP from CAR cells.