Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

Abstract

Fast and efficient homing and engraftment of hematopoietic stem progenitor cells (HSPCs) is crucial for positive clinical outcomes from transplantation. We found that this process depends on activation of the Nlrp3 inflammasome, both in the HSPCs to be transplanted and in the cells in the recipient bone marrow (BM) microenvironment. For the first time we provide evidence that functional deficiency in the Nlrp3 inflammasome in transplanted cells or in the host microenvironment leads to defective homing and engraftment. At the molecular level, functional deficiency of the Nlrp3 inflammasome in HSPCs leads to their defective migration in response to the major BM homing chemoattractant stromal-derived factor 1 (SDF-1) and to other supportive chemoattractants, including sphingosine-1-phosphate (S1P) and extracellular adenosine triphosphate (eATP). We report that activation of the Nlrp3 inflammasome increases autocrine release of eATP, which promotes incorporation of the CXCR4 receptor into membrane lipid rafts at the leading surface of migrating cells. On the other hand, a lack of Nlrp3 inflammasome expression in BM conditioned for transplantation leads to a decrease in expression of SDF-1 and danger-associated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs.

Keywords: Bone marrow sterile inflammation; Complement cascade; Extracellular nucleotides; Nlrp3 inflammasome; Purinergic signaling; Stem cell engraftment; Stem cell homing.

Fig. 1

Expression of Nlrp3, IL-1β, IL-18, caspase 1, ACS, HGMB1, and S100a9 mRNAs in murine BMMNCs and BM-purified SKL cells. Expression of Nlrp3, IL-1β, IL-18, caspase 1, ACS, HGMB1, and S100a9 mRNAs in BMMNCs and BM-purified SKL cells as measured by RT-PCR. Results are combined from three independent purifications of BM and SKL cells isolated from six animals per purification

Fig. 2

Impact of Nlrp3 on the chemotactic activity of murine BMMNCs. The chemotactic responsiveness of BMMNCs from WT or Nlrp3-KO mice to medium supplemented with SDF-1, S1P, or ATP according to the number of CFU-GM clonogenic progenitors. Results are combined from two independent experiments. *p > 0.05

Fig. 3

Defect in short- and long-term engraftment of Nlrp3-KO HSPCs in WT mice Panel A. Lethally irradiated WT mice (9 per group) were transplanted with bone marrow mononuclear cells (BMMNCs) from WT or Nlrp3-KO mice, which had been previously labeled with a PKH67 cell linker. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67⁺ cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs from WT or Nlrp3-KO mice, and 12 days after transplantation femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed for counting the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). *p < 0.05. Panel C. Lethally irradiated mice (9 per group) were transplanted with BMMNCs from WT or Nlrp3-KO mice. White blood cells (left) and platelets (right) were counted at intervals (at 0, 3, 7, 14, 21, and 28 days after transplantation). *p < 0.05.

Fig. 4

Defect in homing and short-term engraftment of HSPCs in Nlrp3-KO mice. Panel A. Lethally irradiated Nlrp3-KO or WT mice (4 mice per group) were transplanted with bone marrow mononuclear cells (BMMNCs) from WT mice, which had been previously labeled with a PKH67 cell linker. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67⁺ cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated Nlrp3-KO or WT mice (5 mice per group) were transplanted with BMMNCs from WT mice, and 12 days after transplantation femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed to count the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). *p < 0.05

Fig. 5

Confocal analysis of membrane lipid rafts in purified murine SKL cells. Panel A. Defective lipid raft formation in murine C57Bl/6 Nlrp3-KO BM-purified SKL cells or wild type BM-purified SKL cells exposed to adenosine (10 μM) or CoPP (50 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml), stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control) but not in SKL cells isolated from Nlrp3-KO animals or SKL cells isolated from WT animals after adenosine and CoPP treatment. Panel B. The chemotactic responsiveness of mBMMNCs untreated or treated with ¹⁰Panx, ^SCPanx, or apyrase in unsupplemented medium or medium supplemented with SDF-1, S1P, or ATP, as determined by counting the number of CFU-GM clonogenic progenitors. Results are combined from two independent experiments. *p > 0.05, **p > 0.01, ***p > 0.001

Fig. 6

The impact of pannexin 1 channel blockade on short- and long-term engraftment of HSPCs in WT mice. Panel A. Lethally irradiated WT mice (9 per group) were transplanted with bone marrow mononuclear cells (BMMNCs) that had been previously labeled with a PKH67 cell linker and then treated with ¹⁰Panx or ^SCPanx. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67⁺ cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with ¹⁰Panx or ^SCPanx, and 12 days after transplantation the femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed for counting the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). *p < 0.05, **p < 0.01, ***p < 0.001. Panel C. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with ¹⁰Panx. White blood cells (above) and platelets (below) were counted at intervals (at 0, 3, 7, 14, 21, and 28 days after transplantation). *p < 0.05. Panel D. Defective lipid raft formation in murine BM-purified SKL cells from C57Bl/6 mice exposed to ¹⁰Panx inhibitory peptide (200 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml) (positive control) or exposed to ¹⁰Panx inhibitory peptide, stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control), but not in SKL cells after ¹⁰Panx inhibitory peptide treatment

Fig. 7

The effect of myeloablative treatment on mRNA and protein expression related to Nlrp3 inflammasome activation, as measured by qRT-PCR and ELISA. Panel A. Expression of IL-1β, IL-18, AIM2, Nlrp1, SDF-1, SCG, caspase 1, Hmgb1, and S100a9 mRNAs in bone marrow mononuclear cells (BMMNCs) isolated from non-irradiated and irradiated (1000 cGy) C57Bl/6 and Nlrp3-KO animals, as measured by qRT-PCR. Results of qRT-PCR were normalized to the β2 microglobulin (β2m) level. The data represent the mean value ± SEM for three independent experiments. Panel B. The level of C5a protein in conditioned media harvested from irradiated WT or Nlrp3-KO BMMNCs, measured by ELISA. The data represent the mean value for two independent experiments. *p < 0.05

Fig. 8

The role of eATP in the homing and engraftment of HSPCs. eATP plays a dual role in the homing of HSPCs to BM. On the one hand, whether autocrine-secreted from transplanted HSPCs (*) or secreted in response to conditioning for transplantation from cells in the BM microenvironment (**), eATP promotes formation of membrane lipid rafts (yellow cap) on the surface of HSPCs, which assemble together the major receptors for chemoattractants (SDF-1, S1P, and eATP) (adapted from 65)