Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) by Downregulating Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) Activity.

Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide an evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs.

The P2X4 purinergic receptor has emerged as a potent regulator of hematopoietic stem/progenitor cell mobilization and homing-a novel view of P2X4 and P2X7 receptor interaction in orchestrating stem cell trafficking.

Recent evidence indicates that extracellular adenosine triphosphate (eATP), as a major mediator of purinergic signaling, plays an important role in regulating the mobilization and homing of hematopoietic stem progenitor cells (HSPCs). In our previous work we demonstrated that eATP activates the P2X7 ion channel receptor in HSPCs and that its deficiency impairs stem cell trafficking. To learn more about the role of the P2X purinergic receptor family in hematopoiesis, we phenotyped murine and human HSPCs with respect to the seven P2X receptors and observed that, these cells also highly express P2X4 receptors, which shows ~50% sequence similarity to P2X7 subtypes, but that P2X4 cells are more sensitive to eATP and signal much more rapidly. Using the selective P2X4 receptor antagonist PSB12054 as well as P2X4-KO mice, we found that the P2X4 receptor, similar to P2X7 receptor, promotes trafficking of HSPCs in that its deficiency leads to impaired chemotaxis of HSPCs in response to a stromal-derived factor 1 (SDF-1) gradient, less effective pharmacological mobilization, and defective homing and engraftment of HSPCs after transplantation into myeloablated hosts. This correlated with a decrease in SDF-1 expression in the BM microenvironment. Overall, our results confirm the proposed cooperative dependence of both receptors in response to eATP signaling. In G-CSF-induced mobilization, a lack of one receptor is not compensated by the presence of the other one, which supports their mutual dependence in regulating HSPC trafficking.

Danger-associated molecular pattern molecules take unexpectedly a central stage in Nlrp3 inflammasome-caspase-1-mediated trafficking of hematopoietic stem/progenitor cells.

Like their homing after transplantation to bone marrow (BM), the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not fully understood, and several overlapping pathways are involved. Several years ago our group proposed that sterile inflammation in the BM microenvironment induced by pro-mobilizing agents is a driving force in this process. In favor of our proposal, both complement cascade (ComC)-deficient and Nlrp3 inflammasome-deficient mice are poor G-CSF and AMD3100 mobilizers. It is also known that the Nlrp3 inflammasome mediates its effects by activating caspase-1, which is responsible for proteolytic activation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) and their release from cells along with several danger-associated molecular pattern molecules (DAMPs). We observed in the past that IL-1ß and IL-18 independently promote mobilization of HSPCs. In the current work we demonstrated that caspase-1-KO mice are poor mobilizers, and, to our surprise, administration of IL-1ß or IL-18, as in the case of Nlrp3-KO animals, does not correct this defect. Moreover, neither Caspase-1-KO nor Nlrp3-KO mice properly activated the ComC to execute the mobilization process. Interestingly, mobilization in these animals and activation of the ComC were both restored after injection of the DAMP cocktail eATP+HGMB1+S100A9, the components of which are normally released from cells in an Nlrp3 inflammasome-caspase-1-dependent manner. In addition, we report that caspase-1-deficient HSPCs show a decrease in migration in response to BM homing factors and engraft more poorly after transplantation. These results for the first time identify caspase-1 as an orchestrator of HSPC trafficking.

Heme Oxygenase 1 (HO-1) as an Inhibitor of Trafficking of Normal and Malignant Hematopoietic Stem Cells - Clinical and Translational Implications.

Evidence indicates that bone marrow (BM)-residing hematopoietic stem/progenitor cells (HSPCs) are released into peripheral blood (PB) after administration of pro-mobilizing drugs, which induce a state of sterile inflammation in the BM microenvironment. In the reverse process, as seen after hematopoietic transplantation, intravenously injected HSPCs home and engraft into BM niches. Here again, conditioning for transplantation by myeloablative chemo- or radiotherapy induces a state of sterile inflammation that promotes HSPC seeding to BM stem cell niches. Therefore, the trafficking of HSPCs and their progeny, including granulocytes and monocytes/macrophages, is regulated by a response to pro-inflammatory stimuli. This responsiveness to inflammatory cues is also preserved after malignant transformation of hematopoietic cells. Results from our laboratory indicate that the responsiveness of hematopoietic cells to pro-inflammatory stimuli is orchestrated by Nlrp3 inflammasome. As reported, HO-1 effectively attenuates intracellular activation of Nlrp3 inflammasome as well as the pro-inflammatory effects of several humoral mediators, including complement cascade (ComC) cleavage fragments that promote migration of hematopoietic cells. Based on this finding, inhibition of HO-1 activity may become a practical strategy to enhance the mobilization and homing of normal HSPCs, and, alternatively, its activation may prevent unwanted spread and in vivo expansion of leukemic cells. Graphical Abstract.

SARS-CoV-2 Entry Receptor ACE2 Is Expressed on Very Small CD45- Precursors of Hematopoietic and Endothelial Cells and in Response to Virus Spike Protein Activates the Nlrp3 Inflammasome.

Angiotensin-converting enzyme 2 (ACE2) plays an important role as a member of the renin-angiotensin-aldosterone system (RAAS) in regulating the conversion of angiotensin II (Ang II) into angiotensin (1-7) (Ang [1-7]). But at the same time, while expressed on the surface of human cells, ACE2 is the entry receptor for SARS-CoV-2. Expression of this receptor has been described in several types of cells, including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which raises a concern that the virus may infect and damage the stem cell compartment. We demonstrate for the first time that ACE2 and the entry-facilitating transmembrane protease TMPRSS2 are expressed on very small CD133+CD34+Lin-CD45- cells in human umbilical cord blood (UCB), which can be specified into functional HSCs and EPCs. The existence of these cells known as very small embryonic-like stem cells (VSELs) has been confirmed by several laboratories, and some of them may correspond to putative postnatal hemangioblasts. Moreover, we demonstrate for the first time that, in human VSELs and HSCs, the interaction of the ACE2 receptor with the SARS-CoV-2 spike protein activates the Nlrp3 inflammasome, which if hyperactivated may lead to cell death by pyroptosis. Based on this finding, there is a possibility that human VSELs residing in adult tissues could be damaged by SARS-CoV-2, with remote effects on tissue/organ regeneration. We also report that ACE2 is expressed on the surface of murine bone marrow-derived VSELs and HSCs, although it is known that murine cells are not infected by SARS-CoV-2. Finally, human and murine VSELs express several RAAS genes, which sheds new light on the role of these genes in the specification of early-development stem cells. Graphical Abstract •Human VSELs and HSCs express ACE2 receptor for SARS-CoV2 entry. •Interaction of viral spike protein with ACE2 receptor may hyperactivate Nlrp3 inflammasome which induces cell death by pyroptosis. •SARS-CoV2 may also enter cells and eliminate them by cell lysis. •What is not shown since these cells express also Ang II receptor they may hyperactivate Nlrp3 inflammasome in response to Ang II which may induce pyroptosis. Our data indicates that Ang 1-7 may have a protective effect.

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

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.

SARS-CoV-2 infection and overactivation of Nlrp3 inflammasome as a trigger of cytokine "storm" and risk factor for damage of hematopoietic stem cells.

The scientific community faces an unexpected and urgent challenge related to the SARS-CoV-2 pandemic and is investigating the role of receptors involved in entry of this virus into cells as well as pathomechanisms leading to a cytokine "storm," which in many cases ends in severe acute respiratory syndrome, fulminant myocarditis and kidney injury. An important question is if it may also damage hematopoietic stem progenitor cells?

Innate immunity orchestrates the mobilization and homing of hematopoietic stem/progenitor cells by engaging purinergic signaling-an update.

Bone marrow (BM) as an active hematopoietic organ is highly sensitive to changes in body microenvironments and responds to external physical stimuli from the surrounding environment. In particular, BM tissue responds to several cues related to infections, strenuous exercise, tissue/organ damage, circadian rhythms, and physical challenges such as irradiation. These multiple stimuli affect BM cells to a large degree through a coordinated response of the innate immunity network as an important guardian for maintaining homeostasis of the body. In this review, we will foc++us on the role of purinergic signaling and innate immunity in the trafficking of hematopoietic stem/progenitor cells (HSPCs) during their egression from the BM into peripheral blood (PB), as seen along pharmacological mobilization, and in the process of homing and subsequent engraftment into BM after hematopoietic transplantation. Innate immunity mediates these processes by engaging, in addition to certain peptide-based factors, other important non-peptide mediators, including bioactive phosphosphingolipids and extracellular nucleotides, as the main topic of this review. Elucidation of these mechanisms will allow development of more efficient stem cell mobilization protocols to harvest the required number of HSPCs for transplantation and to accelerate hematopoietic reconstitution in transplanted patients.

The Nlrp3 inflammasome as a "rising star" in studies of normal and malignant hematopoiesis.

Recent investigations indicate that hematopoiesis is coregulated by innate immunity signals and by pathways characteristic of the activation of innate immunity cells that also operate in normal hematopoietic stem progenitor cells (HSPCs). This should not be surprising because of the common developmental origin of these cells from a hemato/lymphopoietic stem cell. An important integrating factor is the Nlrp3 inflammasome, which has emerged as a major sensor of changes in body microenvironments, cell activation, and cell metabolic activity. It is currently the best-studied member of the inflammasome family expressed in hematopoietic and lymphopoietic cells, including also HSPCs. It is proposed as playing a role in (i) the development and expansion of HSPCs, (ii) their release from bone marrow (BM) into peripheral blood (PB) in stress situations and during pharmacological mobilization, (iii) their homing to BM after transplantation, and (iv) their aging and the regulation of hematopoietic cell metabolism. The Nlrp3 inflammasome is also involved in certain hematological pathologies, including (i) myelodysplastic syndrome, (ii) myeloproliferative neoplasms, (iii) leukemia, and (iv) graft-versus-host disease (GvHD) after transplantation. The aim of this review is to shed more light on this intriguing intracellular protein complex that has become a "rising star" in studies focused on both normal steady-state and pathological hematopoiesis.

Valproic Acid Decreases Endothelial Colony Forming Cells Differentiation and Induces Endothelial-to-Mesenchymal Transition-like Process.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor is a widely used anticonvulsant drug. VPA is also under clinical evaluation to be employed in anticancer therapy, as an antithrombotic agent or a molecule to be used in the stem cells expansion protocols. Since endothelial colony forming cells (ECFC) has been identified as the human postnatal vasculogenic cells involved in thrombotic disorders and serve as a promising source of immature cell for vascular repair, objectives of the present study were to determine how VPA contributes to ECFC commitment and their angiogenic properties. We examined the effect of VPA on ECFC obtained from cord blood by evaluating colony number, proliferation, migration and their sprouting ability in vitro, as well as their in vivo vasculogenic properties. VPA inhibited endothelial differentiation potential from of cord blood derived stem cells associated with decreased proliferation and sprouting activity of cultured ECFC. VPA treatment significantly decreased the vessel-forming ability of ECFC transplanted together with mesenchymal stem cells (MSC) in Matrigel implants in nude mice model. Surprisingly, a microscopic evaluation revealed that VPA induces marked morphological changes from a cobblestone-like EC morphology to enlarged spindle shaped morphology of ECFC. RT-qPCR and a CD31/CD90 flow cytometry analysis confirmed a phenotypic switch of VPA-treated ECFC to mesenchymal-like phenotype. In conclusion, the pan-HDAC inhibitor VPA described for expansion of hematopoietic stem cells and very small embryonic like stem cells cannot be successfully employed for differentiation of endothelial lineage committed ECFC into functional endothelial cells. Our data also suggest that VPA based therapeutics may induce endothelial dysfunction associated with fibrosis that might induce thrombosis recurrence or venous insufficiency.

Extracellular Adenosine Triphosphate (eATP) and Its Metabolite, Extracellular Adenosine (eAdo), as Opposing "Yin-Yang" Regulators of Nlrp3 Inflammasome in the Trafficking of Hematopoietic Stem/Progenitor Cells.

Nlrp3 inflammasome plays a pleiotropic role in hematopoietic cells. On the one hand, physiological activation of this intracellular protein complex is crucial to maintaining normal hematopoiesis and the trafficking of hematopoietic stem progenitor cells (HSPCs). On the other hand, its hyperactivation may lead to cell death by pyroptosis, and prolonged activity is associated with sterile inflammation of the BM and, as a consequence, with the HSPCs aging and origination of myelodysplasia and leukemia. Thus, we need to understand better this protein complex's actions to define the boundaries of its safety window and study the transition from being beneficial to being detrimental. As demonstrated, the Nlrp3 inflammasome is expressed and active both in HSPCs and in the non-hematopoietic cells that are constituents of the bone marrow (BM) microenvironment. Importantly, the Nlrp3 inflammasome responds to mediators of purinergic signaling, and while extracellular adenosine triphosphate (eATP) activates this protein complex, its metabolite extracellular adenosine (eAdo) has the opposite effect. In this review, we will discuss and focus on the physiological consequences of the balance between eATP and eAdo in regulating the trafficking of HSPCs in an Nlrp3 inflammasome-dependent manner, as seen during pharmacological mobilization from BM into peripheral blood (PB) and in the reverse mechanism of homing from PB to BM and engraftment. We propose that both mediators of purinergic signaling and the Nlrp3 inflammasome itself may become important therapeutic targets in optimizing the trafficking of HSPCs in clinical settings.

The Inhibition of CD39 and CD73 Cell Surface Ectonucleotidases by Small Molecular Inhibitors Enhances the Mobilization of Bone Marrow Residing Stem Cells by Decreasing the Extracellular Level of Adenosine.

We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.

The Nlrp3 Inflammasome Orchestrates Mobilization of Bone Marrow-Residing Stem Cells into Peripheral Blood.

Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a "sterile inflammation" in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1ß and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP-Nlrp3-ComC axis in the egress of stem cells into PB.

ATP-Nlrp3 Inflammasome-Complement Cascade Axis in Sterile Brain Inflammation in Psychiatric Patients and its Impact on Stem Cell Trafficking.

Recent evidence indicates that the occurrence of psychiatric disorders in patients is linked to a local "sterile" inflammation of brain or due to a systemic inflammation process that affects the central nervous system. This is supported by the observation that in peripheral blood of psychotic patients are detectable several mediators and markers of inflammation as well as clinical data on correlations between systemic chronic inflammatory processes and psychiatric disorders. This may explain why some reported anti-inflammatory treatment strategies have beneficial effects on ameliorating psychotic events. In this review we will present a concept that aberrant purinergic signaling and increases in extracellular level of adenosine triphosphate (ATP) in the brain parenchyma may lead to activation of Nlrp3 inflammasome in microglia cells and as a consequence microglia released danger associated molecular pattern (DAMP) proteins activate complement cascade (ComC) in mannan binding lectin (MBL) - dependent manner. Activation of ATP-Nlrp3 inflammasome-ComC axis may also orchestrate trafficking of stem cells released from bone marrow into peripheral blood observed in psychotic patients. Based on this, the ATP-Nlrp3 inflammasome-ComC axis may become a target for new therapeutic approaches, which justifies the development and clinical application of efficient anti-inflammatory treatment strategies targeting this axis in psychiatry.

Correction: Cancer from the perspective of stem cells and misappropriated tissue regeneration mechanisms.

The original version of this Article omitted the following from the Acknowledgements.

Correction: Novel evidence that extracellular nucleotides and purinergic signaling induce innate immunity-mediated mobilization of hematopoietic stem/progenitor cells.

Following the publication of this article, the authors noted that the following should be included in the Acknowledgements section: "MA is the recipient of a START scholarship (0785) from FNP". The authors wish to apologise for any inconvenience caused.