Purinergic Signaling and Its Role in Mobilization of Bone Marrow Stem Cells.

Mobilization or egress of stem cells from bone marrow (BM) into peripheral blood (PB) is an evolutionary preserved and important mechanism in an organism for self-defense and regeneration. BM-derived stem cells circulate always at steady-state conditions in PB, and their number increases during stress situations related to (a) infections, (b) tissue organ injury, (c) stress, and (d) strenuous exercise. Stem cells also show a circadian pattern of their PB circulating level with peak in early morning hours and nadir late at night. The number of circulating in PB stem cells could be pharmacologically increased after administration of some drugs such as cytokine granulocyte colony-stimulating factor (G-CSF) or small molecular antagonist of CXCR4 receptor AMD3100 (Plerixafor) that promote their egress from BM into PB and lymphatic vessels. Circulating can be isolated from PB for transplantation purposes by leukapheresis. This important homeostatic mechanism is governed by several intrinsic complementary pathways. In this chapter, we will discuss the role of purinergic signaling and extracellular nucleotides in regulating this process and review experimental strategies to study their involvement in mobilization of various types of stem cells that reside in murine BM.

Bone Marrow-Derived VSELs Engraft as Lung Epithelial Progenitor Cells after Bleomycin-Induced Lung Injury.

BACKGROUND: Alveolar type 2 (AT2) cells and bronchioalveolar stem cells (BASC) perform critical regenerative functions in response to lung damage. Published data show that nonhematopoietic, bone marrow-derived "very small embryonic-like stem cells" (VSELs) can differentiate in vivo into surfactant protein C (SPC)-producing AT2 cells in the lung. Here, we test directly whether VSEL-derived BASC and AT2 cells function to produce differentiated progeny. METHODS: using a reporter mouse in which the H2B-GFP fusion protein is driven from the murine SPC promoter, we tested whether bone marrow-derived VSELs or non-VSEL/nonhematopoietic stem cells (non-VSEL/non-HSCs) can differentiate into AT2 and BASC cells that function as progenitor cells. Immediately following bleomycin administration, WT recipient mice underwent intravenous administration of VSELs or non-VSEL/non-HSCs from SPC H2B-GFP mice. GFP+ AT2 and BASC were isolated and tested for progenitor activity using in vitro organoid assays. RESULTS: after 21 days in vivo, we observed differentiation of VSELs but not non-VSEL/non-HSCs into phenotypic AT2 and BASC consistent with previous data in irradiated recipients. Subsequent in vitro organoid assays revealed that VSEL-derived AT2 and BASC maintained physiological potential for differentiation and self-renewal. CONCLUSION: these findings prove that VSELs produce functional BASC and AT2 cells, and this may open new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury.

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.

Pannexin-1 channel "fuels" by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells.

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

A Novel Evidence That Mannan Binding Lectin (MBL) Pathway of Complement Cascade Activation is Involved in Homing and Engraftment of Hematopoietic Stem Progenitor Cells (HSPCs).

Delayed homing and engraftment of hematopoietic stem progenitor cells (HSPCs) or even failure to engraft at all is significant clinical problem after hematopoietic transplant. Therefore, in order to develop more efficient homing and engraftment facilitating strategies it is important to learn more about this process. Our team has postulated that myeloablative conditioning for transplantation induces in bone marrow (BM) microenvironment a state of sterile inflammation in which elements of innate immunity activated by radio- or chemotherapy conditioning for transplant play an important role. In frame with this claim we reported that a significant role in this process plays activation of complement cascade (ComC). Accordingly, mice that that lack a fifth component (C5) of ComC turned out to engraft poorly with normal syngeneic BM cells as compared to normal control animals. In extension of our previous studies we provide for first time evidence that mannan binding lectin (MBL) pathway is involved in activation of ComC in myeloablated transplant recipient BM and thus plays an important role in homing and engraftment of HSPCs. To support this MBL-KO mice show significant defect in hematopoietic reconstitution after hematopoietic transplantation. This correlates with a decrease in expression of stromal derived factor-1 (SDF-1) and impaired activation of Nlrp3 inflammasome in irradiated BM of these mice.

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.

Novel Evidence that Purinergic Signaling - Nlrp3 Inflammasome Axis Regulates Circadian Rhythm of Hematopoietic Stem/Progenitor Cells Circulation in Peripheral Blood.

We found that circadian changes in ATP level in peripheral blood (PB) activate the Nlrp3 inflammasome, which triggers diurnal release of hematopoietic stem/progenitor cells (HSPCs) from murine bone marrow (BM) into PB. Consistent with this finding, we observed circadian changes in expression of mRNA for Nlrp3 inflammasome-related genes, including Nlrp3, caspase 1, IL-1ß, IL-18, gasdermin (GSDMD), HMGB1, and S100A9. Circadian release of HSPCs from BM into PB as well as expression of Nlrp3-associated genes was decreased in mice in which pannexin 1-mediated secretion of ATP was inhibited by the blocking peptide 10Panx and in animals exposed to the specific small-molecule inhibitor of the Nlrp3 inflammasome MCC950. In addition to HSPCs, a similar decrease in diurnal cell counts was observed for mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). These results shed more light on the complexity of circadian regulation of HSPC release into PB, which is coordinated in a purinergic signaling-, innate immunity-dependent manner. Moreover, in addition to circadian changes in expression of the Nlrp3 inflammasome we also observed diurnal changes in expression of other inflammasomes, including Aim2, Nrp1a, and Nlrp1b.

An Overview of Novel Unconventional Mechanisms of Hematopoietic Development and Regulators of Hematopoiesis - a Roadmap for Future Investigations.

Hematopoietic stem cells (HSCs) are the best-characterized stem cells in adult tissues. Nevertheless, as of today, many open questions remain. First, what is the phenotype of the most primitive "pre-HSC" able to undergo asymmetric divisions during ex vivo expansion that gives rise to HSC for all hemato-lymphopoietic lineages. Next, most routine in vitro assays designed to study HSC specification into hematopoietic progenitor cells (HPCs) for major hematopoietic lineages are based on a limited number of peptide-based growth factors and cytokines, neglecting the involvement of several other regulators that are endowed with hematopoietic activity. Examples include many hormones, such as pituitary gonadotropins, gonadal sex hormones, IGF-1, and thyroid hormones, as well as bioactive phosphosphingolipids and extracellular nucleotides (EXNs). Moreover, in addition to regulation by stromal-derived factor 1 (SDF-1), trafficking of these cells during mobilization or homing after transplantation is also regulated by bioactive phosphosphingolipids, EXNs, and three ancient proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoA), and the fibrinolytic cascade (FibC). Finally, it has emerged that bone marrow responds by "sterile inflammation" to signals sent from damaged organs and tissues, systemic stress, strenuous exercise, gut microbiota, and the administration of certain drugs. This review will address the involvement of these unconventional regulators and present a broader picture of hematopoiesis.

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.

Bioactive Phospholipids Enhance Migration and Adhesion of Human Leukemic Cells by Inhibiting Heme Oxygenase 1 (HO-1) and Inducible Nitric Oxygenase Synthase (iNOS) in a p38 MAPK-Dependent Manner.

Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), have emerged as important mediators regulating the trafficking of normal and cancer cells. While the role of S1P in regulating migration of hematopoietic cells is well established, in this work we compared its biological effects to the effects of C1P, LPC, and LPA. We employed 10 human myeloid and lymphoid cell lines as well as blasts from AML patients. We observed that human leukemic cells express functional receptors for phospholipids and respond to stimulation by phosphorylation of p42/44 MAPK and AKT. We also found that bioactive phospholipids enhanced cell migration and adhesion of leukemic cells by downregulating expression of HO-1 and iNOS in a p38 MAPK-dependent manner but did not affect cell proliferation. By contrast, downregulation of p38 MAPK by SB203580 enhanced expression of HO-1 and iNOS and decreased migration of leukemic cells in vitro and their seeding efficiency to vital organs in vivo after injection into immunodeficient mice. Based on these findings, we demonstrate that, besides S1P, human leukemic cells also respond to C1P, LPC, and LPA. Since the prometastatic effects of bioactive phospholipids in vivo were mediated, at least in part, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, we propose that inhibitors of p38 MAPK or stimulators of HO-1 activity will find application in inhibiting the spread of leukemic cells in response to bioactive phospholipids.

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

Pharmacological mobilization of hematopoietic stem progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) is a result of mobilizing agent-induced "sterile inflammation" in the BM microenvironment due to complement cascade (ComC) activation. Here we provide evidence that ATP, as an extracellular nucleotide secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates the mobilization process. This process is augmented in a P2X7 receptor-dependent manner, and P2X7-KO mice are poor mobilizers. Furthermore, after its release into the extracellular space, ATP is processed by ectonucleotidases: CD39 converts ATP to AMP, and CD73 converts AMP to adenosine. We observed that CD73-deficient mice mobilize more HSPCs than do wild-type mice due to a decrease in adenosine concentration in the extracellular space, indicating a negative role for adenosine in the mobilization process. This finding has been confirmed by injecting mice with adenosine along with pro-mobilizing agents. In sum, we demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs. Although ATP triggers and promotes this process, adenosine has an inhibitory effect. Thus, administration of ATP together with G-CSF or AMD3100 or inhibition of CD73 by small molecule antagonists may provide the basis for more efficient mobilization strategies.

Sterile Inflammation of Brain, due to Activation of Innate Immunity, as a Culprit in Psychiatric Disorders.

Evidence has accumulated that the occurrence of psychiatric disorders is related to chronic inflammation. In support of this linkage, changes in the levels of circulating pro-inflammatory cytokines and chemokines in the peripheral blood (PB) of psychiatric patients as well as correlations between chronic inflammatory processes and psychiatric disorders have been described. Furthermore, an inflammatory process known as "sterile inflammation" when initiated directly in brain tissue may trigger the onset of psychoses. In this review, we will present the hypothesis that prolonged or chronic activation of the complement cascade (ComC) directly triggers inflammation in the brain and affects the proper function of this organ. Based on the current literature and our own work on mechanisms activating the ComC we hypothesize that inflammation in the brain is initiated by the mannan-binding lectin pathway of ComC activation. This activation is triggered by an increase in brain tissue of danger-associated molecular pattern (DAMP) mediators, including extracellular ATP and high-mobility group box 1 (HMGB1) protein, which are recognized by circulating pattern-recognition receptors, including mannan-binding lectin (MBL), that activate the ComC. On the other hand, this process is controlled by the anti-inflammatory action of heme oxygenase 1 (HO-1). In this review, we will try to connect changes in the release of DAMPs in the brain with inflammatory processes triggered by the innate immunity involving activation of the ComC as well as the inflammation-limiting effects of the anti-inflammatory HO-1 pathway. We will also discuss parallel observations that during ComC activation subsets of stem cells are mobilized into PB from bone marrow that are potentially involved in repair mechanisms.

Effect of Inorganic Dietary Selenium Supplementation on Selenoprotein and Lipid Metabolism Gene Expression Patterns in Liver and Loin Muscle of Growing Lambs.

Effect of selenium (Se) supplementation on the selenoprotein and lipid metabolism gene expression patterns in ruminants, especially in lambs is not yet fully understood. The aim of study was to evaluate the effect of Se supplementation on the messenger RNA (mRNA) expression patterns of selected selenoproteins and genes related to lipid metabolism in growing lambs. The experiment was conducted on 48 Polish Merino lambs divided into two groups (n = 24): control (C)-lambs fed with a basal diet (BD) with no Se supplementation, and supplemented (S)-lambs fed with a BD, supplemented with 0.5 mg Se/kg as sodium selenate for 8 weeks. Expression of 12 selenoproteins and six genes related to lipid metabolism was analyzed in the liver and longissimus dorsi (LD) muscle of growing lambs by qPCR. Significant differences were found in the expression of GPX1, GPX2, SEPM, SEPW1, SEP15, SEPGS2, and TXNRD1 in the liver, and GPX1, SEPP1, SEPN1, SEPW1, SEP15, and MSRB1 in the LD muscle between S and C lambs. Se supplementation mainly upregulated SEPW1, SEP15 (P < 0.001; P < 0.01) mRNA expression in the liver, and GPX1, SEPP1, SEPN1, SEPW1 (P < 0.001; P < 0.01) in the muscle of S group. On the other hand, significant decrease in GPX2 (P < 0.01), SEPM (P < 0.001), and SEPHS2 (P < 0.01) mRNA expression levels were observed in the liver of S group of lambs. Se supplementation did not affect PON1, LXRα, and PPARα mRNA expression levels, but a significant increase in mRNA levels of APOE and LPL in the LD muscle (P < 0.05) as well as LPL (P < 0.05) in the liver were noticed in the group of Se supplemented lambs. Our study confirmed that, in lambs, similarly to other species, mRNA expression patterns of several selenoproteins highly depend on dietary Se levels, and their expression is ruled by hierarchical principles and tissue-specific mechanisms. Moreover, the study showed that changes Se intake leads to different levels of genes expression related with lipid metabolism.