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We postulated that mobilization, homing, and engraftment of hematopoietic stem/progenitor cells (HSCPs) is facilitated by a state of sterile inflammation induced in bone marrow (BM) after administration of pro-mobilizing drugs or in response to pre-transplant myeloablative conditioning. An important role in this phenomenon plays purinergic signaling that by the release of extracellular adenosine triphosphate (eATP) activates in HSPCs and in cells in the hematopoietic microenvironment an intracellular pattern recognition receptor (PPR) known as Nlrp3 inflammasome. We reported recently that its deficiency results in defective trafficking of HSPCs. Moreover, it is known that eATP after release into extracellular space is processed by cell surface expressed ectonucleotidases CD39 and CD73 to extracellular adenosine (eAdo) that in contrast to eATP shows an anti-inflammatory effect. Based on data that the state of sterile inflammation promotes trafficking of HSPCs, and since eAdo is endowed with anti-inflammatory properties we become interested in how eAdo will affect the mobilization, homing, and engraftment of HSPCs and which of eAdo receptors are involved in these processes. As expected, eAdo impaired HSPCs trafficking and this occurred in autocrine- and paracrine-dependent manner by direct stimulation of these cells or by affecting cells in the BM microenvironment. We report herein for the first time that this defect is mediated by activation of the A2B receptor and a specific inhibitor of this receptor improves eAdo-aggravated trafficking of HSPCs. To explain this at the molecular level eAdo-A2B receptor interaction upregulates in HSPCs in NF-kB-, NRF2- and cAMP-dependent manner heme oxygenase-1 (HO-1), that is Nlrp3 inflammasome inhibitor. This corroborated with our analysis of proteomics signature in murine HSPCs exposed to eAdo that revealed that A2B inhibition promotes cell migration and proliferation. Based on this we postulate that blockage of A2B receptor may accelerate the mobilization of HSPCs as well as their hematopoietic reconstitution and this approach could be potentially considered in the future to be tested in the clinic. [ABSTRACT FROM AUTHOR]

Introduction. Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking. Material and methods. We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking. Results. We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499. Conclusions. This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs. (Folia Histochemica et Cytobiologica 2022, Vol. 60, No. 3, 280-290). [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection leads to severe lung damage due to pneumonia and, in more severe cases, leads to acute respiratory distress syndrome, or ARDS. This affects the viability of bronchoalveolar cells. An important role in the pathogenesis of these complications is the hyperactivation of the renin-angiotensin-aldosterone (RAA) pathway and induction of cytokine storm that occurs in an Nlrp3 inflammasome-dependent manner. To shed more light on the susceptibility of lung tissue to SARS-CoV-2 infection, we evaluated murine bronchioalveolar stem cells (BASC), alveolar type II cells (AT2), and 3D-derived organoids expression of mRNA encoding genes involved in virus entry into cells, components of RAA, and genes that comprise elements of the Nlrp3 inflammasome pathway. We noticed that all these genes are expressed by lung alveolar stem cells and organoids-derived from these cells. Interestingly, all these cells express a high level of ACE2 that, on the one hand, serves as an entry receptor for SARS-CoV-2 and, on the other, converts angiotensin II into its physiological antagonist, angiotensin 1–7 (Ang 1–7), which has been reported to have a protective role in lung damage. To shed more light on the role of Ang 1–7 on lung tissue, we exposed lung-derived BASC and AT2 cells to this mediator of RAA and noticed that it increases the proliferation of these cells. Based on this, Ang 1–7 could be employed to alleviate the damage to lung alveolar stem/progenitor cells during SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Nox2 or NADPH oxidase generates reactive oxygen species (ROS) that are involved in several physiological and pathological processes regulating hematopoiesis and as reported ROS are involved in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). Based on this and to learn more on the role of Nox2 in trafficking of HSPCs, we performed mobilization studies in Nox2-KO mice and noticed that these animals are poor G-CSF and AMD3100 mobilizers. Next we established hematological chimeras of WT and Nox2-KO mice and performed mobilization studies to learn if the defect in mobilization was HSPCs or BM microenvironment related. Moreover, Nox-2-KO BMMNC show defective homing and engraftment after transplantation into normal syngeneic recipients, and Nox2-KO mice display delayed hematopoietic recovery as recipients of normal BMMNC. To explain this at molecular level there are two main intracellular sources of Nox2-derived ROS. The first source is ROS generated by activation of Nox2 associated cell surface receptors involved in migration of HSPCs and the other source is ROS released from Nox2 present on mitochondria membrane. We learned that both sources of ROS activate intracellular pattern recognition receptor Nlrp3 inflammasome that is pivotal for migration of HSPCs. Accordingly we learned that Nox2-KO mice have almost completely abrogated activation of Nlrp3 inflammasome as assayed by immunofluorescence activation assay. The promigratory effects of Nlrp3 inflammasome correlated with inclusion of receptors for major hematopoietic chemoattractant that are stromal-derivedfactor 1 (SDF-1) and sphingosine-1 phosphate (S1P), CXCR4 and S1P1R receptors respectively -- into cell membrane lipid rafts (MLRs). Inclusion of both receptors into MLRs is required for optimal chemotactic responsiveness of HSPCs to SDF-1 and S1P. This data explains severe defect in HSPCs trafficking we observed in Nox2-KO animals. [ABSTRACT FROM AUTHOR]

Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promote trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs as well as defective expression of these receptors on transplanted HSPCs or in bone marrow (BM) microenvironment of graft recipient mice lead to defective homing, engraftment and delayed hematopoietic reconstitution. We explained these defects as result of an impaired activation of downstream target from P2X4 and P2X7 receptors that is intracellular pattern recognition receptor Nlrp3 inflammasome. P2X receptor family consists of seven purinergic receptors (P2X1-7). We report that one of the members of this receptor family ― P2X1 receptor is highly expressed on HSPCs and promotes migration of leucocytes and lymphocytes. This prompted us to investigate its role in trafficking of more primitive hematopoietic cells. Our data indicates that P2X1 receptor is involved in eATP mediated trafficking of HSPCs, and this occurs in Nlrp3 inflammasome-dependent manner. This study sheds light on novel role of P2X1 receptor in normal hematopoiesis and further supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in mobilization, homing and engraftment of HSPCs. [ABSTRACT FROM AUTHOR]

Significance: The success rate of hematopoietic stem cell transplantation depends mainly on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) followed by the speed of their engraftment in the myeloablated transplant recipient. Therefore, clinical outcomes will significantly benefit from accelerating the homing and engraftment of these cells. This is, in particular, important when the number of cells available for the transplantation of HSPCs is limited. Recent Advances: We postulated that myeloablative conditioning for hematopoietic transplantation by radio- or chemotherapy induces a state of sterile inflammation in transplant recipient peripheral blood (PB) and bone marrow (BM). This state is mediated by activation of the BM stromal and innate immunity cells that survive myeloablative conditioning and respond to danger-associated molecular patterns released from the cells damaged by myeloablative conditioning. As a result of this, several factors are released that promote proper navigation of HSPCs infused into PB of transplant recipient and prime recipient BM to receive transplanted cells. Critical Issues: We will present data that cellular innate immunity arm and soluble arm comprised complement cascade proteins, promoting the induction of the BM sterile inflammation state that facilitates the navigation, homing, and engraftment of HSPCs. Future Directions: Deciphering these mechanisms would allow us to better understand the mechanisms that govern hematopoietic recovery after transplantation and, in parallel, provide important information on how to optimize this process in the clinic by employing small molecular modifiers of innate immunity and purinergic signaling. Antioxid. Redox Signal. 37, 1254–1265. [ABSTRACT FROM AUTHOR]