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In single-cell organisms, extracellular microvesicles (ExMVs) were one of the first cell-cell communication platforms that emerged very early during evolution. Multicellular organisms subsequently adapted this mechanism. Evidence indicates that all types of cells secrete these small circular structures surrounded by a lipid membrane that may be encrusted by ligands and receptors interacting with target cells and harboring inside a cargo comprising RNA species, proteins, bioactive lipids, signaling nucleotides, and even entire organelles "hijacked" from the cells of origin. ExMVs are secreted by normal cells and at higher levels by malignant cells, and there are some differences in their cargo. On the one hand, ExMVs secreted from malignant cells interact with cells in the microenvironment, and in return, they are exposed by a "two-way mechanism" to ExMVs secreted by non-leukemic cells. Therefore, leukemogenesis occurs and progresses in ExMVs enriched microenvironments, and this biological fact has pathologic, diagnostic, and therapeutic implications. We are still trying to decipher this intriguing cell-cell communication language better. We will present a current point of view on this topic and review some selected most recent discoveries and papers., (© 2024. The Author(s).)

The liver-derived circulating in peripheral blood and intrinsic cell-expressed complement known as complosome orchestrate the trafficking of hematopoietic stem/progenitor cells (HSPCs) both during pharmacological mobilization and homing/engraftment after transplantation. Our previous research demonstrated that C3 deficient mice are easy mobilizers, and their HSPCs engraft properly in normal mice. In contrast, C5 deficiency correlates with poor mobilization and defects in HSPCs' homing and engraftment. The trafficking of HSPCs during mobilization and homing/engraftment follows the sterile inflammation cues in the BM microenvironment caused by stress induced by pro-mobilizing drugs or myeloablative conditioning for transplantation. Therefore, to explain deficiencies in HSPC trafficking between C3-KO and C5-KO mice, we evaluated the responsiveness of C3 and C5 deficient cells to low oxidative stress. As reported, oxidative stress in BM is mediated by the activation of purinergic signaling, which is triggered by the elevated level of extracellular adenosine triphosphate (eATP) and by the activation of the complement cascade (ComC). In the current work, we noticed that BM lineage negative cells (lin - ) isolated from C3-KO mice display several mitochondrial defects reflected by an impaired ability to adapt to oxidative stress. In contrast, C5-KO-derived BM cells show a high level of adaptation to this challenge. To support this data, C3-KO BM lin - cells were highly responsive to eATP stimulation, which correlates with enhanced levels of reactive oxygen species (ROS) generation and more efficient activation of intracellular Nlrp3 inflammasome. We conclude that the enhanced sensitivity of C3-KO mice cells to oxidative stress and better activation of the Nox2-ROS-Nlrp3 inflammasome signaling axis explains the molecular level differences in trafficking between C3- and C5-deficient HSPCs., (© 2024. The Author(s).)

Very small embryonic like stem cells (VSELs) are a dormant population of stem cells that, as proposed, are deposited during embryogenesis in various tissues, including bone marrow (BM). These cells are released under steady state conditions from their tissue locations and circulate at a low level in peripheral blood (PB). Their number increases in response to stressors as well as tissue/organ damage. This increase is evident during neonatal delivery, as delivery stress prompts enrichment of umbilical cord blood (UCB) with VSELs. These cells could be purified from BM, PB, and UCB by multiparameter sorting as a population of very small CXCR4 + Lin - CD45 - cells that express the CD34 or CD133 antigen. In this report, we evaluated a number of CD34 + Lin - CD45 - and CD133 + Lin - CD45 - UCB-derived VSELs. We also performed initial molecular characterization of both cell populations for expression of selected pluripotency markers and compared these cells at the proteomic level. We noticed that CD133 + Lin - CD45 - population is more rare and express, at a higher level, mRNA for pluripotency markers Oct-4 and Nanog as well as the stromal-derived factor-1 (SDF-1) CXCR4 receptor that regulates trafficking of these cells, however both cells population did not significantly differ in the expression of proteins assigned to main biological processes., (© 2023 International Society for Advancement of Cytometry.)

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration., (© 2024. The Author(s).)

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries., (© 2024. The Author(s).)

Hematopoiesis is regulated by several mediators such as peptide-based growth factors, cytokines, and chemokines, whose biological effects have been studied for many years. However, several other mediators have been identified recently that affect the fate of hematopoietic stem/progenitor cells (HSPC) as well as non-hematopoietic cells in the bone marrow microenvironment. These new mediators comprise members of purinergic signaling pathways and are active mediators of the soluble arm of innate immunity, the complement cascade (ComC). In this review, we will discuss the coordinated effects of these pathways in regulating the biology of HSPC. Importantly, both purinergic signaling and the ComC are activated in stress situations and interact with specific receptors expressed on HSPC. Evidence has accumulated indicating that some of the purinergic as well as ComC receptors could also be activated intracellularly by intrinsically expressed ligands. To support this recent evidence, our work indicates that the major mediator of purinergic signaling, adenosine triphosphate, and the cleavage product of the fifth component of the ComC (C5), C5a anaphylatoxin, can activate their corresponding receptors expressed on the outer mitochondrial membrane in an autocrine manner. We will also discuss recent evidence that these responses, mediated by purinergic signaling and the ComC network, are coordinated by activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 - reactive oxygen species - NLR family pyrin domain containing 3 (NLRP3) inflammasome (Nox2-ROS-NLRP3) axis., (© 2023. The Author(s).)

A cell's most significant existential task is to survive by ensuring proper metabolism, avoiding harmful stimuli, and adapting to changing environments. It explains why early evolutionary primordial signals and pathways remained active and regulate cell and tissue integrity. This requires energy supply and a balanced redox state. To meet these requirements, the universal intracellular energy transporter purine nucleotide-adenosine triphosphate (ATP) became an important signaling molecule and precursor of purinergic signaling after being released into extracellular space. Similarly, ancient proteins involved in intracellular metabolism gave rise to the third protein component (C3) of the complement cascade (ComC), a soluble arm of innate immunity. These pathways induce cytosol reactive oxygen (ROS) and reactive nitrogen species (RNS) that regulate the redox state of the cells. While low levels of ROS and RNS promote cell growth and differentiation, supra-physiological concentrations can lead to cell damage by pyroptosis. This balance explains the impact of purinergic signaling and innate immunity on cell metabolism, organogenesis, and tissue development. Subsequently, along with evolution, new regulatory cues emerge in the form of growth factors, cytokines, chemokines, and bioactive lipids. However, their expression is still modulated by both primordial signaling pathways. This review will focus on the data that purinergic signaling and innate immunity carry on their ancient developmental task in hematopoiesis and specification of hematopoietic stem/progenitor cells (HSPCs). Moreover, recent evidence shows both these regulatory pathways operate in a paracrine manner and inside HSPCs at the autocrine level., (© 2024. The Author(s).)

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., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Neurodegenerative diseases (NDDs) continue to be a significant healthcare problem. The economic and social implications of NDDs increase with longevity. NDDs are linked to neuroinflammation and activated microglia and astrocytes play a central role. There is a growing interest for stem cell-based therapy to deliver genes, and for tissue regeneration. The promise of mesenchymal stem cells (MSC) is based on their availability as off-the-shelf source, and ease of expanding from discarded tissues. We tested the hypothesis that MSC have a major role of resetting activated microglial cells. We modeled microglial cell lines by using U937 cell-derived M1 and M2 macrophages. We studied macrophage types, alone, or in a non-contact culture with MSCs. MSCs induced significant release of exosomes from both types of macrophages, but significantly more of the M1 type. RNA sequencing showed enhanced gene expression within the exosomes with the major changes linked to the inflammatory response, including cytokines and the purinergic receptors. Computational analyses of the transcripts supported the expected effect of MSCs in suppressing the inflammatory response of M1 macrophages. The inflammatory cargo of M1 macrophage-derived exosomes revealed involvement of cytokines and purinergic receptors. At the same time, the exosomes from MSC-M2 macrophages were able to reset the classical M2 macrophages to more balanced inflammation. Interestingly, we excluded transfer of purinergic receptor transcripts from the co-cultured MSCs by analyzing these cells for the identified purinergic receptors. Since exosomes are intercellular communicators, these findings provide insights into how MSCs may modulate tissue regeneration and neuroinflammation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bone marrow (BM) contains not only hematopoietic stem cells (HSCs) but also some very rare, early development, small quiescent stem cells that, upon activation, may differentiate across germlines. These small cells, named very small embryonic like stem cells (VSELs), can undergo specification into several types of cells including HSCs. Interestingly, murine BM is also home to a "mystery" population of small CD45 + stem cells with many of the phenotypic characteristics attributed to resting HSCs. Since the size of the "mystery" population cells are between that of VSELs and HSCs, and because CD45 - VSELs can be specified into CD45 + HSCs, we hypothesized that the quiescent CD45 + "mystery" population could be a missing developmental link between VSELs and HSCs. To support this hypothesis, we showed that VSELs first became enriched for HSCs after acquiring expression of the CD45 antigen already expressed on "mystery" stem cells. Moreover, VSELs freshly isolated from BM similar to the "mystery" population cells, are quiescent and do not reveal hematopoietic potential in in vitro and in vivo assays. However, we noticed that CD45 + "mystery" population cells, similar to CD45 - VSELs, became specified into HSCs after co-culture over OP9 stroma. We also found that mRNA for Oct-4, a pluripotency marker that is highly expressed in VSELs, is also detectable in the "mystery" population cells, albeit at a much lower level. Finally, we determined that the "mystery" population cells specified over OP9 stroma support were able to engraft and establish hematopoietic chimerism in lethally irradiated recipients. Based on these results, we propose that the murine BM "mystery" population could be an intermediate population between BM-residing VSELs and HSCs already specified for lympho-hematopoietic lineages., (© 2023. The Author(s).)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Hematopoietic and immune cells originate from a common hematopoietic/lymphopoietic stem cell what explains that these different cell types often share the same receptors and respond to similar factors. Moreover, the common goal of both lineages is to ensure tissue homeostasis under steady-state conditions, fight invading pathogens, and promote tissue repair. We will highlight accumulating evidence that innate and adaptive immunity modulate several aspects of hematopoiesis within the hormetic zone in which the biological response to low exposure to potential stressors generally is favorable and benefits hematopoietic stem/progenitor cells (HSPCs). Innate immunity impact on hematopoiesis is pleiotropic and involves both the cellular arm, comprised of innate immunity cells, and the soluble arm, whose major component is the complement cascade (ComC). In addition, several mediators released by innate immunity cells, including inflammatory cytokines and small antimicrobial cationic peptides, affect hematopoiesis. There are intriguing observations that HSPCs and immune cells share several cell-surface pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and cytosol-expressed NOD, NOD-like, and RIG-I-like receptors and thus can be considered "pathogen sensors". In addition, not only lymphocytes but also HSPCs express functional intracellular complement proteins, defined as complosome which poses challenging questions for further investigation of the intracellular ComC-mediated intracrine regulation of hematopoiesis., (© 2021. The Author(s).)

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-1β (IL-1β) 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-1β 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-1β 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., (© 2021. The Author(s).)

Hematopoietic stem/progenitor cells (HSPCs) express receptors for complement cascade (ComC) cleavage fragments C3a and C5a and may respond to inflammation-related cues by sensing pathogen-associated molecular pattern molecules (PAMPs) released by pathogens as well as non-infectious danger associated molecular pattern molecules (DAMPs) or alarmin generated during stress/tissue damage sterile inflammation. To facilitate this HSPCs are equipped with C3a and C5a receptors, C3aR and C5aR, respectively, and express on the outer cell membrane and in cytosol pattern recognition receptors (PPRs) that sense PAMPs and DAMPs. Overall, danger-sensing mechanisms in HSPCs mimic those seen in immune cells, which should not surprise as hematopoiesis and the immune system develop from the same common stem cell precursor. This review will focus on the role of ComC-derived C3a and C5a that trigger nitric oxide synthetase-2 (Nox2) complex to release reactive oxygen species (ROS) that activate important cytosolic PRRs-Nlrp3 inflammasome, which orchestrates responsiveness of HSPCs to stress. Moreover, recent data indicate that in addition to circulating in peripheral blood (PB) activated liver-derived ComC proteins, a similar role plays ComC expressed and intrinsically activated in HSPCs known as "complosome". We postulate that ComC triggered Nox2-ROS-Nlrp3 inflammasome responses, if they occur within non-toxic to cells' "hormetic range of activation", positively regulate HSCs migration, metabolism, and proliferation. This sheds a new light on the immune-metabolic regulation of hematopoiesis., (© 2023. The Author(s).)

Purpose of Review: Hematopoiesis is co-regulated by innate immunity, which is an ancient evolutionary defense mechanism also involved in the development and regeneration of damaged tissues. This review seeks to shed more light on the workings of the Nlrp3 inflammasome, which is an intracellular innate immunity pattern recognition receptor and sensor of changes in the hematopoietic microenvironment, and focus on its role in hematopoieisis., Recent Findings: Hematopoietic stem progenitor cells (HSPCs) are exposed to several external mediators of innate immunity. Moreover, since hemato/lymphopoietic cells develop from a common stem cell, their behavior and fate are coregulated by intracellular innate immunity pathways. Therefore, the Nlrp3 inflammasome is functional both in immune cells and in HSPCs and affects hematopoiesis in either a positive or negative way, depending on its activity level. Specifically, while a physiological level of activation regulates the trafficking of HSPCs and most likely maintains their pool in the bone marrow, hyperactivation may lead to irreversible cell damage by pyroptosis and HSPC senescence and contribute to the origination of myelodysplasia and hematopoietic malignancies., Summary: Modulation of the level of Nrp3 inflammasome activation will enable improvements in HSPC mobilization, homing, and engraftment strategies. It may also control pathological activation of this protein complex during HSPC senescence, graft-versus-host disease, the induction of cytokine storms, and the development of hematopoietic malignancies., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

There are concepts in science that need time to overcome initial disbelief before finally arriving at the moment when they are embraced by the research community. One of these concepts is the biological meaning of the small, spheroidal vesicles released from cells, which are described in the literature as microparticles, microvesicles, or exosomes. In the beginning, this research was difficult, as it was hard to distinguish these small vesicles from cell debris or apoptotic bodies. However, they may represent the first language of cell-cell communication, which existed before a more specific intercellular cross-talk between ligands and receptors emerged during evolution. In this review article, we will use the term "extracellular microvesicles" (ExMVs) to refer to these small spheroidal blebs of different sizes surrounded by a lipid layer of membrane. We have accepted an invitation from the Editor-in-Chief to write this review in observance of the 20th anniversary of the 2001 ASH Meeting when our team demonstrated that, by horizontal transfer of several bioactive molecules, including mRNA species and proteins, ExMVs harvested from embryonic stem cells could modify hematopoietic stem/progenitor cells and expand them ex vivo. Interestingly, the result that moved ExMV research forward was published first in 2005 in Leukemia, having been previously rejected by other major scientific journals out of simple disbelief. Therefore, the best judge of a new concept is the passage of time, although the speed of its adoption is aided by perseverance and confidence in one's own data. In this perspective article, we will provide a brief update on the current status of, hopes for, and likely future of ExMV research as well as therapeutic and diagnostic applications, with a special emphasis on 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.

Evidence suggests a role of the immune system in the pathogenesis of a number of mental conditions, including schizophrenia (SCH). In terms of physiology, aside from its crucial protective function, the complement cascade (CC) is a critical element of the regeneration processes, including neurogenesis. Few studies have attempted to define the function of the CC components in SCH. To shed more light on this topic, we compared the levels of complement activation products (CAP) (C3a, C5a and C5b-9) in the peripheral blood of 62 patients with chronic SCH and disease duration of ≥ 10 years with 25 healthy controls matched for age, sex, BMI and smoking status. Concentrations of all the investigated CAP were elevated in SCH patients. However, after controlling for potential confounding factors, significant correlations were observed between SCH and C3a (M = 724.98 ng/mL) and C5a (M = 6.06 ng/mL) levels. In addition, multivariate logistic regression showed that C3a and C5b-9 were significant predictors of SCH. There were no significant correlations between any CAP and SCH symptom severity or general psychopathology in SCH patients. However, two significant links emerged between C3a and C5b-9 and global functioning. Increased levels of both complement activation products in the patient group as compared to healthy controls raise questions concerning the role of the CC in the etiology of SCH and further demonstrate dysregulation of the immune system in SCH patients.

Background: Ischemic heart disease, often caused by an acute myocardial infarction (AMI) is one of the leading causes of morbidity and mortality worldwide. Despite significant advances in medical and procedural therapies, millions of AMI patients progress to develop heart failure every year., Methods: Here, we examine the combination therapy of human mesenchymal stromal cells (MSCs) and endothelial colony-forming cells (ECFCs) to reduce the early ischemic damage (MSCs) and enhance angiogenesis (ECFCs) in a pre-clinical model of acute myocardial infarction. NOD/SCID mice were subjected to AMI followed by transplantation of MSCs and ECFCs either alone or in combination. Cardiomyocyte apoptosis and cardiac functional recovery were assessed in short- and long-term follow-up studies., Results: At 1 day after AMI, MSC- and ECFC-treated animals demonstrated significantly lower cardiomyocyte apoptosis compared to vehicle-treated animals. This phenomenon was associated with a significant reduction in infarct size, cardiac fibrosis, and improvement in functional cardiac recovery 4 weeks after AMI., Conclusions: The use of ECFCs, MSCs, and the combination of both cell types reduce cardiomyocyte apoptosis, scar size, and adverse cardiac remodeling, compared to vehicle, in a pre-clinical model of AMI. These results support the use of this combined cell therapy approach in future human studies during the acute phase of ischemic cardiac injury., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Very small embryonic-like stem cells (VSELs) are a dormant population of development early stem cells deposited in adult tissues that as demonstrated contribute to tissue/organ repair and regeneration. We postulated developmental relationship of these cells to migrating primordial germ cells (PGCs) and explained the quiescent state of these cells by the erasure of differently methylated regions (DMRs) at some of the paternally imprinted genes involved in embryogenesis. Recently, we reported that VSELs began to proliferate and expand in vivo in murine bone marrow (BM) after exposure to nicotinamide (NAM) and selected pituitary and gonadal sex hormones. In the current report, we performed proteomic analysis of VSELs purified from murine bone marrow (BM) after repeated injections of NAM + Follicle-Stimulating Hormone (FSH) that in our previous studies turned out to be an effective combination to expand these cells. By employing the Gene Ontology (GO) resources, we have performed a combination of standard GO annotations (GO-CAM) to produce a network between BM steady-state conditions VSELs (SSC-VSELS) and FSH + NAM expanded VSELs (FSH + NAM VSELs). We have identified several GO biological processes regulating development, organogenesis, gene expression, signal transduction, Wnt signaling, insulin signaling, cytoskeleton organization, cell adhesion, inhibiting apoptosis, responses to extra- and intracellular stimuli, protein transport and stabilization, protein phosphorylation and ubiquitination, DNA repair, immune response, and regulation of circadian rhythm. We report that VSELs express a unique panel of proteins that only partially overlapped with the proteome of BM - derived hematopoietic stem cells (HSCs) and hematopoietic mononuclear cells (MNCs) and respond to FSH + NAM stimulation by expressing proteins involved in the development of all three germ layers. Thus, our current data supports further germ-lineage origin and multi germ layer differentiation potential of these cells., (© 2022. The Author(s).)

Proliferation, metabolism, and migration of hematopoietic stem/progenitor cells (HSPCs) are coordinated by receptors expressed on outer cell membranes that are integrated into microdomains, known as membrane lipid rafts (MLRs). These structures float freely in the cell membrane bilayer and are enriched in cholesterol and sphingolipids for their functional integrity. Receptors, if expressed in MLRs, have prolonged occupancy on the cell surface and enhanced signaling power. Based on this, we have become interested in the regulation of synthesis of MLRs components in HSPCs. To address this, we tested the effect of selected factors that promote proliferation or migration and their potential involvement in the synthesis of MLRs components in HSPCs. Based on our previous research showing that HSPCs from Nox2-KO and Nlrp3-KO mice display a profound defect in MLRs formation, we focused on the role of Nox2-ROS-Nlrp3 inflammasome in regulating lipogenesis in HSPCs. We found that while at steady state conditions, Nox2-derived ROS is required for a proper expression of enzymes regulating lipogenesis, during inflammation, this effect is augmented by Nlrp3 inflammasome. Thus, our data sheds new light on the regulation of lipogenesis in HSPCs and the involvement of the Nox2-ROS-Nlrp3 inflammasome axis that differently regulates lipogenesis at steady state conditions and in response to inflammation, modulating MLRs-mediated responsiveness of these cells to external stimuli., (© 2022. The Author(s).)

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.

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 A 2B receptor and a specific inhibitor of this receptor improves eAdo-aggravated trafficking of HSPCs. To explain this at the molecular level eAdo-A 2B 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 A 2B inhibition promotes cell migration and proliferation. Based on this we postulate that blockage of A 2B 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., (© 2022. The Author(s).)

The mechanisms that regulate egress of hematopoietic stem/progenitor cells (HSPCs) into peripheral blood (PB) in response to stress, inflammation, tissue/organ injury, or administration of mobilization-inducing drugs are still not well understood, and because of the importance of stem cell trafficking in maintaining organism homeostasis, several complementary pathways are believed to be involved. Our group proposes that mobilization of HSPCs is mainly a result of sterile inflammation in the bone marrow (BM) microenvironment in response to pro-mobilizing stimuli and that during the initiation phase of the mobilization process BM-residing cells belonging to the innate immunity system, including granulocytes and monocytes, release danger-associated molecular pattern molecules (DAMPs, also known as alarmins), reactive oxygen species (ROS), as well as proteolytic and lipolytic enzymes. These factors together orchestrate the release of HSPCs into PB. One of the most important DAMPs released in the initiation phase of mobilization is extracellular adenosine triphosphate, a potent activator of the inflammasome. As a result of its activation, IL-1β and IL-18 as well as other pro-mobilizing mediators, including DAMPs such as high molecular group box 1 (Hmgb1) and S100 calcium-binding protein A9 (S100a9), are released. These DAMPs are important activators of the complement cascade (ComC) in the mannan-binding lectin (MBL)-dependent pathway. Specifically, Hmgb1 and S100a9 bind to MBL, which leads to activation of MBL-associated proteases, which activate the ComC and in parallel also trigger activation of the coagulation cascade (CoaC). In this review, we will highlight the novel role of the innate immunity cell-expressed NLRP3 inflammasome, which, during the initiation phase of HSPC mobilization, couples purinergic signaling with the MBL-dependent pathway of the ComC and, in parallel, the CoaC for optimal release of HSPCs. These data are important to optimize the pharmacological mobilization of HSPCs.

Although regenerative and inflammatory processes are involved in the etiopathogenesis of many psychiatric disorders, their roles are poorly understood. We investigate the potential role of stem cells (SC) and factors influencing the trafficking thereof, such as complement cascade (CC) components, phospholipid substrates, and chemokines, in the etiology of schizophrenia. We measured sphingosine-1-phosphate (S1P), stromal-derived factor 1 (SDF-1), and CC cleavage fragments (C3a, C5a, and C5b-C9; also known as the membrane attack complex) in the peripheral blood of 49 unrelated patients: 9 patients with ultra-high risk of psychosis (UHR), 22 patients with first-episode psychosis (FEP), and 18 healthy controls (HC). When compared with the HC group, the UHR and FEP groups had higher levels of C3a. We found no significant differences in hematopoietic SC, very small embryonic-like stem cell (VSEL), C5a, S1P, or SDF-1 levels in the UHR and FEP groups. However, among FEP patients, there was a significant positive correlation between VSELs (CD133+) and negative symptoms. These preliminary findings support the role of the immune system and regenerative processes in the etiology of schizophrenia. To establish the relevance of SC and other factors affecting the trafficking thereof as potential biomarkers of schizophrenia, more studies on larger groups of individuals from across the disease spectrum are needed.

We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, trans-endothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs., (© 2022. The Author(s).)

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.

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.

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.

Scientists and health professionals are exhaustively trying to contain the coronavirus disease 2019 (COVID-19) pandemic by elucidating viral invasion mechanisms, possible drugs to prevent viral infection/replication, and health cares to minimize individual exposure. Although neurological symptoms are being reported worldwide, neural acute and long-term consequences of SARS-CoV-2 are still unknown. COVID-19 complications are associated with exacerbated immunoinflammatory responses to SARS-CoV-2 invasion. In this scenario, pro-inflammatory factors are intensely released into the bloodstream, causing the so-called "cytokine storm". Both pro-inflammatory factors and viruses may cross the blood-brain barrier and enter the central nervous system, activating neuroinflammatory responses accompanied by hemorrhagic lesions and neuronal impairment, which are largely described processes in psychiatric disorders and neurodegenerative diseases. Therefore, SARS-CoV-2 infection could trigger and/or worse brain diseases. Moreover, patients with central nervous system disorders associated to neuroimmune activation (e.g. depression, Parkinson's and Alzheimer's disease) may present increased susceptibility to SARS-CoV-2 infection and/or achieve severe conditions. Elevated levels of extracellular ATP induced by SARS-CoV-2 infection may trigger hyperactivation of P2X7 receptors leading to NLRP3 inflammasome stimulation as a key mediator of neuroinvasion and consequent neuroinflammatory processes, as observed in psychiatric disorders and neurodegenerative diseases. In this context, P2X7 receptor antagonism could be a promising strategy to prevent or treat neurological complications in COVID-19 patients.