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 dangerassociated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs.

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from CML and AML patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (RT-PCR) and protein level (FACS) and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 MAPK and AKT. We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (e.g., as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating expansion of leukemia/lymphoma cells when the ComC becomes activated.

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.

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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.Electronic supplementary materialThe online version of this article (10.1007/s12015-019-09918-y) contains supplementary material, which is available to authorized users.

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.

The utility in clinical practice of a recently developed and validated predictive model for venous thromboembolism (VTE) events in lymphoma patients, known as the thrombosis lymphoma (ThroLy) score, is unknown. We evaluated the association of ThroLy with VTE in patients treated for diffuse large B‐cell lymphoma (DLBCL) or Hodgkin lymphoma (HL) undergoing ambulatory first‐line chemotherapy. Retrospective analyses were performed on 428 patients (median age 50), 241 were newly diagnosed DLBCL, and 187 had HL. During initial chemotherapy, 64 (15%) patients developed VTE. According to the ThroLy, 322 (75.2%) patients were considered low risk, 88 (20.6%) patients had intermediate risk and 18 (4.2%) patients high risk for VTE development. Patients with DLBCL were more often in the high‐risk ThroLy group and had more VTE events than HL. VTE occurred in; 38.9% (n = 7) high‐risk patients, 29.5% (n = 26) intermediate risk, and 9.6% (n = 31) low risk according to the ThroLy score. However, in multivariate analysis, high ThroLy (OR 5.13; 95% CI: 1.83‐14.36, P = .002), intermediate ThroLy (OR 3.96; 95% CI: 2.19‐7.17, P < .001), and aggressive lymphoma‐DLBCL (OR 1.91; 95% CI: 1.05‐3.47, P = .034) were all significantly associated with development of VTE, 48% of the VTE events occurred in the low‐risk ThroLy score group (the ROC AUC (95% CI) 0.40‐0.70 and C statistic‐0.55). In our study, the ThroLy score was not a suitably accurate model for predicting VTE events in patients at higher risk of VTE. Further research should be conducted to identify new biomarkers that will predict these events and to establish a new VTE risk assessment model.

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 ten 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.