Novel Evidence That Alternative Pathway of Complement Cascade Activation is Required for Optimal Homing and Engraftment of Hematopoietic Stem/progenitor Cells

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. Graphical Abstract

Novel Evidence That Alternative Activation Pathway of Complement Cascade (ComC) Regulates Optimal Homing and Engraftment of Hematopoietic Stem/Progenitor Cells (HSPCs) in Reactive Oxygen Species (ROS) - Nlrp3 Inflammasome-Dependent Manner

Background. We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required not only for mobilization of hematopoietic stem progenitor cells (HSPCs) but also for their proper homing and engraftment after transplantation to bone marrow (BM) (Leukemia 2012; 26:106-16). The ComC consists of zymogen proteins that become activated in a cascade-mediated manner by the i) classical, ii) mannan-binding lectin (MBL), or iii) alternative pathway. However, it is not clear which of these pathways plays a crucial role in inducing state of sterile inflammation in recipient BM conditioned for transplantation. Interestingly, the alternative pathway of ComC activation, in contrast to the other two pathways, is not triggered by antibodies or specific structures expressed on the surface of invading microorganisms, but is continuously activated and "ticking" by the spontaneous hydrolysis of the third component of the ComC (C3), which is the most abundant complement protein present in blood plasma. This process of C3 hydrolysis is hyperactivated in response to tissue/organ damage and changes the structure of C3 in order to promote binding of factor B (FB) that initiates the amplification process by which more C3b molecules and C3b-Bb convertases are created responsible for activation of the ComC. We also recently demonstrated that homing and engraftment of HSPCs to BM is mediated by activation of innate immunity pattern recognition receptor Nlrp3 inflammasome (Leukemia 2020; 34:1512-1523). On the other hand reactive oxygen species (ROS) are known activators of Nlrp3 inflammasome. Hypothesis. We hypothesized that sensitive and "continuously ticking" in peripheral blood (PB) alternative pathway of ComC activation facilitates homing and engraftment of HSPCs in response to myeloablation triggered sterile inflammation of hematopoietic microenvironment in donor BM. We also hypothesized that this depends on the activation of the Nlrp3 inflammasome in ROS-dependent manner. Materials and Methods. Activation of ComC and Nlrp3 inflammasome in BM of conditioned for hematopoietic transplantation by lethal irradiation mice was evaluated by C5a ELISA assay and immunofluorescence glow assay measuring activation of Nlrp3 inflammasome product that is activated caspase-1, respectively. We also run shotgun proteomic analysis with BM conditioned media and BMMNCs extracts. The release of ROS was measured by a colorimetric assay. To assess the involvement of alternative pathway of ComC activation, we performed homing and engraftment experiments in wild-type (WT) and FB-deficient mice, that have defect in alternative pathway of ComC activation. FB-KO and WT animals were transplanted with WT BMMNC. We also analyzed changes in the BM microenvironment in response to lethal irradiation in WT and FB-KO mice at mRNA and protein level. Results. We demonstrate for a first time that conditioning for transplantation by myeloablative irradiation induces in BM state of sterile inflammation reflected by activation of ComC, the release of reactive oxygen species (ROS), and activation of Nlrp3 inflammasome in ROS-dependent manner. Moreover, as compared to WT animals, all these pathways were significantly inhibited in FB-KO mice. As a result of this FB-KO animals displayed defective homing and engraftment after transplantation of WT BMMNC. This correlated at molecular level by the decreased expression of cell adhesion molecules and group of structural proteins involved in so called "docking structures" necessary for cell migration and homing as well as for Nlrp3 inflammasome complex and caspase 1, 2, 4 and 6. Moreover, FB-KO mice demonstrated lower level of SDF-1 and KL in BM after myeloablative conditioning for transplantation. Conclusions. We provide for a first time an evidence that myeloablative conditioning for transplantation by lethal irradiation activates in ComC-ROS-Nlrp3 inflammasome - dependent manner a state of sterile inflammation in the BM microenvironment, required for optimal homing and engraftment. This data also explains this phenomenon at molecular level and provides an evidence for a crucial involvement of alternative pathway of ComC activation. Disclosures No relevant conflicts of interest to declare.

Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

DDX41 is needed for pre-and post-natal hematopoietic stem cell differentiation in mice

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms. DDX41 binds to DNA/RNA hybrids and interacts with spliceosome components. How it affects hematopoiesis is still unclear. Using a knockout mouse model, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation. Lack of DDX41 particularly affected myeloid progenitor development, starting at embryonic day 13.5. Transplantation of DDX41-deficient fetal liver and adult bone marrow (BM) cells were unable to rescue mice from lethal irradiation after transplantation. DDX41 knockout stem cells were also defective in ex vivo colony forming assays. RNASeq analysis of lineage-negative, cKit+Sca1+ cells isolated from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered in DDX41 knockout cells. Furthermore, altered splicing of genes involved in key biological processes were observed in cells lacking DDX41. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through its regulation of gene expression programs and splicing.

Serotonin Targeting Using Common Antidepressants Induces Rapid Recovery of Cytopenia

Background. Hematopoiesis is a highly regulated system where multiple, yet undiscovered, factors orchestrate the self-renewal of bone marrow stem cells and their differentiation into blood cells. Following acute stresses like infections, inflammation, chemotherapy or radiation, the hematopoietic system quickly adapts by a process termed "emergency" or "stress" hematopoiesis. For instance, switches from steady state to emergency granulopoiesis or emergency erythropoiesis have been described in response to infection or bleeding. We recently identified, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. Furthermore, pharmacologic restoration of serotonin levels using selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, a common antidepressant, was demonstrated to rescue the anemic phenotype in mice models. Here, we hypothesized that serotonin also has a role on other hematopoietic lineages, and that the serotonergic system could be a valuable therapeutic target in radiation or chemotherapy-induced cytopenia. Moreover, as our previous work suggested a cross-talk between serotonin and erythropoietin, we postulate that serotonin could act in cooperation with known hematopoietic growth factors. Material and method. For mice models, we submitted C57BL/6 wild-type 8-10 weeks old mice to sub-lethal irradiation and monitored hematopoietic recovery through complete blood counts. We compared mice treated with fluoxetine administrated orally (~ 20 mg/kg/day in drinking water) to a control group, with or without hematopoietic growth factors. For the retrospective human cohort, we used a computerized database to identify patients who underwent autologous hematopoietic stem cell transplantation (ASCT) in the adult hematology department of Necker hospital between 2008 and 2018. We compared 22 patients treated with an SSRI to 66 controls, matched according to number of injected CD34+ cells/kg, age, sex, conditioning chemotherapy, pathology, depth of response before transplantation, previous lines of chemotherapy, and year of transplantation. The study was conducted according to the Declaration of Helsinki. Results. First, we confirmed that, following sub-lethal irradiation, pharmacologic restoration of 5-HT levels in mice using fluoxetine improved normalization of the erythroid lineage (at day 17 post-irradiation, mean hemoglobin was 8.1 versus 2.8 g/dL in the fluoxetine versus control group (p=0.0002)). Second, the data revealed that the use of SSRI lead to a more rapid restoration of the leukocytes and platelets levels (at day 17 post-irradiation, mean leukocyte level was 1740 versus 314/mm3 (p<0.0001) and mean platelet level was 216 versus 43/mm3 (p=0.002) in the fluoxetine versus control group, respectively). Third, we observed an additive effect between fluoxetine and Granulocyte-Colony Stimulating Factor (G-CSF) on the recovery of the three myeloid lineages (at day 17 post-irradiation, mean hemoglobin was 10.5 versus 4.0 g/dL (p<0.0001), mean leukocyte level was 2900 versus 900/mm3 (p<0.0001) and mean platelet level was 308 versus 84/mm3 (p=0.0009) in the fluoxetine + G-CSF versus control group, respectively). Finally, analysis of the in vivo murine model under steady state condition showed that without any hematopoietic stress, fluoxetine did not impact hematopoiesis. In human, analysis of the retrospective ASCT cohort demonstrated that patients treated with SSRI had a more rapid neutrophil recovery than matched control patients (mean duration of neutropenia <500/mm3 was 12.2 days in the SSRI group and 14.26 days in the control group, p=0.0216). SSRI therapy was not associated with a higher rate or quicker relapses of underlying malignant hemopathy: mean progression free survival was 36 months in both groups. Conclusion. In this work, we report a previously unknown role of SSRI in the hematopoietic recovery of cytopenia, both after sub-lethal irradiation in mice and after autologous hematopoietic stem cell transplantation in human. We also observed a significant cooperation between SSRI and G-CSF on the three myeloid lineages. We propose that the serotonergic system could be a valuable therapeutic target in stress hematopoiesis such as in therapy-induced aplasia in patients. Disclosures Hermine: AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. OffLabel Disclosure: Fluoxetine is a selective serotonin reuptake inhibitor for oral administration. It is an antidepressant approved for major depressive disorder, obsessive compulsive disorder, bulimia nervosa and panic disorder. We believe its role on serotonin can improve hematopoiesis.

NOD2 Supports Crypt Survival and Epithelial Regeneration after Radiation-Induced Injury

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules. To assess crypt survival, a clonogenic microcolony assay was performed with 15 Gy of X-ray irradiation. The fractional crypt survival rate (46.0 ± 15.5% vs. 24.7 ± 9.2%, p < 0.01) and fractional EdU-positive crypt survival rate (29.8 ± 14.5% vs. 9.79 ± 4.37%, p = 0.015) were significantly decreased in the NOD2−/− mice compared with the wild-type (WT) mice at 3.5 days after irradiation. To evaluate intestinal epithelial regeneration capability, organoid reconstitution assays were performed. Small bowel crypts of the WT and NOD2−/− mice were isolated and seeded into Matrigel for 3D culture. In the organoid reconstitution assays, the number of organoids formed did not differ between the NOD2−/− and WT mice. Organoid formation ability was also assessed after exposure to 5 Gy irradiation. Organoid formation ability was significantly decreased in the NOD2−/− mice compared with the WT ones after exposure to 5 Gy irradiation (33.2 ± 5.9 vs. 19.7 ± 8.8/well, p < 0.01). NOD2 supports crypt survival after potentially lethal irradiation damage and is associated with intestinal epithelial regeneration.

TLR5 signaling in murine bone marrow induces hematopoietic progenitor cell proliferation and aids survival from radiation

Key Points Flagellin activates TLR5 signaling in mouse bone marrow and induces hematopoietic progenitor cell proliferation. Flagellin-induced MPP3 cells aid the survival of mice exposed to lethal irradiation.