Artigo Acesso aberto Revisado por pares

Cobalt protoporphyrin IX increases endogenous G‐ CSF and mobilizes HSC and granulocytes to the blood

2019; Springer Nature; Volume: 11; Issue: 12 Linguagem: Inglês

10.15252/emmm.201809571

ISSN

1757-4684

Autores

Agata Szade, Krzysztof Szade, Witold Nowak, Karolina Bukowska-Strakovà, Lucie Muchová, Monika Gońka, Monika Żukowska, Maciej Cieśla, Neli Kachamakova‐Trojanowska, Marzena Rams‐Baron, A. Ratuszna, Józef Dulak, Alicja Józkowicz,

Tópico(s)

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Resumo

Article11 November 2019Open Access Transparent process Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood Agata Szade Corresponding Author Agata Szade [email protected] orcid.org/0000-0002-0575-4659 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Krzysztof Szade Krzysztof Szade orcid.org/0000-0002-7227-4276 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Witold N Nowak Witold N Nowak orcid.org/0000-0002-1526-3511 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Karolina Bukowska-Strakova Karolina Bukowska-Strakova orcid.org/0000-0001-9181-439X Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Department of Clinical Immunology and Transplantology, Institute of Pediatrics, Jagiellonian University Medical College, Krakow, Poland Search for more papers by this author Lucie Muchova Lucie Muchova orcid.org/0000-0003-2082-1060 Fourth Department of Internal Medicine and Institute of Medical Biochemistry and Laboratory Medicine, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic Search for more papers by this author Monika Gońka Monika Gońka Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Monika Żukowska Monika Żukowska orcid.org/0000-0002-2832-2146 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Maciej Cieśla Maciej Cieśla orcid.org/0000-0002-8460-1991 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Neli Kachamakova-Trojanowska Neli Kachamakova-Trojanowska orcid.org/0000-0002-3226-0726 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Marzena Rams-Baron Marzena Rams-Baron A. Chelkowski Institute of Physics, University of Silesia, Chorzow, Poland Silesian Center for Education and Interdisciplinary Research, Chorzow, Poland Search for more papers by this author Alicja Ratuszna Alicja Ratuszna A. Chelkowski Institute of Physics, University of Silesia, Chorzow, Poland Silesian Center for Education and Interdisciplinary Research, Chorzow, Poland Search for more papers by this author Józef Dulak Józef Dulak orcid.org/0000-0001-5687-0839 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Alicja Józkowicz Alicja Józkowicz orcid.org/0000-0002-7317-260X Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Agata Szade Corresponding Author Agata Szade [email protected] orcid.org/0000-0002-0575-4659 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Krzysztof Szade Krzysztof Szade orcid.org/0000-0002-7227-4276 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Witold N Nowak Witold N Nowak orcid.org/0000-0002-1526-3511 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Karolina Bukowska-Strakova Karolina Bukowska-Strakova orcid.org/0000-0001-9181-439X Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Department of Clinical Immunology and Transplantology, Institute of Pediatrics, Jagiellonian University Medical College, Krakow, Poland Search for more papers by this author Lucie Muchova Lucie Muchova orcid.org/0000-0003-2082-1060 Fourth Department of Internal Medicine and Institute of Medical Biochemistry and Laboratory Medicine, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic Search for more papers by this author Monika Gońka Monika Gońka Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Monika Żukowska Monika Żukowska orcid.org/0000-0002-2832-2146 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Maciej Cieśla Maciej Cieśla orcid.org/0000-0002-8460-1991 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Neli Kachamakova-Trojanowska Neli Kachamakova-Trojanowska orcid.org/0000-0002-3226-0726 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Marzena Rams-Baron Marzena Rams-Baron A. Chelkowski Institute of Physics, University of Silesia, Chorzow, Poland Silesian Center for Education and Interdisciplinary Research, Chorzow, Poland Search for more papers by this author Alicja Ratuszna Alicja Ratuszna A. Chelkowski Institute of Physics, University of Silesia, Chorzow, Poland Silesian Center for Education and Interdisciplinary Research, Chorzow, Poland Search for more papers by this author Józef Dulak Józef Dulak orcid.org/0000-0001-5687-0839 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Alicja Józkowicz Alicja Józkowicz orcid.org/0000-0002-7317-260X Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Search for more papers by this author Author Information Agata Szade *,1, Krzysztof Szade1, Witold N Nowak1, Karolina Bukowska-Strakova1,2, Lucie Muchova3, Monika Gońka1, Monika Żukowska1, Maciej Cieśla1,7, Neli Kachamakova-Trojanowska1,4, Marzena Rams-Baron5,6, Alicja Ratuszna5,6, Józef Dulak1,4 and Alicja Józkowicz1 1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 2Department of Clinical Immunology and Transplantology, Institute of Pediatrics, Jagiellonian University Medical College, Krakow, Poland 3Fourth Department of Internal Medicine and Institute of Medical Biochemistry and Laboratory Medicine, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic 4Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland 5A. Chelkowski Institute of Physics, University of Silesia, Chorzow, Poland 6Silesian Center for Education and Interdisciplinary Research, Chorzow, Poland 7Present address: Division of Molecular Hematology, Lund University, Lund, Sweden *Corresponding author: Tel: +48 12 6646024; E-mail: [email protected] EMBO Mol Med (2019)11:e09571https://doi.org/10.15252/emmm.201809571 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation. Synopsis Recombinant G-CSF is the mobilizing factor used for treating neutropenia and prior to harvesting hematopoietic stem cells for transplantation. This article describes cobalt protoporphyrin IX as a new efficient mobilizing factor upstream of G-CSF. Cobalt protoporphyrin IX (CoPP) increases the concentration of endogenous G-CSF, IL-6 and MCP-1 and induces the mobilization of cells from the bone marrow to the blood. CoPP mobilizes higher number of mature granulocytes and functional HSC than exogenous recombinant G-CSF. Transplantation of CoPP-mobilized cells leads to faster hematopoietic recovery and higher donor chimerism compared to transplantation of G-CSF-mobilized cells. G-CSF neutralization inhibits the CoPP-induced mobilization. CoPP-induced mobilization is independent of Nrf2/HO-1 axis. Introduction Porphyrins are macrocyclic compounds essential for plants, bacteria, and animals, found in molecules such as chlorophylls and cytochromes (Chandra et al, 2000). Porphyrins form complexes with metals to generate metalloporphyrins. Often the bound metal ions determine the unique properties of the metalloporphyrins. For example, only Fe-protoporphyrin IX (heme) is a substrate for heme oxygenase-1 (HO-1), but other protoporphyrins such as tin protoporphyrin IX (SnPP) can inhibit HO-1 enzymatic activity (Schulz et al, 2012) (Fig 1A and B). Cobalt protoporphyrin (CoPP) is the inducer of Nrf-2/HO-1 axis, both in vitro and in vivo. Thus, CoPP is considered as a potential inducer of HO-1 where it may have therapeutic advantages (Shan et al, 2006). Figure 1. CoPP increases number of granulocytes and upregulates a set of cytokinesC3H mice were injected with CoPP, SnPP, or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 h after the last injection. A. Chemical structures of heme (HO-1 substrate) and CoPP (HO-1 in vivo inducer). B. Heme degradation reaction catalyzed by HO-1. C. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography. D. Total leukocyte and red blood cell count in PB. CoPP increases the number of major WBC types. Granulocyte and monocyte percentages are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP. E. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box). F. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6, and IL-5. Data information: Results are shown as mean + SEM, one-way ANOVA with Bonferroni post-test, n = 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Download figure Download PowerPoint Heme oxygenase-1 is an enzyme which degrades heme into carbon monoxide (CO), iron ions, and biliverdin (Fig 1B), which is subsequently reduced to bilirubin (Tenhunen et al, 1968). The idea of activation of HO-1 for therapeutic purposes is based on its broad anti-inflammatory properties (Ryter et al, 2006). HO-1 also influences the maturation and activity of myeloid cells. Specific deletion of HO-1 in myeloid lineage (Lyz-Cre:Hmox1fl/fl) partially blocks differentiation of myeloid progenitors toward macrophages (Wegiel et al, 2014). We showed that lack of HO-1 also affects granulopoiesis (Bukowska-Strakova et al, 2017). HO-1−/− mice have more granulocytes in the peripheral blood (PB), what is connected with increased myelocyte proliferation in the bone marrow (BM). Moreover, CoPP may inhibit the maturation of dendritic cells (Chauveau et al, 2005), but this effect seems to be HO-1 independent (Mashreghi et al, 2008). Given this rationale, we sought to study how pharmacological induction of HO-1 by CoPP can influence the function of myeloid lineage. Unexpectedly, we observed that CoPP efficiently mobilizes granulocytes as well as hematopoietic stem and progenitor cells to the PB in mice. Because of constant need to improve clinical mobilization strategies, we investigated the effects induced by CoPP as a potential new approach to meet this requirement. During mobilization, hematopoiesis is enhanced, and large numbers of cells are released from the BM to the blood, including stem and immature hematopoietic cells (Lapidot & Petit, 2002). Mechanism of mobilization is complex and involves several cell populations and cytokine pathways (Duhrsen et al, 1988; Lapidot & Petit, 2002; Tay et al, 2017), with G-CSF (granulocyte colony-stimulating factor) as one of the best characterized mobilizing factors (Souza et al, 1986; Lapid et al, 2008). G-CSF acts on the BM myeloid progenitors, driving their proliferation and differentiation toward granulocytes (Metcalf & Nicola, 1983). Apart from G-CSF, many other agents, such as stromal cell-derived factor 1α (Hattori et al, 2001; Devine et al, 2008), stem cell factor (Andrews et al, 1992), interleukin 6 (IL-6) (Pojda & Tsuboi, 1990), IL-8 (Laterveer et al, 1995), Groβ (Pelus & Fukuda, 2006; Fukuda et al, 2007), or granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gianni et al, 1989) can act as mobilizing factors (reviewed in (Lapid et al, 2008)). Recently, Hoggatt et al (2018) reported rapid mobilization of highly engrafting stem cells with a single injection of Groβ and AMD3100 combination. Current progress in basic science concerning cell mobilization has already been successfully translated into clinical practice (Bronchud et al, 1987; Sheridan et al, 1992). Pharmacological mobilization is of outstanding importance for the prevention or treatment of neutropenia (Kelly & Wheatley, 2009; Lyman et al, 2010) and for the transplantation of hematopoietic stem cells (HSC). The success of BM transplantation depends on the collection of sufficient number of HSC (Kondo et al, 2008). Nowadays, the source of transplantable HSC is not necessarily BM itself, but rather the PB after mobilization of HSC into the circulation (Cashen et al, 2007; To et al, 2011). Recombinant human G-CSF is widely used for mobilization purposes (Mehta et al, 2015). Despite improvements in the treatment protocols, in some patients application of G-CSF is inefficient. Among healthy donors, G-CSF mobilization fails in 5–30%, but in high-risk patients, the failure rate reaches even up to 60% (Ferraro et al, 2011; To et al, 2011). Patients who fail to mobilize HSC in response to G-CSF might be treated additionally with plerixafor (To et al, 2011); however, the price of this drug is sometimes a major obstacle—single dose costs several thousand dollars. Therefore, to improve the treatment efficiency, we need new agents with additional activities. These can include modulating of extracellular matrix (Saez et al, 2014), phosphorylation of signaling proteins (Wang et al, 2016), inhibition of proteasome (Ghobadi et al, 2014), or induction of endogenous G-CSF (Hoggatt & Pelus, 2014). Here, we describe the previously unknown mobilizing properties of CoPP and compare its efficiency to the standard mobilizing factor, G-CSF. Results CoPP treatment increases leukocyte numbers in the blood CoPP and SnPP are commonly used as activator and inhibitor of HO-1, respectively (Fig 1B) (Ryter et al, 2006). As expected, administration of CoPP to C3H mice resulted in a 2.6-fold increase in HO-1 activity in the liver, whereas SnPP decreased HO-1 activity by 2.4 times (Fig 1C). Along with increased HO-1 activity, mice treated with CoPP had increased absolute number of all types of leukocytes (WBC) in the blood, with a visible shift toward myeloid lineage (Fig 1D). Erythrocyte parameters were unaffected by CoPP treatment (Fig 1D). To examine whether the observed leukocytosis was linked with changes in cytokine profile in plasma, we performed Luminex screen on 32 cytokines (Fig 1E). CoPP increased concentrations of the set of cytokines (Fig 1E,F) that includes IL-6, monocyte chemoattractant protein 1 (MCP-1, CCL2), interferon γ-induced protein 10 (IP-10, CXCL10), IL-5, and to the greatest extent G-CSF (Fig 1F). However, CoPP did not increase the other CSFs—M-CSF, GM-CSF, and IL-3 (Appendix Fig S1). SnPP, HO-1 inhibitor, did not influence the complete blood cell count or any of the analyzed cytokines (Fig 1D–F, Appendix Fig S1). Both CoPP and G-CSF mobilize myeloid cells but differ in T-cell mobilization and upregulation of cytokines in plasma As the treatment of mice with CoPP increased G-CSF concentration in plasma (Fig 1F), and G-CSF is known clinical mobilizing agent, we directly compared the effects of G-CSF and CoPP administration. We injected the mice with G-CSF or CoPP once a day for 5 days and analyzed myeloid and hematopoietic stem/progenitor cells in PB and BM by flow cytometry. Both G-CSF and CoPP increased the number of CD45+ cells in PB (Fig 2A). Although both in G-CSF- and CoPP-treated mice the highest increase was observed for granulocytes, G-CSF mobilized 1.6 times more total granulocytes than CoPP. The numbers of monocytes were similarly increased after G-CSF and CoPP. Importantly, only G-CSF treatment led to the increase in PB lymphocytes, mainly T cells (Fig 2A). Figure 2. Both G-CSF and CoPP mobilize myeloid cells, but only G-CSF mobilizes T cellsC57BL6xFVB mice were injected with G-CSF, CoPP, or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. A. Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase in granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP. B. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF. C. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF. D, E. Treatment with G-CSF and CoPP (D) decreases percentage of Ly6G+ macrophages in the BM and (E) increases relative spleen weight. F. viSNE maps of CD11b+ CD11c− blood cells, colored by SSC value. Three independent experiments were performed, and the results were pooled. Data information: Results are shown as mean + SEM, one-way ANOVA with Bonferroni post-test, n = 7 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Download figure Download PowerPoint Next, we compared cytokine concentrations in plasma using Luminex assay (Fig 2B). Consistently with the previous experiment, CoPP induced high levels of endogenous G-CSF, IL-6, and MCP-1 (Fig 2C). CoPP treatment also elevated KC (keratinocyte-derived cytokine, CXCL1), IP-10, and MIG (monokine induced by interferon γ, CXCL9). In contrast, G-CSF did not significantly induce any of these cytokines (Fig 2B). Analyzing the other features related to cell mobilization (Platzbecker et al, 2001; Winkler et al, 2010), we found that mice treated with both G-CSF and CoPP had decreased numbers of CD11b+ F4/80+ MHC IIlow Ly6G+ macrophages in the BM (Fig 2D) and enlarged spleens (Fig 2E). Concluding, both CoPP and G-CSF mobilize efficiently myeloid cells, but result in different mobilization of lymphocytes. CoPP induces several cytokines together with endogenous G-CSF that are not elevated during mobilization with recombinant G-CSF. CoPP mobilizes granulocytes with mature phenotype Although both G-CSF and CoPP efficiently mobilize granulocytes, we observed that these cells have a distinct phenotype. Multiparameter analysis revealed that myeloid cells from the mice treated with CoPP are more similar to the cells from the control mice than to the cells from the G-CSF-treated mice (Fig 2F). viSNE maps showed increased density of the granular cells in the CoPP group compared with the control. We also observed a population of cells with intermediate granularity in mice treated with G-CSF that was not visible in the control mice (Fig 2F). Accordingly, 2D flow cytometry plots confirm that CoPP mobilizes granulocytes that are more granular (higher SSC parameter) and have higher expression of Ly6G comparing to granulocytes mobilized by G-CSF (Fig 3A). Granulocytes mobilized by CoPP phenotypically resemble the mature granulocytes in control mice, while these mobilized by G-CSF show immature phenotype, with lower granularity and Ly6G expression, typical for early differentiation stages in BM. Altogether, although mice treated with G-CSF had a higher total number of granulocytes in the blood, these were mainly immature (Ly6Gmid SSCmid), whereas CoPP treatment increased the number of mature cells (Ly6Ghi SSChi; Fig 3B). Figure 3. CoPP and G-CSF mobilize granulocytes with different phenotypeC57BL/6 mice were treated with G-CSF, CoPP, or solvent controls (NaCl, DMSO) for five consecutive days. Samples were collected 6 h after the last injection. A. Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes. B, C. Relative abundance of different granulocyte phenotypes in blood (B) or BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF. D. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli. Data information: Results are shown as mean + SEM, one-way ANOVA with Bonferroni post-test, n = 7 mice per group (B, C) or two-way ANOVA with Bonferroni post-test, n = 6 mice per group (D). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Download figure Download PowerPoint Next, we analyzed the composition of myeloid cell populations in the BM. Both G-CSF and CoPP treatment increased the frequency of granulocytes with the immature phenotype, (CD11b+ CD11c− Ly6Clow Ly6G+ and CD11b+ CD11c− Ly6Clow SSCmed Ly6Gmed), but the increase after G-CSF was more pronounced. Percentage of granulocytes with the mature phenotype (CD11b+ CD11c− Ly6Clow SSChi Ly6Ghi) was decreased after G-CSF, but not affected by CoPP (Fig 3C). To verify the functional properties of mobilized granulocytes, we checked the production of reactive oxygen species (ROS). For this purpose, G-CSF- and CoPP-mobilized or control PB was incubated with N-formylmethionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA) or opsonized Escherichia coli and subjected to rhodamine 123 (DHR 123) staining (Fig 3D). There was a higher percentage of ROS-producing cells in the blood of G-CSF- or CoPP-treated mice than in control mice, after stimulation with E. coli, with similar tendency after PMA treatment. We did not observe any differences between groups after stimulation with fMLP. Interestingly, mobilized granulocytes with immature phenotype seemed to be able to produce ROS after stimulation with PMA (CoPP- and G-CSF-mobilized cells) and E. coli (G-CSF-mobilized cells, Appendix Fig S2). Concluding, granulocytes mobilized either by G-CSF or by CoPP are at least as functional as the steady-state granulocytes in tested conditions. CoPP mobilizes more HSPC than G-CSF As G-CSF also mobilizes hematopoietic stem and progenitor cells (Lapidot & Petit, 2002), we analyzed HSPC populations in the blood and BM of mice treated with G-CSF and CoPP. CoPP increased percentage and number of total HSPC pool defined as c-Kit+ Lin− Sca-1+ (KLS) cells in the blood (Appendix Fig S3A). In animals treated with G-CSF, the increase in KLS cells was visible, although not statistically significant when all four groups were compared together (one-way ANOVA with Bonferroni post-test). We further characterized mobilized KLS pool using CD34 and SLAM markers: CD48 and CD150, which enable to define HSC (KLS CD48−CD150+), MPP (multipotent progenitors, KLS CD48−CD150−), and HPC (hematopoietic progenitors, KLS CD48+CD150− and KLS CD48+CD150+) populations (Fig 4A) (Oguro et al, 2013). CoPP treatment mobilized more HSC, MPP, and HPC than the treatment with G-CSF (Fig 4B). Of note, only small proportion of KLS cells mobilized by G-CSF and CoPP were CD34 negative (Appendix Fig S3B), what is consistent with the previous observation, that G-CSF-mobilized HSC (in contrast to steady-state HSC) are CD34+ (Tajima et al, 2000). Next, we compared how G-CSF and CoPP affect HSPC in BM. Only CoPP treatment significantly increased the percentage of KLS cells in BM (Appendix Fig S3A), but this increase was restricted to more differentiated HPC fraction and was not observed among LT-HSC (long-term HSC; KLS CD48−CD150+CD34−) and MPP populations (Fig 4C). Figure 4. CoPP mobilizes more HSPC than G-CSF in C57BL/6xFVB mice A. Gating strategy of KLS (c-Kit+Lin−Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors), and HPC (hematopoietic progenitors). B. CoPP mobilizes higher numbers of HSC (KLS CD48−CD150+), MPP (KLS CD48−CD150−), HPC-1 (KLS CD48+CD150−), and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB. C. Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction. In contrast, frequency of MPP tends to decrease. D. Scheme of common myeloid progenitor (CMP) differentiation toward granulocytes/monocytes, erythrocytes, and platelets. E. The number of KLS− (c-Kit+Lin−Sca-1−) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage-committed progenitors, GMP (granulocyte-macrophage progenitors, KLS− CD48+CD150−CD34+) and MEP (megakaryocyte–erythroid progenitors, KLS− CD48+CD150+CD34−), were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS− CD48−CD150−CD34−). F. Both G-CSF and CoPP treatments decrease the KLS− percentage in the BM; however, the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Data information: Results are shown as mean + SEM, one-way ANOVA with Bonferroni post-test, n = 7 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Download figure Download PowerPoint Cobalt protoporphyrin mobilization resulted in higher number of more committed progenitors (Fig 4D) in PB: granulocyte-macrophage progenitors (GMP) and megakaryocyte–erythroid progenitors (MEP). The increase in erythroid progenitors (EP) number was similar in both groups. In contrast, both G-CSF and CoPP decreased percentage of committed progenitors c-Kit+Lin−Sca-1− (KLS−) in BM; however, the decrease after CoPP was less pronounced (Fig 4E). Further characterization of committed progenitors with CD34, CD48, and CD150 markers showed that MEP and EP are similarly decreased after G-CSF and CoPP. Only G-CSF, but not CoPP decreased percentage of GMP in BM, that did not change after CoPP treatment (Fig 4E). Together with the observation that there was a higher number of mobilized GMP by CoPP, it might suggest that CoPP or its downstream effectors affect GMP proliferation. CoPP mobilizes functional HSPC Cobalt protoporphyrin increased the number of HSPC in the PB more efficiently than G-CSF. To verify whether CoPP-mobilized HSPC are functional, we transplanted the mobilized PB mononuclear cells (PBMC) and checked their hematopoietic reconstitution potential. We treated the green fluorescent protein (GFP)-expressing mice (C57BL/6-Tg(UBC-GFP)30Scha/J) with CoPP, G-CSF, or NaCl (Fig EV2). At the fifth day of the treatment, we collected the blood and transplanted 5 × 106 PBMC to the lethally irradiated GFP− recipient mice, together with 105 GFP− BM-derived competitor cells. Two independent experiments were performed. Donor mice (C57BL/6-Tg(UBC-GFP)30Scha/J) are on C57BL6 genetic background, which is described as poor HSPC mobilizing strain (Roberts et al, 1997). Thus, we anal

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