Characterization of hematopoietic stem cells in the circulation

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate into multiple cell lineages, giving rise to all blood components and immune cells, during the entire life of an individual. HSCs are localized in the bone marrow inside specialized compartments named “hematopoietic niches”. The niche contains stromal cells of mesenchymal origin, as is the case of adipocytes and osteoblasts as well as endothelial cells and cells of hematopoietic origin such as macrophages or megakaryocytes (1). All of these cells produce and deposit elements in the extracellular matrix but also secrete local hematopoietic cytokines that can induce or inhibit the proliferation and differentiation of progenitor cells. Early studies described that some of these HSCs are found travelling through the circulation of the organism (2). Additionally, the release of HSCs from the BM into peripheral blood follows circadian patterns, i.e. their numbers oscillate between day and night (3). In the present thesis we have analyzed whether HSCs in the circulation (named here circulating HSCs) have any physiological function and the mechanisms through which cHSCs are released into bloodstream. We have found that circulating HSC have a myeloid bias and are important for the repopulation of damaged niches. In addition, we found that multiple clones of these cHSCs enter the bloodstream and contribute to the regeneration of hematopoiesis in remote niches. We have found that the chemokine receptor CXCR2 is expressed in HSCs and is important for their homeostatic egress into the circulation. Genetic deficiency of Cxcr2 prevents the release of HSCs and the repopulation of remote damaged niches and gives rise to hematopoietic defects with age. Correspondingly, we have identified a population of perivascular cells inside the BM that express the chemokine ligand CXCL1 and could be key in the signaling of cHSC egress, and ultimately in preserving hematopoietic health through life.El estudio ha sido financiado por el proyecto SAF2015-65607-R otorgados al Dr. Andrés Hidalgo por el Ministerio de Economía, Industria y Competitividad (MEIC). Por su parte, Dª Itziar Cossío Cuartero ha sido beneficiaria de una beca FPI (SAF2015-65607-R). El CNIC recibe financiación del MEIC y la Fundación Pro-CNIC.N

The investors of the microfinance sector

El sector microfinanciero ha experimentado un importante desarrollo en los últimos años favorecido por el impulso de la inversión socialmente responsable y también, por el exceso de liquidez que ha habido en el sistema financiero global. Las denominadas Instituciones Microfinancieras son los agentes clave de este desarrollo y su doble objetivo de rentabilidad no sólo social, sino financiera; les ha configurado como la principal vía de inversión, si bien, se han desarrollado otros canales y productos financieros directos o indirectos. El espectro de inversores, tradicionalmente públicos, también se ha ampliado, incorporándose aquellos de carácter comercial e incluso especulativo en su búsqueda de nuevas alternativas de inversión atractiva. De este modo, se ha abierto un nuevo campo financiero, no exento del debate ético por el objetivo primordial que hay detrás del sector microfinanciero: la reducción de la pobreza en el mundo.The microfinance sector has had an important development in the last years ought to the growth of the socially responsible investment and also, by the excess of global financial liquidity. Although there are other channels and other financial products, direct and indirect, what we called MicroFinancial Institutions have been the key agents for this development and their double objectives on profitability, social and financial has enable them to become the principal way to invest. The investment range that traditionally has been public has expanded into commercial and speculative investors in search of new attractive investment alternatives. So, a new financial area has opened; including the ethical discussion on the main objective behind the microfinancial sector: Reduction of poverty in the world

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression.We thank K. Tasken, J. Saarela and the NCMM at the University of Oslo (UiO), S. Kanse (UiO) and B. Smedsrød (UiT), for access to facilities. We acknowledge Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital (Bergen, Norway) and R. Hovland for karyotyping, FISH, translocation and DNA analyses of AML and MDS patients included in this study, and Department of Pathology, Oslo University Hospital (Oslo, Norway) and S. Spetalen for deep sequencing. L.M. Gonzalez, L.T. Eliassen, X. Zhang, M. Ristic and other members of L. Arranz group, O.P. Rekvig, R. Doohan, L.D. Håland, M.I. Olsen, A. Urbanucci, J. Landskron, K.B. Larsen, R.A. Lyså and UiT Advanced Microscopy Core Facility, UiO and UiT Comparative Medicine Units, for assistance. P. Garcia and S. Mendez-Ferrer for providing NRASG12D and Nes-gfp mice, respectively. P. Garcia and L. Kurian for careful reading of the manuscript. E. Tenstad (Science Shaped) for artwork in schematics. We would also like to thank the AML and MDS patients, and healthy volunteers, who donated biological samples. Our work is supported by a joint meeting grant of the Northern Norway Regional Health Authority, the University Hospital of Northern Norway (UNN) and UiT (Strategisk-HN06-14), Young Research Talent grants from the Research Council of Norway, (Stem Cell Program, 247596; FRIPRO Program, 250901), and grants from the Norwegian Cancer Society (6765150), the Northern Norway Regional Health Authority (HNF1338-17), and the Aakre-Stiftelsen Foundation (2016/9050) to L.A. Vav-Cre NRASG12D experiments were supported by NIH grant R01CA152108 to J.Z.S

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health.This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505)

Caracterización de las células madre hematopoyéticas en la circulación

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 12-03-2021Esta tesis tiene embargado el acceso al texto completo hasta el 12-09-2022Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate into multiple cell lineages, giving rise to all blood components and immune cells, during the entire life of an individual. HSCs are localized in the bone marrow inside specialized compartments named “hematopoietic niches”. The niche contains stromal cells of mesenchymal origin, as is the case of adipocytes and osteoblasts as well as endothelial cells and cells of hematopoietic origin such as macrophages or megakaryocytes (1). All of these cells produce and deposit elements in the extracellular matrix but also secrete local hematopoietic cytokines that can induce or inhibit the proliferation and differentiation of progenitor cells. Early studies described that some of these HSCs are found travelling through the circulation of the organism (2). Additionally, the release of HSCs from the BM into peripheral blood follows circadian patterns, i.e. their numbers oscillate between day and night (3). In the present thesis we have analyzed whether HSCs in the circulation (named here circulating HSCs) have any physiological function and the mechanisms through which cHSCs are released into bloodstream. We have found that circulating HSC have a myeloid bias and are important for the repopulation of damaged niches. In addition, we found that multiple clones of these cHSCs enter the bloodstream and contribute to the regeneration of hematopoiesis in remote niches. We have found that the chemokine receptor CXCR2 is expressed in HSCs and is important for their homeostatic egress into the circulation. Genetic deficiency of Cxcr2 prevents the release of HSCs and the repopulation of remote damaged niches and gives rise to hematopoietic defects with age. Correspondingly, we have identified a population of perivascular cells inside the BM that express the chemokine ligand CXCL1 and could be key in the signaling of cHSC egress, and ultimately in preserving hematopoietic health through lif

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized antiphospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R- T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.This work was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación [grant number SAF2014-52048-R (to A.V.)]; the Consejería de Educación de la Comunidad de Madrid [grant number S2011/BMD-2349 (to A.V.)]; the CSIC program i-COOP+2014 [grant number COOPA20053 (to A.V.)] and the European Commission [grant number PITN-GA-2011-289033 (to A.V.)]; the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program [grant number PITN-GA-2011-289033 (to S.R.S.)]; the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela [grant number CDCH-UCV 03-00-6057-2005 (to V.S.)]; and [grant number PG-03-8728-2013 (to G.B.)]; and the Fondo Nacional de Ciencia, Tecnología e Innovación [grant number P-2011000884 (to G.B.)].Peer Reviewe

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.This work was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación [grant number SAF2014-52048-R (to A.V.)]; the Consejería de Educación de la Comunidad de Madrid [grant number S2011/BMD-2349 (to A.V.)]; the CSIC program i-COOP+ 2014 [grant number COOPA20053 (to A.V.)] and the European Commission [grant number PITNGA-2011-289033 (to A.V.)]; the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program [grant number PITN-GA-2011-289033 (to S.R.S.)]; the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela [grant number CDCH-UCV 03-00-6057-2005 (to V.S.)]; and [grant number PG-03-8728-2013 (to G.B.)]; and the Fondo Nacional de Ciencia, Tecnología e Innovación [grant number P-2011000884 (to G.B.)