Project description:The advent of recent cutting-edge technologies has allowed the discovery and characterization of novel hematopoietic progenitors, including SSCloCD66b+CD15+CD11b-CD49dhiproNeu1s and SSChiCD66b+CD15+CD11b-CD49dintproNeus2s, CD66b+CD15+CD11b+CD49d+CD101-preNeus and Lin-CD66b+CD117+CD71+eNePs along human neutropoiesis process. In this research field, we recently identified CD66b-CD38+CD64dimCD115-, CD34+ and CD34dim/-cells exclusively committed to the neutrophil lineage [which we renamed as CD34+ and CD34dim/- neutrophil-committed progenitors (NCPs)], representing the earliest neutrophil precursors identifiable and sorted by flow cytometry. Moreover, based on their differential CD34 and CD45RA expression, we could identify four populations of NCPs, namely: CD34+CD45RA-/NCP1s, CD34+CD45RA+/NCP2s, CD34dim/-CD45RA+/NCP3s and CD34dim/-CD45RA-/NCP4s. This said, a very recent study by Ikeda and coworkers (PMID: 36862552) reported that neutrophil precursors termed either neutrophil progenitors (NePs) or “early neutrophil-committed progenitors” would generate immunosuppressive neutrophil-like CXCR1+CD14+CD16-monocytes. Hence, presuming that NePs/alias “early neutrophil-committed progenitors” correspond to NCPs, the selective neutrophil-commitment that we attributed to NCPs is contradicted by Ikeda and coworkers’ paper. In this study, by performing a more analytical reevaluation at the phenotypic and molecular levels of the cells generated by NCP2s and NCP4s (selected as representatives of NCPs), we categorically exclude that NCPs generate neutrophil-like CXCR1+CD14+CD16-monocytes. Rather, we provide substantial evidence indicating that the cells generated by NePs/alias “early neutrophil-committed progenitors” are neutrophilic cells at different stage of maturation, displaying moderate levels of CD14, instead of neutrophil-like CXCR1+CD14+CD16-monocytes as pointed by Ikeda and coworkers. Hence, the conclusion that NePs/alias “early neutrophil-committed progenitors” aberrantly differentiate into neutrophil-like monocytes derives, in our opinion, from data misinterpretation
Project description:During systemic inflammation, different neutrophil subsets are mobilized to the blood circulation. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright) based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but the underlying mechanism is largely unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes that are relevant for their functions. Neutrophil subsets were isolated by FACS sorting from the blood of healthy volunteers who were administered a single dose of lipopolysaccharide (LPS). The transcriptome was determined by microarray. The mobilized neutrophil subsets were characterized by specific transcriptome profiles reflecting their phase in neutrophil lifespan. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. This was confirmed by stimulation of peripheral neutrophils with IFNgamma. These cells acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that the suppressive phenotype of the neutrophil subset is induced by IFNgamma. Specific stimulation of neutrophils might have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease. After LPS infusion, blood was taken at t=0 and t=4 hours. Neutrophils were FACS sorted based on CD16 and CD62L expression. Gene expression of neutrophil subsets was assessed relative to t=0 as control.
Project description:In this study we report the identification of a unipotent human neutrophil-committed progenitors (NCPs) within bone marrow (BM) CD34+ and CD34dim/- cells. We show that NCPs can be either CD45RA+ or CD45RA-. By scRNA-seq experiments, we could uncover that NCPs actually consist of four cell clusters (c1-c4), substantially matching with the phenotypic identification of NCPs based on CD34, but not CD45RA, expression. c1-c4 cells were found to be at different maturation levels, aligned along two developmental neutrophil trajectories, as well as characterized by specific gene profiles.
Project description:Technological advances have greatly improved our knowledge of myelopoiesis, for instance with the discovery of granulocyte-monocyte-DC progenitors (GMDPs), monocyte-DC progenitors (MDPs), common dendritic progenitors (CDPs) and common monocyte progenitors (cMoPs) based on sophisticated flow cytometry approaches. Concomitantly, little progress has been made to characterize the very early phases of human neutropoiesis, despite the recently reported eNePs, PM w/o eNePs, ProNeus, preNeus, all of them being, however, CD66b+neutrophil progenitors. More recently, we identified four SSCloLin-CD66b-CD45dimCD34+/CD34dim/-CD64dimCD115-cells as the earliest precursors specifically committed to the neutrophil lineage present in human bone marrow (BM), which we called neutrophil-committed progenitors (NCPs), namely from NCP1s to NCP4s. In this study, we report the isolation and characterization of two new SSChiCD66b-CD64dimCD115-NCPs that, by phenotypic, flow cytometric SSC, transcriptomic, maturation and immunohistochemistry properties/features, stand after NCP4s but precede the CD66b+Promyelocytes (PMs) during the neutropoiesis cascade. These cells, similarly to SSCloCD45RA+NCP2s/NCP3s and SSCloCD45RA-NCP1s/NCP4s, exhibit phenotypic differences in CD45RA expression levels and, therefore, were named as SSChiCD45RA+NCP5s and SSChiCD45RA-NCP6s. In addition, NCP5s result more immature than NCP6s, as determined by both their cell differentiation and proliferative potential, and by their transcriptomic and phenotypical features. Finally, by examining whether NCPs and all other CD66b+neutrophil precursors are altered in representative hematological malignancies, we found that, in patients with chronic-phase chronic myeloid leukemia (CP-CML), but not with systemic mastocytosis (SM), there is an increased frequency of NCP4s and all downstream neutrophil progenitors, particularly, the CD45RA-cells, like NCP6s, suggesting their involvement in CML pathogenesis. Altogether, our data advance our knowledge of human neutropoiesis.
Project description:Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.
Project description:During systemic inflammation, different neutrophil subsets are mobilized to the blood circulation. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright) based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but the underlying mechanism is largely unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes that are relevant for their functions. Neutrophil subsets were isolated by FACS sorting from the blood of healthy volunteers who were administered a single dose of lipopolysaccharide (LPS). The transcriptome was determined by microarray. The mobilized neutrophil subsets were characterized by specific transcriptome profiles reflecting their phase in neutrophil lifespan. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. This was confirmed by stimulation of peripheral neutrophils with IFNgamma. These cells acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that the suppressive phenotype of the neutrophil subset is induced by IFNgamma. Specific stimulation of neutrophils might have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease.