Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses

Monocytes can have important effects on the polarization and expansion of lymphocytes and may contribute to shaping primary and memory T-cell responses in humans and mice. However, their precise contribution in terms of cellular subsets and the molecular mechanisms involved remains to be determined. Mouse monocytes originate from a bone marrow progenitor, the macrophage and DC precursor (MDP), which also gives rise to conventional dendritic cells through a separate differentiation pathway. Mouse monocytes may be grouped in different functional subsets. The CD115(+) Gr1(+) 'inflammatory' monocyte subset can give rise not only to immunostimulatory 'TipDCs' in infected mice but also to immunosuppressive 'myeloid-derived suppressor cells' in tumor-bearing mice. CD115(+) Gr1(+) monocytes can also contribute to the renewal of several resident subsets of macrophages and DCs, such as microglia and Langerhans cells, in inflammatory conditions. The CD115(+) Gr1(-) 'resident' monocyte subset patrols blood vessels in the steady state and extravasates during infection with Listeria monocytogenes or in the healing myocardium. CD115(+) Gr1(-) monocytes are responsible for an early and transient inflammatory burst during Lm infection, which may play a role in the recruitment of other effector cells and subsequently differentiate toward 'M2'-like macrophages that may be involved in wound healing. More research will no doubt confirm the existence of more functional subsets, the developmental relationship between mouse subsets as well as the correspondence between mouse subsets and human subsets of monocytes. We will discuss here the potential roles of monocytes in the immune response, the existence of functional subsets and their relationship with other myeloid cells, including dendritic cells.     

Lineage development of hematopoietic stem and progenitor cells

Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.     

Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.     

Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells

Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA^– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2^– CCR9⁺ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.Subject terms: Innate immunity, Haematopoietic stem cells     

Developmental biology of zebrafish myeloid cells

The zebrafish (Danio rerio) has emerged as an informative vertebrate model for developmental studies, particularly those employing genetic approaches such as mutagenesis and screening. Zebrafish myelopoiesis has recently been characterized, paving the way for the experimental strengths of this model organism to contribute to an improved understanding of the genetic regulation of myeloid development. Zebrafish have a multi-lineage myeloid compartment with two types of granulocyte (heterophil/neutrophil and eosinophil granulocytes), and monocyte/macrophages, each with characteristic morphological features and histochemical staining properties. Molecular markers have been characterised for various myeloid cell types and their precursor cells, for example: stem cells (scl, hhex, lmo2), myeloid lineage precursors (spi1/pu.1, c/ebp1), heterophil granulocytes (mpx/mpo), macrophages (L-plastin, fms). In zebrafish, the sites of early myeloid and erythroid commitment are anatomically separated, being located in the rostral and caudal lateral plate mesoderm respectively. Functional macrophages appear before cells displaying granulocytic markers. By the second day of life, cells expressing granulocyte- and macrophage-specific genes are scattered throughout the embryo, but tend to aggregate in the ventral venous plexus, which may be a site of their production or a preferred site for their residence. Even in early embryos, macrophages are phagocytically active, and granulocytes participate in acute inflammation. Equipped with an understanding of the developmental biology of these various myeloid cells and a set of tools for their identification and functional study, we will now be able to exploit the experimental strengths of this model organism to better understand the genetic regulation of myelopoiesis.     

人造血干/祖细胞多态性的研究:Ⅲ.骨髓CD34~ 造血干/祖细胞功能亚群的分析

CD 34~ 造血干/祖细胞(HSC/HPC)是十分异质性的,由多个不同功能亚群所构成,在分化方向与重建造血等方面差异显著。本文对正常人骨髓CD 34~ HSC/HPC各亚群进行了较全面的分析。首先以阳性选择策略,采用Isolex~(TM) 50免疫磁球分选术富集骨髓CD 34~ HSC/HPC,其纯度>90%,随之采用免疫荧光抗体双标记二维流式细胞仪对其测定,发现高纯度的CD 34~ HSC/HPC可分为八个不同亚群:1.CD 34~ /CD 71~-(23.4%—56.6%)与CD 34~ /CD 71~ (33.4%—66.6%);2.CD34~ /CD 45~-(80.8%—82.5%)与CD34~ /CD 45~ (8.1%—11.2%);3.CD 34~ /CD 33~-(20.4%—80.6%)与CD 34~ /CD 33~ (14.6%—64.8%);4.CD 34~ /DR~-(6.3%—11.0%)与CD 34~ /DR~ (82.8%—85.5%)。用免疫胶体金——免疫桥酶联组化双染色对上述亚群进一步分析的结果与流式细胞仪的高度一致,为研究各亚群的功能与生物学特性提供了坚实基础     

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation

Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra‐cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule‐generated forces, in the early commitment to the myeloid lineage.     

Rosiglitazone Promotes Bone Marrow Adipogenesis to Impair Myelopoiesis under Stress

Objective

The therapeutic use of thiazolidinediones (TZDs) causes unwanted hematological side effects, although the underlying mechanisms of these effects are poorly understood. This study tests the hypothesis that rosiglitazone impairs the maintenance and differentiation of hematopoietic stem/progenitor cells, which ultimately leads to hematological abnormalities.

Methods

Mice were fed a rosiglitazone-supplemented diet or a normal diet for 6 weeks. To induce hematopoietic stress, all mice were injected once with 250 mg/kg 5-fluorouracil (5-Fu) intraperitoneally. Next, hematopoietic recovery, hematopoietic stem/progenitor cells (HSPCs) subsets, and myeloid differentiation after 5-Fu treatment were evaluated. The adipogenesis induced by rosiglitazone was assessed by histopathology and oil red O staining. The effect of adipocytes on HSPCs was studied with an in vitro co-culture system.

Results

Rosiglitazone significantly enhanced bone marrow adipogenesis and delayed hematopoietic recovery after 5-Fu treatment. Moreover, rosiglitazone inhibited proliferation of a granulocyte/monocyte progenitor (GMP) cell population and granulocyte/macrophage colony-stimulating factor (GM-CSF) colonies, although the proliferation and mobilization of Lin-c-kit+Sca-1+ cells (LSK) was maintained following hematopoietic stress. These effects could be partially reversed by the selective PPARγ antagonist BADGE. Finally, we demonstrated in a co-culture system that differentiated adipocytes actively suppressed the myeloid differentiation of HSPCs.

Conclusion

Taken together, our results demonstrate that rosiglitazone inhibits myeloid differentiation of HSPCs after stress partially by inducing bone marrow adipogenesis. Targeting the bone marrow microenvironment might be one mechanism by which rosiglitazone impairs stress-induced hematopoiesis.     

HIV-1 utilizes the CXCR4 chemokine receptor to infect multipotent hematopoietic stem and progenitor cells

HIV infection is characterized by gradual immune system collapse and hematopoietic dysfunction. We recently showed that HIV enters multipotent hematopoietic progenitor cells and establishes both active cytotoxic and latent infections that can be reactivated by myeloid differentiation. However, whether these multipotent progenitors include long-lived hematopoietic stem cells (HSCs) that could establish viral reservoirs for the life of the infected person remains unknown. Here we provide direct evidence that HIV targets long-lived HSCs and show that infected HSCs yield stable, multilineage engraftment in a xenograft model. Furthermore, we establish that the capacity to use the chemokine receptor CXCR4 for entry determines whether a virus will enter multipotent versus differentiated progenitor cells. Because HSCs live for the life span of the infected person and are crucial for hematopoietic health, these data may explain the poor prognosis associated with CXCR4-tropic HIV infection and suggest HSCs as long-lived cellular reservoirs of latent HIV.     

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a proto-oncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.     

Deep diving in the blood stem cell‐ome

Defining the functional distinctions between cells comprising the bone marrow has yielded fundamental insights into lineage ordering and drivers of blood cell production. A novel, highly granular and multi‐dimensional molecular characterization of functional subsets of hematopoietic stem‐ and progenitor cells recently published in Cell Stem Cell (Cabezas‐Wallscheid et al, 2014 ) will serve as a landmark and treasure trove for unanticipated insights into basic biology and the development of future targeted medicine.     

人造血干／祖细胞多态性的研究：Ⅷ.人正常骨髓CD34^＋造血细胞体视学的特征

人ＣＤ３４＋造血细胞是具有高度自我更新,多向分化及重建长期造血与免疫学功能的独特体细胞。为系统探索ＣＤ３４＋造血细胞的形态,细胞化学及超微结构特征,新近我们设计组合并建立了ＣＩＭＳ－１００ＦＡＣＳ４４０无菌二次分选术,可使所获ＣＤ３４＋５造血的纯度达１００％。     

Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies

Lymphoid and myeloid cells isolated from second trimester fetal lymphoid organs were characterized by utilizing a panel of monoclonal antibodies that define human lineage-restricted, differentiation, histocompatibility, and activation antigens. At distinct gestational stages, the appearance of morphologically identifiable lymphoid and myeloid cells paralleled the appearance of cells expressing definable lymphoid and myeloid antigens. The proportion of cells in fetal liver, bone marrow, and spleen that expressed histocompatibility, myeloid, and B cell antigens increased with fetal maturation. In contrast, even the earliest fetal thymuses studied were of a phenotype no different than that seen during later stages of ontogeny. Although the cellular lineage of most fetal hematopoietic cells could be identified by this panel of reagents, a considerable number of fetal liver and bone marrow cells did not express any of these antigens, suggesting the possibility that they might represent early hematopoietic progenitor cells. These studies support the notion that the adult cellular phenotype is the result of both an orderly acquisition of differentiation antigens and the migration of these primitive cellular populations to specific fetal organs. Identification of hematopoietic progenitors in fetal tissues may facilitate the identification and isolation of early lymphoid and myeloid progenitor cells in adults.     

A common pathway for dendritic cell and early B cell development

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.     

Molecular and functional characterization of early human hematopoiesis

Through improvements in xenograft assay methods and in the identification of novel cell surface markers, significant progress has been made in our understanding of the human hematopoietic stem and progenitor hierarchy. The isolation of clonally pure populations of stem cells and early progenitors opens the way to carry out gene expression profiling studies to uncover the molecular regulators of each developmental step and to gain insight into the process of lineage commitment in human hematopoiesis.     

The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models

Chronic inflammation drives the development of atherosclerosis, and details regarding the involvement of different leukocyte subpopulations in the pathology of this disease have recently emerged. This Review highlights the surprising contribution of granulocyte subsets and mast cells to early atherogenesis and subsequent plaque instability, and describes the complex, double-edged role of monocyte, macrophage and dendritic-cell subsets through crosstalk with T cells and vascular progenitor cells. Improved understanding of the selective contributions of specific cell types to atherogenesis will pave the way for new targeted approaches to therapy.