scholarly journals A transgenic line that reports CSF1R protein expression provides a definitive marker for the mouse mononuclear phagocyte system

2020 ◽  
Author(s):  
Kathleen Grabert ◽  
Anuj Sehgal ◽  
Katharine M. Irvine ◽  
Evi Wollscheid-Lengeling ◽  
Derya D. Ozdemir ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Mononuclear Phagocyte ◽  
Mononuclear Phagocyte System ◽  
Lymphoid Tissues ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Monocytes And Macrophages ◽  
Macrophage Populations

AbstractThe proliferation, differentiation and survival of cells of the mononuclear phagocyte system (MPS, progenitors, monocytes, macrophages and classical dendritic cells) is controlled by signals from the macrophage colony-stimulating factor receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters but none is both universal and lineage-restricted. Here we report the development and characterization of a novel CSF1R reporter mouse. A Fusion Red (FRed) cassette was inserted in-frame with the C-terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells (HSC), arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly-expressed in marrow monocytes and common myeloid progenitors (CMP) but significantly lower in granulocyte-macrophage progenitors (GMP). In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169+ resident macrophages and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations including classical dendritic cells. Whole mount imaging of non-lymphoid tissues in mice with combined CSF1R-FRed/Csf1r-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.

scholarly journals The Mononuclear Phagocyte System: The Relationship between Monocytes and Macrophages

2019 ◽  
Vol 40 (2) ◽  
pp. 98-112 ◽  
Author(s):  
David A. Hume ◽  
Katharine M. Irvine ◽  
Clare Pridans
Keyword(s):  
Mononuclear Phagocyte ◽  
Mononuclear Phagocyte System ◽  
Monocytes And Macrophages ◽  
The Relationship

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80

Development ◽  
1991 ◽  
Vol 112 (2) ◽  
pp. 517-526 ◽  
Author(s):  
L. Morris ◽  
C.F. Graham ◽  
S. Gordon
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Mononuclear Phagocyte ◽  
Cellular Interactions ◽  
Fetal Tissues ◽  
Prominent Component ◽  
Inflammatory Stimuli ◽  
Monocytes And Macrophages ◽  
Formed Part ◽  
Haemopoietic Cells

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

Effects of B7.2−/− Mature Dendritic Cells on Tolerance Induction to Alloantigens in Fetal Mice Following In Utero Transplantation with Lineage Depleted Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2134-2134
Author(s):  
Swati Bhattacharyya ◽  
Morton J. Cowan
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
In Utero ◽  
Relative Role ◽  
Skin Grafts ◽  
Hematopoietic Stem ◽  
Fetal Mice

Abstract In utero hematopoietic stem cell transplantation (IUT) has the potential to cure a variety of marrow stem cell defects without using marrow ablative therapy. However IUT for diseases other than SCID has been unsuccessful. To better understand the barriers to successful IUT we wanted to define the role of the B7.1/B7.2 co-stimulatory molecules in inducing tolerance to allogeneic donor bone marrow cells in the fetal murine recipient. We studied the relative role of B7.1 and B7.2 expression on dendritic cells (DC) on engraftment and in generating donor specific tolerance in fetal mice. Mature DC (mDC) from B7.1−/− or B7.2−/− donors and wild type (wt) lineage depleted (lin−) C57Bl/6 (B6) bone marrow (BM) were injected into gestational day (GD) 14 Balb/c fetuses. Recipients of lin− wt BM and B7.1−/− mDC had a significantly lower survival (47.4%, p<0.01) associated with mild-moderate GvHD compared to the recipients of B7.2−/− mDC and lin− BM (82.3%) where none developed GvHD. Engraftment results in blood at 6 weeks post IUT showed, B7.1−/− recipients had multilineage engraftment (4.7±0.8% T cells and 5.7± 1.1% granulocytes) in their blood, but by 12 weeks, only donor CD3+ (predominantly CD8+) cells (2.1±1.3%) were present. The percent H2Kb+ (donor) T cells (predominantly CD4+) in the blood of recipients of lin− wt BM and B7.2−/− was 11.8±8.5% at 6 weeks p<0.001 and 6.5±2.5% at 12 weeks, p=0.006. The circulating donor CD4+ cells were Th2 (CD4+CD25−IL4+IL10+) and Treg (CD4+CD25+IL4−IL10−). Both fractions inhibited the T cell proliferative response in the MLR. Long term engraftment in thymic tissues was found in the tolerant recipients of lin− wt BM and B7.2−/− mDC (13.4±8.3% donor CD3+ T cells). We also found prolonged (rejection by day 36) acceptance of donor skin grafts in 7 of 12 recipients of B7.2−/− mDC and 2 of 5 recipients of B7.2−/− mDC and lin−BM. All third party C3H grafts were rejected by day 14 and 80% of the Balb/c (self) skin grafts were permanently accepted. We hypothesized that tolerized animals would behave similarly to recipients of megadoses of syngeneic BM with an increase in multilineage engraftment. We injected a total of 200x106 male wt B6 lin− BM cells over 5 days into adult IUT recipients of B7.1−/− or B7.2−/− mDC ± lin− wt BM and wt age-matched allogeneic and syngeneic (female) controls. Mice that had received B7.2−/− mDC + lin− BM in utero showed multi-lineage engraftment in the blood. In contrast, the in utero recipients of B7.1−/− mDC + lin− BM showed no significant engraftment (p<0.05). In conclusion, donor DC costimulatory molecules significantly affect survival, engraftment and GvHD; and these responses to B7.2−/− mDC and lin− BM appear to be mediated by both Th2 and Treg donor cells.

Human Conventional and Plasmacytoid Dendritic Cells Can Originate from Both Lymphoid and Myeloid Progenitors in a New Humanized Mouse System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2273-2273
Author(s):  
Fumihiko Ishikawa ◽  
Tadafumi Iino ◽  
Hiroaki Niiro ◽  
Shuro Yoshida ◽  
Toshihiro Miyamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Dendritic Cell ◽  
Plasmacytoid Dendritic Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Transcription Profiles ◽  
Human Dendritic Cells ◽  
Myeloid Progenitors

Abstract Dendritic cells play a key role in host defense by presenting exogenous antigens to T cells. Two dendritic cell subsets, conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), express distinct repertoire of Toll-like-receptors and recognize different antigens. We previously reported that murine cDCs and pDCs differentiate via either the myeloid or the lymphoid pathway (Shigematsu et al. Immunity ). It is, however, still unclear whether human cDCs and pDCs develop from myeloid, lymphoid or both lineages. In order to analyze the in vivo differentiation of human dendritic cells, we employed the newly-developed xenotrasplant assay system which utilizes newborn NOD-scid/IL2rgnull mice (Ishikawa et al., Blood, in press). Transplantation of 104 Lin-CD34+CD38- hematopoietic stem cells into sublethally irradiated newborn NOD-scid/IL2rgnull mice resulted in generation of all hematopoietic and lymphoid components for a long-term via physiological intermediates such as common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). We found that in this system, dendritic cell subcomponents such as hCD11c+hIL3Ralow cDCs and hCD11c-hIL3Rahigh pDCs, efficiently developed in recipients’ bone marrow, spleen and peripheral blood. To elucidate the origin of human mDCs and pDCs, we purified CMP or CLP from the cord blood, and transplanted these cells into sublethally irradiated newborn NOD-scid/IL2rgnull mice via facial vein. At 4-6 weeks post-transplantation, CMP gave rise only to myeloid cells such as erythroid cells, platelets and granulocytes, while CLP exclusively generated T, B and NK cells. Interestingly, in either mouse group injected with CMP or CLP, cDCs and pDCs were easily detected in the spleen and in the bone marrow. Phenotypic and RT-PCR analyses of purified CMP- or CLP-derived DCs revealed that DCs possessed similar phenotypic characteristics, and transcription profiles in TLR families, BDCA antigens and costimulation molecules, irrespective of their lineage origin. Thus, human cDCs and pDCs develop through both myeloid and lymphoid pathways as in case of mouse hematopoiesis. Further characterization of DCs of different lineage origin is currently performed by microarray analyses in order to find genes specifically expressed in each DC subset.

Myeloid Dendritic Cells Contribute to Hematopoietic Progenitor Mobilization by G-CSF.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2319-2319
Author(s):  
Teerawit Supakorndej ◽  
Mahil Rao ◽  
Daniel Link
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Cell Population ◽  
Diphtheria Toxin ◽  
Myeloid Dendritic Cells ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
The Mean ◽  
Bone Marrow Chimeras

Abstract Abstract 2319 Granulocyte-colony stimulating factor (G-CSF) is the prototypic agent used to mobilize hematopoietic stem and progenitor cells (HSPCs) into the blood where they can then be harvested for stem cell transplantation. G-CSF acts in a non-cell-intrinsic fashion to induce HSPC mobilization. We recently showed that G-CSF signaling in a CD68+ monocyte/macrophage lineage cell within the bone marrow initiates the HSPC mobilization cascade (Christopher et al., 2011). Consistent with this finding, two other groups showed that ablation of monocytes/macrophages induces HSPC mobilization (Winkler et al., 2010; Chow et al., 2011). CD68 marks a heterogeneous cell population that includes monocytes, macrophages, myeloid dendritic cells, and osteoclasts. To further define the relevant cell population(s) for HSPC mobilization by G-CSF, we first examined the role of osteoclasts. Receptor activator of NF-kappaB (RANK) signaling is required for osteoclast development. Osteoprotegerin (OPG) is a decoy receptor for RANK ligand, and treatment with OPG-Fc (a stabilized form of OPG) results in osteoclast ablation in mice. We treated mice with 100 μg of OPG-Fc and documented complete osteoclast ablation by histomorphometry. Osteoclast ablation did not result in constitutive HSPC mobilization, nor did it affect G-CSF-induced HSPC mobilization. To further assess the role of osteoclasts, we transplanted RANK−/− fetal liver cells into irradiated Csf3r−/− (G-CSF receptor deficient) recipients. Since RANK is required for osteoclast development, the osteoclasts in these bone marrow chimeras lack the G-CSFR, while other hematopoietic cells (including monocytes/macrophages) are G-CSFR sufficient. Again, G-CSF-induced HSPC mobilization in these mice was normal. Based on these data, we conclude that osteoclasts are dispensable for HSPC mobilization by G-CSF. We next quantified changes in monocytic/macrophage cell populations in the bone marrow after G-CSF treatment (250 μg/kg per day for 5 days) using a novel multi-color flow cytometry assay that includes CD115, F4/80, MHC class II, Gr-1, B220, and CD11c. Using this assay, we observed a significant decrease in macrophages (11.8 ± 3.6-fold) and, surprisingly, myeloid dendritic cells (MDCs; 5.5 ± 1.2-fold) in the bone marrow with G-CSF treatment. To further assess the role of MDCs, we used transgenic mice expressing the diphtheria toxin receptor under the control of the CD11c promoter (CD11c-DTR) to conditionally ablate MDCs. To avoid systemic toxicity, we transplanted CD11c-DTR bone marrow into congenic wild type recipients prior to MDC ablation. The resulting bone marrow chimeras were treated with diphtheria toxin (DT; 400 ng per day for 6 days), which resulted in a 92% reduction in MDCs. Ablation of MDCs resulted in a significant increase in colony-forming cells in the blood and spleen (figure 1A). Moreover, MDC ablation significantly increased mobilization of colony-forming cells and c-Kit+lineage−Sca-1+ (KLS) cells by G-CSF (figures 1B and 1C). Taken together, these data suggest that myeloid dendritic cells, but not osteoclasts, contribute to HSPC mobilization by G-CSF. Figure 1. HSPC mobilization in CD11c-DTR mice. CD11c-DTR bone marrow chimeras were treated with diphtheria toxin (DT) alone, G-CSF alone, or DT plus G-CSF. The number of CFU-C (A & B) or KLS cells (C) in the blood and spleen are shown. Data represent the mean ± SEM of 10–11 mice pooled from two independent experiments. *p < 0.05; **p < 0.001; ***p < 0.0001. Figure 1. HSPC mobilization in CD11c-DTR mice. CD11c-DTR bone marrow chimeras were treated with diphtheria toxin (DT) alone, G-CSF alone, or DT plus G-CSF. The number of CFU-C (A & B) or KLS cells (C) in the blood and spleen are shown. Data represent the mean ± SEM of 10–11 mice pooled from two independent experiments. *p < 0.05; **p < 0.001; ***p < 0.0001. Disclosures: No relevant conflicts of interest to declare.

Development of thymic and splenic dendritic cell populations from different hemopoietic precursors

Blood ◽  
2001 ◽  
Vol 98 (12) ◽  
pp. 3376-3382 ◽  
Author(s):  
Li Wu ◽  
Angela D'Amico ◽  
Hubertus Hochrein ◽  
Meredith O'Keeffe ◽  
Ken Shortman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Dendritic Cell ◽  
Cell Populations ◽  
Lymphoid Tissues ◽  
Hemopoietic Precursors ◽  
Surface Molecules ◽  
Splenic Dendritic Cell ◽  
Antigen Presenting ◽  
Macrophage Precursors

Abstract The antigen-presenting dendritic cells (DCs) found in mouse lymphoid tissues are heterogeneous. Several types of DCs have been identified on the basis of the expression of different surface molecules, including CD4, CD8α, and DEC-205. Previous studies by the authors showed that the mouse intrathymic lymphoid-restricted precursors (lin−c-kit+Thy-1lowCD4low) can produce DCs in the thymus and spleen upon intravenous transfer, suggesting a lymphoid origin of these DCs. In the current study, the potential for DC production by the newly identified bone marrow (BM) common lymphoid precursors (CLPs), common myeloid precursors (CMPs), and committed granulocyte and macrophage precursors was examined. It was found that both the lymphoid and the myeloid precursors had the potential to produce DCs. All the different DC populations identified in mouse thymus and spleen could be produced by all these precursor populations. However, CLPs produced predominantly the CD4−CD8α+ DCs, whereas CMPs produced similar numbers of CD4−CD8α+ and CD4+CD8α− DCs, although at different peak times. On a per cell basis, the CLPs were more potent than the CMPs at DC production, but this may have been compensated for by an excess of CMPs over CLPs in BM. Overall, this study shows that the expression of CD8α does not delineate the hemopoietic precursor origin of DCs, and the nature of the early precursors may bias but does not dictate the phenotype of the DC product.

scholarly journals Transcriptional network analysis of transcriptomic diversity in resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system

2020 ◽  
Author(s):  
Kim M. Summers ◽  
Stephen J. Bush ◽  
David A. Hume
Keyword(s):  
Dendritic Cells ◽  
Network Analysis ◽  
Single Cell ◽  
Cell Types ◽  
Mononuclear Phagocyte ◽  
The Body ◽  
Mononuclear Phagocyte System ◽  
Primary Data ◽  
Analysis Tool ◽  
Rna Seq

AbstractThe mononuclear phagocyte system (MPS) is a family of cells including progenitors, circulating blood monocytes, resident tissue macrophages and dendritic cells (DC) present in every tissue in the body. To test the relationships between markers and transcriptomic diversity in the MPS, we collected from NCBI-GEO >500 quality RNA-seq datasets generated from mouse MPS cells isolated from multiple tissues. The primary data were randomly down-sized to a depth of 10 million reads and requantified. The resulting dataset was clustered using the network analysis tool Graphia. A sample-to-sample matrix revealed that MPS populations could be separated based upon tissue of origin. Cells identified as classical DC subsets, cDC1 and cDC2, and lacking Fcgr1 (CD64), were centrally-located within the MPS cluster and no more distinct than other MPS cell types. A gene-to-gene correlation matrix identified large generic co-expression clusters associated with MPS maturation and innate immune function. Smaller co-expression gene clusters including the transcription factors that drive them showed higher expression within defined isolated cells, including macrophages and DC from specific tissues. They include a cluster containing Lyve1 that implies a function in endothelial cell homeostasis, a cluster of transcripts enriched in intestinal macrophages and a generic cDC cluster associated with Ccr7. However, transcripts encoding many other putative MPS subset markers including Adgre1, Itgax, Itgam, Clec9a, Cd163, Mertk, Retnla and H2-a/e (class II MHC) clustered idiosyncratically and were not correlated with underlying functions. The data provide no support for the concept of markers of M2 polarization or the specific adaptation of DC to present antigen to T cells. Co-expression of immediate early genes (e.g. Egr1, Fos, Dusp1) and inflammatory cytokines and chemokines (Tnf, Il1b, Ccl3/4) indicated that all tissue disaggregation protocols activate MPS cells. Tissue-specific expression clusters indicated that all cell isolation procedures also co-purify other unrelated cell types that may interact with MPS cells in vivo. Comparative analysis of public RNA-seq and single cell RNA-seq data from the same lung cell populations showed that the extensive heterogeneity implied by the global cluster analysis may be even greater at a single cell level with few markers strongly correlated with each other. This analysis highlights the power of large datasets to identify the diversity of MPS cellular phenotypes, and the limited predictive value of surface markers to define lineages, functions or subpopulations.

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