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Next Generation Sequencing of transcriptomes to reveal the role of HDAC3 in regulating the development of LMPP and CD115- CDP and bone marrow pDCs

ABSTRACT: Hdac3 is highly expressed in pDCs, and its deficiency led to substantially impaired development of pDCs, but not cDCs in bone marrow (BM) and spleen. Meanwhile, the hematopoietic progenitors with DC differentiation potential, including CMP, CLP and CD115+ CDP were significantly decreased. Although the number of HSC and CD115− CDP were increased, their differentiation potential toward pDCs were abolished. Further investigation demonstrated that HDAC3 was required for the differentiation of pDC from progenitors at different differentiation stages.

ORGANISM(S): Mus musculus

PROVIDER: GSE197207 | GEO | 2022/05/27

REPOSITORIES: GEO

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Next Generation Sequencing of transcriptomes to reveal the role of HDAC3 in regulating the development of LMPP and CD115- CDP and bone marrow pDCs

Project description:Next Generation Sequencing of transcriptomes to reveal the role of HDAC3 in regulating the development of LMPP and CD115- CDP and bone marrow pDCs

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Project description:Purpose: The goals of this study are to compare CD115- monocytes to CD115+ monocytes from naïve and tumor-bearing mice (EL4) with high-throughput data analysis. Methods: CD115- monocytes and CD115+ monocytes were sorted by flow cytomentry separatly. Total RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and residual DNA was removed with a DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Samples were loaded onto a 1% agarose gel before electrophoresis. DNA-free conditions were confirmed by SYBR Gold staining (Invitrogen). The quantity and the quality of RNA was evaluated by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). RNA-Seq libraries were prepared with 1 μg of total RNA from two biological replicates of each group using TruSeq Stranded mRNA LT Sample Prep Kit and analyzed by Illumina Sequencer (both from Illumina, San Diego, CA, USA). Results: We found 1,723 differetially expressed genes (DEGs) between CD115- monocytes and CD115+ monocytes (cutoff value was set at 2-fold). DEGs of interest are invoved in granulocyte, transcription factors, mononuclear phagocytes, and angiogenesis. Conclusions: Our study represents the first detailed DEGs analysis of CD115- monocytes and CD115+ monocytes from naïve and tumor-bearing mice. Our results show that CD115- monocytes may have a closer relationship with neutrophils or PMN-MDSCs than the CD115+ monocytes. Also, our overall transcriptomic analysis further suggests that CD115- M-MDSCs and monocytes are separate subsets of monocytic cells.

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Project description:Committed precursors of conventional dendritic cells (pre-cDCs) derived from the common DC progenitor which differentiate into cDC subpopulations in peripheral tissues have been identified, but committed precursors for plasmacytoid DCs (pDCs) have not been found. Here we show that CDP-derived ‘CCR9- MHCIIlow BST2+ Siglec-H+ pDCs from murine bone marrow which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state. Upon adoptive transfer the fate of CCR9- pDC-like precursors is governed by the tissues they enter. In the bone marrow and liver most transferred CCR9- pDC-like precursors differentiate into CCR9+ pDCs, whereas in peripheral lymphoid organs, lung and intestine they can give rise to CCR9+ pDCs and cDCs. Thus, CCR9- pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential, whose final differentiation depends on tissue-specific factors allowing adaptation to local requirements. Total RNA obtained from CCR9- pDC-like common DC progenitors and CCR9+ pDCs was compared for differential gene expression. 3 independent isolations were performed for the 2 samples. 6 arrays were run in total.

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Genome-wide maps of H3K27ac in HDAC3 knockout bone marrow pDCs compared with control pDCs

Project description:Genome-wide maps of H3K27ac in HDAC3 knockout bone marrow pDCs compared with control pDCs

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Two distinct subsets are identified from the peritoneal myeloid mononuclear cells expressing both CD11c and CD115

Project description:The peritoneal cavity is home to various immune cells. Previous studies have investigated the heterogenous nature of peritoneal myeloid mononuclear cells. However, up to this date, there are no clear criteria to distinguish peritoneal macrophages and dendritic cells (DCs). In the present study, we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14-CD206- and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent antigen presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14-CD206- cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14-CD206- cells generate partially activated T cells and possess a greater ability to induce regulatory T cells under TGF-β and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14-CD206- cells are responsive to the treatment of FLT3L and GM-CSF, the numbers of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of transcriptomes reveals that the gene expression profile of MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with that of SPMs. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, (i) MHCII+CD11c+CD115+CD14-CD206- cells closely related to DCs and (ii) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.

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