Project description:During immune-mediated severe aplastic anemia (SAA) monocytes (CD11b+ Ly6C+ Ly6G-) signficantly increase by both frequency and number within the bone marrow. We isolated BM monocytes from F1 hybrid mice (C57BL/6;Balb/c) induced with SAA via the splenocyte-transfer model. We utilized single-cell RNA sequencing (scRNA-seq) to analyze the heterogenity of BM monocytes during SAA.
Project description:The two immune cell populations Myeloid-derived suppressor cells (MDSCs), monocytes (MONO) and neutrophils (PMNs) are difficult to differentiate because of shared surface marker expression. Here we utilize the integrin receptor CD11b combined with conventional Ly6G and Ly6C expression to more accurately separate cellular populations via FACS. Then we apply high-throughput RNA Sequencing to Ly6G+Ly6C+CD11bhigh MDSC, Ly6G+Ly6C+CD11blow PMN and Ly6G-Ly6C+ monocyte populations. A total of 6,466 genes were significantly differentially expressed in MDSCs vs. monocytes, whereas only 297 genes were significantly different between MDSCs and PMNs. A number of genes implicated in cell cycle regulation were identified, and in vivo EdU labeling revealed that over 75% of MDSCs proliferated locally at the site of S. aureus biofilm infection.
Project description:Murine monocytes (MC) are classified into Ly6Chigh and Ly6Clow MC. Ly6Chigh MC is the pro-inflammatory subset and the counterpart of human CD14++CD16+ intermediate MC which contributes to systemic and tissue inflammation in various metabolic disorders, including hyperhomocysteinemia (HHcy). This study aims to explore molecule signaling mediating MC subset differentiation in HHcy and control mice.Mouse white blood cell were prepared from peripheral blood and stained with antibody against CD11b, Ly6G and Ly6C and subjected for flow cytometry cell sorting. CD11b+Ly6G- cells were selected as MC. MC subsets (CD11b+Ly6G-Ly6Chigh, and CD11b+Ly6G-Ly6Clow) were sorted based on Ly6C levels. The quantification of MC was used flow cytometry analysis for Ly6Chigh and Ly6Clow MC in CT and Cbs-/-. Then, 100 ng mRNA were obtained from 100,000 sorted cells of CT and Cbs-/- (HHcy) mice. Around 30 million reads were achived and 16,487 normalized genes per sample by mRNA-Seq analysis.
Project description:Bulk RNA-seq data of Lin-CD34+ hematopoietic stem and progenitor cells derived from bone marrow of healthy donors and untreated aplastic anemia patients
Project description:To investigate the functional properties of Ly6G+ DC, we employed GeneChip analysis to compare the gene expression profiles between Ly6G+ DC and Ly6C- DC.
Project description:Clonal hematopoiesis was investigated in patients with aplastic anemia using next-generation sequencing and single-nucleotide polymorphism (SNP) array-based karyotyping.
Project description:Phenotypic transition of myeloid cells into distinct lineages in vivo is important in pathogen response. To monitor immune cell phenotype transitions in vivo, we developed a quantitative temporal in vivo proteomics (QTiPs) platform, performing multiplexed (10-plex) tandem-mass-tag (TMT)-based mass spectrometry on sorted cells collected from their in situ microenvironment during infection. We temporally characterized a poorly understood, virus-driven CD11b+,Ly6G-,Ly6Chigh-low myeloid cell population throughout an acute phase of infection in both the site of infection and bone marrow. QTiPs, in combination with phenotypic, functional and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+,Ly6G-,Ly6Chigh-low cells in anti-viral immune response and viral clearance. Most importantly, the highly time-resolved QTiPs dataset showed the transition of CD11b+,Ly6G-,Ly6Chigh-low cells into M2-like macrophages which displayed increased antigen presentation capacities and bioenergetic demands late in infection. Our QTiPs approach precisely captures myeloid cell-macrophage transition in this population, and it is a novel platform for measuring temporospatial proteome transitions in vivo.