Project description:We aimed to study the impact of bacteria-derived metabolites in solid organ transplantation. The short-chain fatty acid butyrate was administered using a new drug-delivery method developed by Wang et al. (https://doi.org/10.1038/s41551-022-00972-5). This system is based on a micelle platform that carries and protects butyrate (ButM) from degradation in the upper gastrointestinal tract, to lower distal portions of the gut. Mice (adult B6 females) were treated for 21 days with ButM (100mg/mL) or 1x PBS. Splenic Ly6Chi CD11b+ monocytes were sorted for bulk RNAseq using the Takara SMART-Seq mRNA LP and Unique Dual Index kits. Libraries were sequenced using the Illumina NovaSeq 6000 platform.
Project description:We recently reported that tumor-specific Th9 cells are less susceptible to exhaustion, fully cytolytic to tumor cells, and possess long-term persistence capacity because of their unique hyperproliferative T cell feature. However, these observations only revealed how tumor-specific Th9 cells eradicate antigen-positive tumor cells. Given the unmet need to develop effective ACT strategies with tumor-specific Th9 cells for solid tumors with the heterogeneity in antigen expression, the CD11b+CD11c–Ly6Chi inflammatory monocytes were flow-sorted from Th9 treated mice, and been further analyzed for microarray.
Project description:Differentially expressed genes of CD11b+Gr-1+ immature myeloid cells (IMCs) in the bone marrow and colonic tumor setting of histidine decarboxylase (HDC)-KO mice were examined by microarray (Affymetrix Mouse 430.2 array). Myeloid differentiation-related candidate genes were sought to be isolated and functionally studied. Total RNA of HDC-expressing CD11b+Gr-1+ IMCs of bone marrow were extracted from HDC-EGFP and HDC-EGFP/HDC-KO mice (3 mice in each group). CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) of colon tumor were sorted from 10-12 colon tumors of WT and HDC-KO mice (5 mice in each group), and pooled to extract total RNA for microarray studies. Two technical replicates for each of the four groups. Four sets of comparisons were performed to screen for upregulated or downregulated genes in the HDC-KO CD11b+Gr-1+ IMCs or MDSCs (experiment group) compared to the WT group: (1) HDC-expressing CD11b+Gr-1+ IMCs of bone marrow of HDC KO mice compared to bone marrow IMCs of WT mice; (2) CD11b+Gr-1+ MDSCs in tumors of HDC-KO mice compared to WT mice; (3) CD11b+Gr-1+ MDSCs of WT colon tumors compared to IMCs in the WT bone marrow; and (4) CD11b+Gr-1+ MDSCs of colon tumors of HDC-KO mice compared to IMCs in the bone marrow of HDC-KO mice.
Project description:To understand the underlying cause for the observed apoptosis in E2f1-3 deficient myeloid cells. We compared gene expression profiles of Cd11b+ sorted myeloid cells isolated from bone marrow of control (E2F1-/- ) and experimental (Mxcre;E2F1-/-2-/-3f/f ) mice. RNA was extracted from Cd11b cells from bone marrow of eight-week-old mice after FACS sorting with FITC- cojugated Cd11b antibody.
Project description:Dendritic cells (DCs) and macrophages (MPs) are important for immunological homeostasis in the colon. We found that F4/80hi CX3CR1hi (CD11b+CD103-) cells account for 80% of mouse colonic lamina propria (cLP) MHC-IIhi cells. Both CD11c+ and CD11c- cells within this population were identified as MPs based on multiple criteria, including a MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs, based on phenotypic and functional analysis and comprise three separate CD11chi cell populations: CD103+CX3CR1-CD11b- DCs, CD103+CX3CR1-CD11b+ DCs and CD103-CX3CR1intCD11b+ DCs. In non-inflammatory conditions, Ly6Chi monocytes differentiated primarily into CD11c+, but not CD11c- MPs. In contrast, during colitis, Ly6Chi monocytes massively invaded the colon and differentiated into pro-inflammatory CD103-CX3CR1intCD11b+ DCs, which produced high levels of IL-12, IL-23, iNOS and TNF. These findings demonstrate the dual capacity of Ly6Chi blood monocytes to differentiate into either regulatory MPs or inflammatory DCs in the colon, and that the balance of these immunologically antagonistic cell types is dictated by microenvironmental conditions. FACS sorted expression from normal controls
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.
Project description:Differentially expressed genes of CD11b+Gr-1+ immature myeloid cells (IMCs) in the bone marrow and colonic tumor setting of histidine decarboxylase (HDC)-KO mice were examined by microarray (Affymetrix Mouse 430.2 array). Myeloid differentiation-related candidate genes were sought to be isolated and functionally studied.
Project description:To understand the underlying cause for the observed apoptosis in E2f1-3 deficient myeloid cells. We compared gene expression profiles of Cd11b+ sorted myeloid cells isolated from bone marrow of control (E2F1-/- ) and experimental (Mxcre;E2F1-/-2-/-3f/f ) mice.
Project description:mRNA expression profiles between Ym1+Ly6Chi monocytes and Ym1-Ly6Chi monocytes from LPS-treated mice were analyzed by RNA-sequencing
Project description:Epidermal Growth Factor Receptor (EGFR) is a critical factor regulating numerous aspects of cell biology. Though it is widely recognized as an oncogene, EGFR has been implicated in cardiovascular health, and has been shown to have unique cell specific roles in the heart. Macrophages and other myeloid cells play key roles in normal cardiac physiology and post injury pathological outcomes. Myeloid cell EGFR has been implicated in disease, though its role in the normal or failing heart has yet to be established. Here, we crossed EGFR floxed mice with LysM Cre transgenics to specifically delete EGFR in Cd11b+ myeloid cells. We then isolated non-cardiomyocytes and subject them to Cd11b microbeads and subsequent column concentration to obtain cardiac Cd11b+ non-myocyte myeloid cells. We used samples from floxed EGFR controls, and EGFR specific myeloid knockouts. In comparisson to control, myeloid EGFR knockouts had 763 differential transcripts when consdering p adjusted less than 0.05 and log2foldchange greater than absolute value 1.5.