Project description:Inflammatory monocyte (MC) subset differentiation is a major feature in tissue inflammatory and atherosclerosis. The underlying molecular mechanism remains unclear. This study aims to explore molecule targets and signaling which determinate immunological features in MC subsets. ApoE-/- mice were fed a normal chow diet and switched to a high-fat (HF) diet at age 8 weeks and maintained on a HF diet for 12-14 weeks. The control C57BL/6 mice were fed a normal chow diet throughout. Blood Ly6Chigh and Ly6Clow MC subsets from control and ApoE-/- mice were isolated by flow cytometry sorting and subjected for bulk high-throughput RNA-sequencing.
Project description:Gene profiling of CNS-derived microglia vs splenic CD11b+Ly6C+ monocyte subsets deom adult mice Gene array identified 1572 genes that were enriched in microglia vs. 611 monocyte enriched genes with a greater than 5-fold difference (P<0.001). Gene profiling of CD11b+CD45Low microglia isolated from the CNS and CD11b+Ly6C+ monocyte subsets isolated from the spleen of naM-CM-/ve adult mice.
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:Currently, it is unclear if all monocyte subsets exhibit osteoclastogenic potential. Furthermore, the role of lineage determining TFs in regulating OC differentiation on a genome-wide scale remains poorly understood. In this study, we utilized a novel Irf8 conditional knockout (Irf8 cKO) mouse model to characterize the importance of IRF8 in OC progenitor development and epigenetic regulation of OC differentiation. To identify global transcriptional program governing the osteoclastogenic potential of Ly6Chi, Ly6Cint, and Ly6C– monocytes, and how it may be further influenced by IRF8 deficiency, we performed RNA-seq on sorted Ly6Chi, Ly6Cint, and Ly6C– cells from WT and Irf8 cKO mice. Cells were analyzed prior to (BMMs) and 4 days after RANKL stimulation (OCs). Here we show that WT Ly6Chi and Ly6Cint monocytes developmentally contain OC-specific transcripts, which are augmented upon RANKL stimulation. Additionally, in the absence of IRF8, OC-specific transcripts in all three monocyte subsets are primed to robustly respond to RANKL stimulation.
Project description:Currently, the epigenetic mechanisms governing the osteoclast differentiation process remains poorly understood. To investigate the combinatorial activity of TFs, cis-regulatory elements, and the enhancer landscape that establish and maintain osteoclast transcriptional identity, we profiled histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) and PU.1 and IRF8 binding by ChIP-seq. RNA-seq data from sorted Ly6Chi, Ly6Cint, and Ly6C– cells were overlaid with ChIP-seq data to identify epigenetic changes specific to each subset and genotype. Our data suggest that the Ly6Chi inflammatory monocytes and Ly6Cint cells are transcriptionally and epigenetically programmed to differentiate in mature osteoclasts when compared to Ly6C– anti-inflammatory monocytes. A combinatorial activity of TFs and cis-regulatory elements in the vicinity of several osteoclast-specific genes are responsible for establishing and maintaining osteoclast identity in these subsets. In Irf8 cKO mice, several promoters and enhancers established during the progenitor stage may be critical for initiating early lineage commitment and priming monocyte subsets to differentiate into robust osteoclasts.
Project description:Gamma-delta (gd) T cells from pooled mouse lymph nodes and spleens were isolated and FACS-sorted for Vg1+Ly6C-, Vg1+Ly6C+, Vg4+Ly6C- and Vg4+Ly6C+ subsets of bulk CD27+ gdT cells.
Project description:Monocytes and their lineage descendants serve as a central defense system against infection and injury but if uncontrolled can also promote an excessive pathological inflammatory response. Therefore a current research goal is to understand how the organism controls the number and function of monocytes and how these variables can be tailored in therapy. Considering the evidence that monocytes are heterogeneous and exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. Here we investigate the differential expression of mRNA among murine steady-state monocyte subsets. Blood was drawn from healthy C57BL/6 donors. For each biological replicate 1000 monocytes of the Ly-6Chi and Ly-6Clo phenotype were isolated from the blood sample through fluorescence activated cell sorting.