Project description:Identification of genes differentially expressed between human CD14+CD16- and CD16+ monocyte-derived macrophages generated in the presence of either GM-CSF (termed GM14 and GM16, respectively) or M-CSF (termed M14 and M16, respectively)
Project description:Identification of genes differentially expressed between human CD14+CD16- and CD16+ monocyte-derived macrophages generated in the presence of either GM-CSF (termed GM14 and GM16, respectively) or M-CSF (termed M14 and M16, respectively) Human peripheral CD14+CD16- and CD16+ blood monocytes from three independent healthy donors (D1, D2 and D3) were isolated by positive selection from peripheral blood mononuclear cells (PBMC) using magnetic separation systems (MACS, Miltenyi Biotec). Briefly, PBMC were first incubated with MACS anti-CD56 antibody conjugated to paramagnetic microbeads in order to eliminate the NK (CD16+) cell fraction. NK-depleted PBMC were further incubated with MACS anti-CD16 antibody to isolate CD16+ monocytes. CD56-CD16- PBMC were finally incubated with MACS anti-CD14 antibody to obtain the CD14+CD16- monocyte fraction. Monocytes were cultured for 7 days in medium containing either GM-CSF or M-CSF. Total RNA from each condition was extracted using the RNeasy kit (Qiagen) and hybridized to an Agilent Human Whole Genome (4x44) Oligo Microarray. All experimental procedures were performed following manufacturer instructions.
Project description:Comparison of the transcriptome of CD14+ human monocytes and CD14+ human monocyte-derived macrophages generated in the presence of M-CSF (M-MØ) or GM-CSF (GM-MØ).
Project description:Comparison of the transcriptome macrophages derived from CD14+ human monocyte-derived macrophages generated in the presence of M-CSF (M-Mphage) or GM-CSF (GM-Mphage) and MTX.
Project description:Macrophage phenotypic and functional heterogeneity derives from tissue-specific transcriptional signatures shaped by the local microenvironment. GM-CSF drives the generation of human monocyte-derived macrophages with a potent pro-inflammatory activity upon stimulation. One-carbon metabolism (OCM) is a complex network of biosynthetic pathways that includes de novo biosynthesis of purines and thymidylate, amino acid metabolism, and methylation reactions. We explored the molecular impact of blocking OCM with the anti-folate pemetrexed (PMX) on the gene expression profile in GM-CSF-primed human monocyte derived macrophages.
Project description:Macrophage phenotypic and functional heterogeneity derives from tissue-specific transcriptional signatures shaped by the local microenvironment. GM-CSF drives the generation of human monocyte-derived macrophages with a potent pro-inflammatory activity upon stimulation. Janus Kinase (JAK) inhibitors are small molecules that reversibly inhibit JAK activity and their subsequent intracellular signaling and have become the treatment of choice for diseases with an inflammatory or immune basis. We explored the molecular impact of blocking GM-CSFR-JAK2-STAT5 axis with the Janus Kinase (JAK) inhibitors Upadacitinib or Baricitinib on the gene expression profile in GM-CSF-primed human monocyte derived macrophages.
Project description:Human monocyte-derived macrophages (MDM) from HDM-allergic donors were compared to MDM from healthy donors. CD14+ monocytes were isolated from donated blood and differentiated in the presence of GM-CSF and TGFb (alveolar-like MDM) for 7 days before havest of RNA
Project description:Monocyte maturation program into macrophages (MΦ) is well-defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicate in vitro the human monocyte “waterfall” process by first generating tissue-like inflammatory monocytes, and then shifted them towards MΦ. Purified CD14+CD16- monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNg and IL23 differentiated into CD14+CD163- cells displayed a monocyte-like morphology. The in vitro generated inflammatory CD14+CD163- cells (Infl mo-like), like the CD14+CD163- mo-like cells that accumulate in inflamed colon of Crohn’s disease patients, promoted IL-1β-dependent memory Th17 and Th17/Th1 responses. Next, in vitro generated Infl mo-like cells converted to functional CD163+ MΦ following exposure to TGFβ and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163+ MΦ and MΦ detected in various inflamed non-lymphoid and lymphoid diseased tissues. Our findings propose a two-step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulating this process might open therapeutic avenues for chronic inflammatory disorders. Classical CD14+CD16- human monocytes were sorted and cultured for 6 days with GM-CSF+IFNg+IL23, leading to the generation of CD163- d6 cells. Following the 6 days culture with GM-CSF+IFNg+IL23, TGFβ+IL10 were added for another 6 days. This gave rise to the CD163+ d12 and CD163- d12 populations. We used microarray (Clariom D, Affymetrix) to observe molecular differences in the 3 in vitro generated populations: (1) CD163- d6 (2) CD163+ d12 (3) CD163- d12.
