Danger signal initiates differentiation of monocytes into osteopontin‑producing macrophages III
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ABSTRACT: Purpose: We report the application of single-cell RNA sequencing for profiling the genexpression of different monocyte types in order to characterize monocytes that differentiate into macrophages under danger signal in comparison to conventional GM-CSF and M-CSF macrophages Methods: Monocytes freshly isolated from PBMCs were differentiated into GM-CSF, M-CSF, and danger-signal induced macrophages. Two million cells of each macrophage population were tagged accordingly with TotalSeq™‑B Hashtags. Single cell RNA sequencing was performed on all macrophage populations in order to characterize cells from each population. ScRNAseq analysis was performed with the R package Seurat on 16470 barcodes after filtering. Results: Cluster analysis reveals distinct separation of all three macrophage populations. Conclusion: The clear difference in overall gene expression profile suggests that macrophages exposed to danger signal differentiate into a novel macrophage type in contrast to conventional GM-CSF and M-CSF monocytes.
Project description:Monocytes can differentiate into macrophages or dendritic cells. When treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) monocytes differentiate into macrophage-like cells. Here, we report that pharmacological blockade of the nuclear receptor PPARγ in monocytes turns GM-CSF into a potent inducer of dendritic cell (Mo-DC) differentiation. Remarkably, simultaneous blockade of PPARγ and mTORC1 in the presence of GM-CSF promoted the differentiation of Mo-DCs with a stronger phenotypic stability and immunogenic profile when compared with canonical Mo-DCs differentiated by treatment with GM-CSF and IL-4. Moreover, and in contrast with the observations made with GM-CSF and IL-4, blockade of PPARγ and mTORC1 was shown to be able to induce the differentiation of monocyte-derived macrophages (Mo-Macs) into Mo-DCs. Transcriptional profiling performed at either early time points, as well as at the end of the differentiation process, revealed marked differences in the gene expression signature between Mo-DCs induced by GM-CSF and IL-4 and Mo-DCs induced by GM-CSF in the presence of PPARγ and/or mTORC1 inhibitors, thus suggesting diverging differentiation pathways. Our observations might contribute, not only to a better understanding of the mechanisms involved in Mo-DCs differentiation but also to improving the efficacy of both, DC vaccines and therapies focusing on the modulation of myeloid cell functions.
Project description:Caspases, which are key effectors of apoptosis, have demonstrated non-apoptotic functions. One of these functions is the differentiation into macrophages of peripheral blood monocytes exposed to Colony-Stimulating Factor-1 (CSF1). Conversely, GM-CSF induces the differentiation of monocytes into macrophages in a caspase-independent manner. Macrophages generated by CSF1 and GM-CSF have distinct polarity. Macrophage polarization plays an important role in the pathogenesis of diverse human diseases as cancer, leading us to explore if caspase inhibition would affect macrophage polarization. To explore the role of caspases in CSF1 differentiation, we used human monocytes sorted from buffy coats treated by cytokines. We reported that caspase inhibition delays the ex vivo differentiation of peripheral blood monocytes exposed to CSF1 and modifies the phenotype of generated macrophages, e.g. cell shape, surface markers. Moreover, by RNAseq, we observed that the macrophages generated in presence of CSF1 and QVD are different from CSF1-treated monocytes and from GM-CSF-treated monocytes. Cell cycle and focal adhesion-related pathway genes were selectively down-regulated. This study confirms the importance of caspase activation in CSF1 differentiation.
Project description:Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels inhibit many lipopolysaccharide (LPS) -elicited macrophage inflammatory responses, the effects of elevated cAMP on macrophage differentiation are not well understood. We have differentiated monocytes to macrophages in the presence of GM-CSF or GM-CSF + FSK to elevate cAMP levels and determine its effects on differentiation. Microarray analysis was performed separately on samples from 3 human donors: 3 control and 3 forskolin-treated samples.
Project description:Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels inhibit many lipopolysaccharide (LPS) -elicited macrophage inflammatory responses, the effects of elevated cAMP on macrophage differentiation are not well understood. We have differentiated monocytes to macrophages in the presence of GM-CSF or GM-CSF + FSK to elevate cAMP levels and determine its effects on differentiation.
Project description:Human and murine studies showed that granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaM). We used microarray technology and functional assays to characterize GMaM in vitro and used a mouse model of colitis to study GMaM functions in vivo. Peripheral blood monocytes where cultured 16 h with media containing AB serum (control monocytes) or media containing 10 ng/mL GM-CSF and AB serum (GM-CSF activated monocytes). In three independent experiments, total RNA from GMaM and control monocytes was isolated and processed for microarray hybridization.
Project description:<p>Macrophages play a critical role in the inflammatory response and tumor development. Macrophages are primarily divided into pro-inflammatory M1-like and anti-inflammatory M2-like macrophages based on their activation status and functions. <em>In vitro</em> macrophage models could be derived from mouse bone marrow cells stimulated with two types of differentiation factors: GM-CSF (GM-BMDMs) and M-CSF (M-BMDMs), to represent M1-and M2-like macrophages, respectively. Since macrophage differentiation requires coordinated metabolic reprogramming and transcriptional rewiring in order to fulfill their distinct roles, we combined both transcriptome and metabolome analysis, coupled with experimental validation, to gain insight into the metabolic status of GM-and M-BMDMs. The data revealed higher levels of the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and urea and ornithine production from arginine in GM-BMDMs, and a preference for glycolysis, fatty acid storage, bile acid metabolism, and citrulline and nitric oxide (NO) production from arginine in M-BMDMs. Correlation analysis with the proteomic data showed high consistency in the mRNA and protein levels of metabolic genes. Similar results were also obtained when compared to RNA-seq data of human monocyte derived macrophages from the GEO database. Furthermore, canonical macrophage functions such as inflammatory response and phagocytosis were tightly associated with the representative metabolic pathways. In the current study, we identified the core metabolites, metabolic genes, and functional terms of the two distinct mouse macrophage populations. We also distinguished the metabolic influences of the differentiation factors GM-CSF and M-CSF, and wish to provide valuable information for <em>in vitro</em> macrophage studies. </p>
Project description:Human and murine studies showed that granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaM). We used microarray technology and functional assays to characterize GMaM in vitro and used a mouse model of colitis to study GMaM functions in vivo.
Project description:This study aims to characterize the transcriptional profile of Granulocyte-macrophage colony-stimulating factor induced macrophages (GM-MÃ) and M-CSF macrophages (M-MÃ) and to investigate in situ a subset of genes and their products specific to each phenotype in human atherosclerosis plaques 18 RNG/MRC two-colour oligonucleotide microarrays (Le Brigand et al. 2006) were used to generate global mRNA expression profiles for GM-CSF-induced, M-CSF-induced, and peritoneal macrophages. The microarray experiments were conducted either as a common reference (peritoneal-like macrophages) design or using a direct design by hybridizing Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-MÃ) and CSF-induced human monocyte-derived macrophages(M-MÃ) on the same arrays
Project description:In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs, whereas STAT5 tetramer deficiency yields a macrophage phenotype. In the dextran sulfate sodium model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and supplementation with a sustained concentration of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. Thus, this study suggests a protective role of STAT5 tetramers in inflammatory bowel disease through the regulation of arginine metabolism. We previously reported that STAT5 tetramerization in monocytes is detrimental in autoimmune-mediated neuroinflammation. This study highlights the opposing role of STAT5 tetramers in inflammatory bowel disease.
Project description:Systemic GM-CSF promotes myelopoiesis and inflammation and GM-CSF blockade is being evaluated as treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is however required for monocytes to differentiate into alveolar macrophages (AM) that control alveolar homeostasis and dampen inflammation. By mapping cross-species AM development stages to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of proinflammatory macrophages. In a multi-center, open-label, randomized, controlled trial in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides rationale for further testing of inhaled GM-CSF as non-invasive treatment to improve alveolar gas exchange and simultaneously boost anti-viral immunity in COVID-19