Project description:The relative contribution of polarized macrophages to the maintenance of tolerance is unknown. We examined their roles by in vivo adoptive transfer immunotherapy of M0, M1 and M2a macrophages as pre-treatment of colitis. In other experiments, M2a macrophages were used as pre-treatment or treatment of established colitis followed by immunotherapy with nTreg cells. Survival, weight gain, tissue infiltration, iTreg and Th17 cell development, T cell activation, and the frequency of proinflammatory cytokines were used as outcome measurements. Pre-treatment with M2a but not M1 macrophages increased the development of iTreg and Th17 cells. M2a macrophages used as pre-treatment or in treatment of established colitis allowed for successful therapy with nTreg cells. M1 and M2a macrophages have distinct gene expression profiles. M0, M1 and M2a macrophages were cell sorted and were used to generate total RNA for each array set, which was labeled and hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips in accordance to the manufacturerâ??s protocol. Three sets of arrays were performed, and the results were averaged. The subset of probe sets whose expression increased or decreased by two-fold or more relative to M0 cells as a common standard was identified and used for further analysis.
Project description:In this study, we used human THP-1-derived macrophages as an immune cell model, and systematically performed glycoproteomics of three subtypes of macrophages by StrucGP.To study the N‑Glycoproteomic of them, the intact glycopeptides were first enriched and identified using triplicate mass spectrometry (MS)-based glycoproteomic approaches, then StrucGP was used for subsequent analysis. In total three subtypes of macrophages, these intact glycopeptides consist of 253 N-linked glycan structures at 652 unique glycosites from 372 N-glycoproteins, along with their PSM information. A total of 135, 163, and 201 N-glycan structures were identified from M0, M1, and M2 macrophages, respectively.
Project description:Bone marrow derived macrophages from 8-week-old C57BL/6 mice was stimulated ex vivo with or without LPS and IFN-γ for 24 hours. Each group has two replicates.
Project description:While naive BMDMs without further treatment are referred to as M0-like macrophages, which can be polarized to either M1-like or M2-like macrophages,To assess the consequence of PTEN binding on macrophages, we profiled the global transcriptome of M0-like BMDMs with and without PTEN treatment.
Project description:The relative contribution of polarized macrophages to the maintenance of tolerance is unknown. We examined their roles by in vivo adoptive transfer immunotherapy of M0, M1 and M2a macrophages as pre-treatment of colitis. In other experiments, M2a macrophages were used as pre-treatment or treatment of established colitis followed by immunotherapy with nTreg cells. Survival, weight gain, tissue infiltration, iTreg and Th17 cell development, T cell activation, and the frequency of proinflammatory cytokines were used as outcome measurements. Pre-treatment with M2a but not M1 macrophages increased the development of iTreg and Th17 cells. M2a macrophages used as pre-treatment or in treatment of established colitis allowed for successful therapy with nTreg cells. M1 and M2a macrophages have distinct gene expression profiles.
Project description:Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers and unusual lymphadenopathies, with infiltrating CD68+, S100+ and CD1a– histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically-activated (M1) and alternatively-activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and immunodeficiencies (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients. Moreover, higher levels of IFNg were found in the serum of both HS patients when compared to HC. Gene-ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms "type I IFN", "IFNg signalling pathway" and "immune responses" as the top 3 most significant terms for monocytes. Finally, the transcriptomic profiles of M0 and M1 macrophages, from patient HS2, were similar to the transcriptomic profile of tissue biopsies taken from patients with RDD-like lymphadenopathies. Monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and with a unique dysregulated IFNgsignalling. These findings may help to find better therapeutic options for this rare disorder.
Project description:Macrophages are effector cells of the innate immune system and undergo phenotypical changes in response to organ injury and repair. They are most often classified as proinflammatory M1 and anti-inflammatory M2 macrophages. Protein arginine deiminase (PAD) enzymes, that catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification, are expressed in macrophages. However, the substrate scope of the PADs and their role in the immune cells remain not well defined. This study aims to investigate the role of PADs in the THP-1 macrophage polarization to M1 and M2 phenotype and identify the citrullinated proteins, and the modified Arg that are associated with this biological switch using mass spectrometry. Our study showed that PAD2, and to a lesser extent PAD1 and PAD4 were dominantly expressed in M1 macrophages. We showed that inhibiting PADs by BB-Cl-amidine decreased the macrophage’s polarization to M1 and increased to M2 phenotype. The process was mediated by the downregulation of proteins involved in NF-kβ pathway. Silencing of PAD2 confirmed activation to M2 macrophages through upregulation of the antiviral innate immune response and interferon signaling. 192 novel citrullination sites that belong to inflammation, cell death and DNA/RNA processing pathways were identified in M1 and M2 macrophages.
Project description:In order to assess whether differences in the transcriptomic profile of human tumor-associated macrophages vs. alveolar macrophages from adjacent non-tumor-tissue (GSE162669) were caused by factors secreted by tumor cells, primary human monocyte-derived macrophages (MDMs) were polarized towards a TAM-like phenotype by cultivating them in tumor cell-conditioned medium. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro polarized TAM-like macrophages. Other polarization schemes (M1: LPS/IFN-gamma, M2: IL10 or IL4) did not lead to similar transcriptomic changes. For details, see Hoppstädter et al., 2021 (doi:10.1016/j.ebiom.2021.103578).
Project description:Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Although the importance of glycolytic and oxidative metabolism in M1 and M2 macrophages, respectively, is well established, our knowledge of metabolic checkpoints controlling these effector states is limited. Here we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the conversion of cytosolic pyruvate to mitochondrial acetyl-CoA by pyruvate dehydrogenase, functions as a metabolic checkpoint in M1 macrophages. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). The therapeutic potential of attenuation of pro-inflammatory responses by PDK inhibition was tested, both genetically and pharmacologically, in obesity-induced insulin resistance, a disease process in which M1 macrophages contribute to adipose tissue inflammation and insulin resistance. Taken together, these studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages.
Project description:Unstimulated (M0), M1-polarized (GM-CSF, LPS, IFNγ-stimulated), and M2-polarized (M-CSF, IL-4-stimulated) canine blood-derived macrophages were generated in vitro and investigated for differences in their transcriptome to create a basis for future investigations upon the role of macrophage polarization in dogs, a species, which has emerging importance for translational research.