Project description:To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites Trypanosoma cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen Leishmania mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. Trypanosoma cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. Keywords: Bone marrow macrophage response to intracellular parasites and cytokines We analyzed a series MEEBO arrays on which were hybed RNA amplified from bone marrow-derived macrophages from C57BL/6 mice. Macrophages infected with L. mexicana or T. cruzi or stimulated by LPS, IFNG, IL-4, IL-10, TNF, IFNB, or IL-17 were compared to one another as well as to uninfected, unstimulated control macrophages. All experiments were performed over a 24 h timecourse with timepoints taken at 2 h, 6 h, 12 h, and 24 h.
Project description:Candida auris has been globally recognized as a multidrug-resistant human fungal pathogen that contributes for the worldwide occurrence of nosocomial outbreaks. It has been reported that C. auris was able to avoid neutrophil attack, suggestive of an impaired innate immune response. Whether C. auris evades the innate immune recognition of BMDM (bone marrow derived macrophage) remains poorly understood, and as for well-known Candida species -C. albicans, it can trigger immune response. To determine whether occurs difference between immune response stimulated by C. auris or C. albicans, we performed mRNA-seq of BMDM stimulated by C. auris or C. albicans.
Project description:Mouse bone marrow-derived macrophages (BMDM) grown in macrophage colony-stimulating factor (CSF-1) have been used widely in studies of macrophage biology and the response to toll-like receptor agonists. We investigated whether similar cells could be derived from the domestic pig. Cultivation of pig bone marrow cells for 5-7 days in presence of rhCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, CD163, CD172a), are potent phagocytic cells and produced tumor necrosis factor (TNF) in response to lipopolysaccharide (LPS). Bone marrow cells could be stored frozen and thawed, providing a renewable resource. We profiled gene expression in pig BMDM from outbred animals (Large-White Landrace F1cross) responding to LPS using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely-resembled human than mouse macrophages, and included genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20) and the vitamin D3-converting enzyme Cyp27B1. Conversely, pig BMDM, like human macrophages, did not induce genes involved in arginine metabolism, nor did they produce nitric oxide. The data establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control. RNA for gene expression analysis was collected at time points 0, 2, 7 and 24 hours post LPS stimulation (100ng/ml). Each time point included BMDM from the same three pigs and each cell culture was replicated. The replicate of the pig3_24h was not suitable for RNA analysis. Therefore, a total of 23 microarrays were hybridized.
Project description:Mouse bone marrow-derived macrophages (BMDM) grown in macrophage colony-stimulating factor (CSF-1) have been used widely in studies of macrophage biology and the response to toll-like receptor agonists. We investigated whether similar cells could be derived from the domestic pig. Cultivation of pig bone marrow cells for 5-7 days in presence of rhCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, CD163, CD172a), are potent phagocytic cells and produced tumor necrosis factor (TNF) in response to lipopolysaccharide (LPS). Bone marrow cells could be stored frozen and thawed, providing a renewable resource. We profiled gene expression in pig BMDM from outbred animals (Large-White Landrace F1cross) responding to LPS using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely-resembled human than mouse macrophages, and included genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20) and the vitamin D3-converting enzyme Cyp27B1. Conversely, pig BMDM, like human macrophages, did not induce genes involved in arginine metabolism, nor did they produce nitric oxide. The data establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.
Project description:Immortalised cell lines analogous to their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here we report that immortalisation of bone marrow-derived macrophages using the J2-virus resulted in the loss of a protein of interest, MSR1, in wild-type cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry-based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), with comparison to the immortalised bone marrow-derived macrophages (iBMDMs), as well as primary BMDMs. This revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and IFN signalling. J774A.1 cells were determined to be the most similar to the gold standard primary BMDM model, with BMA3.1A7 cells the least similar due to the reduction in abundance of several proteins related closely to macrophage function. This comprehensive proteomic data offers valuable insights into the selection of specific macrophage cell lines for cell signalling and inflammation research.
Project description:To get insight into TRIM33 functions, TRIM33 ChIP-seq was carried out in murine macrophage cell line (RAW) and in bone marrow-derived macrophages (BMDM). The results showed that, in addition to its role in hematopoietic differentiation, TRIM33 may modulate PU.1 transcriptional activity during macrophage development and/or activation.To characterize the role of TRIM33 in macrophages, we bred TRIM33fl/fl mice with Lyz-Cre mice where the Cre recombinase gene is under the regulatory sequences of the Lyz gene that is expressed only in mature myeloid cells. Bone marrow cells from LyzCre/Trim33+/+ mice and LyzCre/Trim33flox/flox mice were differentiated in macrophages and treated during 0h, 4h, 12h and 24h with LPS. Using ChIP-seq, we provide a link between TRIM33 binding and H3K4me3 spreading on inflammatory genes in macrophages. Chromatin immunoprecipitations of TRIM33 and H3K4Me3 followed by multiparallel sequencing performed in murine bone marrow-derived macrophages (BMDM).
Project description:To reveal the molecular mechanisms underlying DRP1-mediated macrophage reprogramming during hypoxia serum starvation (HSS) an in vitro ischemia model, we performed bulk RNA-seq analysis on mouse bone marrow derived macrophages (BMDM) upon HSS-induced DRP1 mediated macrophage reprogramming.