Genome wide transcriptional analysis of tissue macrophages and bone marrow derived macrophages (BMDMs)
ABSTRACT: Tissue macrophages from peritoneal cavity, lung, liver, spleen, small intestine and adipose tissue and M-CSF derived bone marrow derived macrophages (BMDMs) were determined for gene expression. Overall design: Macrophages from six different tissues and BMDMs were compared for gene expression.
Project description:Tissue macrophages from peritoneal cavity, lung, liver, spleen, small intestine and adipose tissue and M-CSF derived bone marrow derived macrophages (BMDMs) were determined for gene expression. Macrophages from six different tissues and BMDMs were compared for gene expression.
Project description:Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing I?B? phosphorylation and inhibiting expression of TNF?, CCL2, interleukin-1?, and interleukin-6. The activity of EI-tPA was replicated by activated ?2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.
Project description:Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.
Project description:Primary murine bone marrow derived macrophages (BMDMs) of Setdb2GT/GT and WT mice were stimulated with polyI:C and harvested at 0, 2 and 8h. Everything with biological triplicates (=cells from 3 individual mice per condition)
Project description:Alpk1-deficient mice demonstrate exacerbated colitis and increased IL-12/Th1 response upon challenge with an intestinal pathobiont, Helicobacter hepaticus (Hh). Hematopoietic compartment is driving the pathogenic phenotype in this animal model, and Alpk1 is highly expressed in myeloid cells (macrophages and dendritic cells). Alpk1 deficiency has a recessive phenotype, since Alpk1+/- (heterozygous) mice show the same phenotype as the wild type mice. Mouse bone-marrow derived macrophages (BMDMs) show elevated IL-12 production in Alpk1-/- mice in response to stimulation with Hh. Since the molecular mechanism of how Alpk1 deficiency affects macrophage response to Hh is unknown, we aimed to characterise global changes in gene expression in Alpk1+/- vs Alpk1-/- bone-marrow differentiated cells (BMDMs). Cells were isolated from bone marrow of Alpk1+/- and Alpk1-/- (mixture of bone marrows from three mice per genotype) and plated in BMDM differentiation medium (RPMI, 10% FCS, penicillin and streptomycin, 50 micro beta-mercapthoethanol, 20 ng/ml recombinant mouse GM-CSF(Peprotech)), 7 million cells in per 10 sm uncoated TC dish in 10 ml of medium for eight days, extra 10 ml of medium was added to plates at day 4, before collection and replating in 96-well plates, 150 thousand cells/200 microliters of differentiation medium per well in technical triplicates per genotype/stimulation condition (R1-R3 labels of the samples). The following day BMDMs were stimulated with MOI of 10 of Hh and 10ng/ml of mouse IFNg (Peprotech) (Alpk1+/- BMDMs – het _Hh_8h vs Alpk1-/- BMDMs – alpk1_Hh_8h) or IFNg only (het _nonstim_8h vs alpk1_nonstim_8h) before lysis for RNA extraction using Quick-RNA kit from Zymo Research. Purified RNA was submitted to the Welcome Trust Centre for Human Genetics (Oxford) for RNA-Sequencing
Project description:To recruit phagocytes, apoptotic cells characteristically release ATP, which functions as a “danger” signal. Here, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as NR4A and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into AdoR A2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a “calm down” signal. If apoptotic cells produce “danger” or “anti-danger” signal(s), we rationalized that such signals would activate gene expression in macrophages. To investigate this possibility, we examined the effect of the culture supernatant from apoptotic cells on macrophage gene expression by using microarrays. For mouse BMDMs, bone marrow cells from female C57BL/6J mice at 8 weeks of age were cultured for more than 7 days with DMEM containing 10% FCS supplemented with mouse M-CSF. We used adherent cells as BMDMs in the study. W3 cells, mouse T cell line expressing Fas, were treated with human Fas ligand at 37°C for 30 min to induce apoptosis. The cells were then washed and re-suspended at a concentration of 1 × 107 cells/ml with RPMI containing 1% FCS, and further incubated for 60 min at 37°C. Following Fas ligand treatment, more than 90% of the W3 cells were Annexin V positive, and only small percentage were positive for both Annexin V and propidium iodide (PI). The culture supernatant was collected from apoptotic W3 cells. Next, BMDMs were incubated with medium (BMDMs-Medium) or apoptotic W3 cell supernatant (BMDMs-Apoptotic cell supernatant) for 1 h. Total RNA was extracted from the cells and hybridized on Affymetrix microarrays.
Project description:The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B2, prostaglandin (PG) E2, and PGD2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE2/cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.
Project description:Identification of pro- and anti-inflammatory pathways induced in M-CSF differentiated bone-marrow derived macrophages (BMDMs) after 3 h stimulation with two different TLR2 agonists, Helicobacter hepaticus polysacharide and Pam3CSK4 (75 ng/ml), using TSB (Tryptone Soya Broth) medium as a control
Project description:We report that cavity macrophages from the pericardial, pleural and peritoneal cavities have distinct expression profile from cardiac macrophages Overall design: Comparison of sorted macrophages from the pericardial cavity, pleural cavity, peritoneal cavity, and heart under baseline conditions. 4 replicates per cavity or tissue were included.
Project description:Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE-/- mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206<sup>high</sup>CD11c<sup>low</sup> profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206<sup>high</sup>CD11c<sup>low</sup> macrophages in advanced versus early atherosclerotic disease in ApoE-/- mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes.