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: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:TLR ligands consistently induce expression of two Hes family members Hes1 and Hey1 in macrophages.To evaluate the effects of these two factors on inflammatory responses, we generated mice lacking both Hes1 and Hey1 (DKO). WT and DKO BMDMs were then untreated or exposed to LPS for 3 hours, and microarray was performed to examine global gene expression profiles to identify Hes1 and Hey1-regulated inflammatory genes Examination of Hes1 and Hey1-regulated inflammatory genes in macrophages
Project description:We reported exosome-guided phenotype switches between M1- and M2-polarized BMDMs. M1- or M2-polarized BMDMs were successfully reprogrammed to M2- or M1-phenotype via the treatment of exosomes obtained from M2- or M1-polarized BMDMs. In this uploaded information, the exosomes from M1- and M2-polarized BMDMs were analyzed by high-throughput sequencing.
Project description:We report the application of sequencing technology for high-throughput profiling of mRNA m6A methylation in mice BMDMs with or without PBS treatment. By obtaining over thirty billion bases of sequence from m6A antibody immunoprecipitated mRNA, we generated m6A methylation maps of mouse BMDMs in the presence or absence of LPS. We find that SOCS1 mRNA methylation is robustly enhanced by LPS treatment in WT BMDMs, leading to the increase of SOCS1 expression. However, LPS challenge fails to increase SOCS1 expression in the M14-/- BMDMs, indicating m6A modification plays a cirital role in the SOCS1 expression. This study provides a framework for the application of comprehensive mRNA m6A profiling towards characterization of BMDMs.
Project description:TLR ligands consistently induce expression of two Hes family members Hes1 and Hey1 in macrophages.To evaluate the effects of these two factors on inflammatory responses, we generated mice lacking both Hes1 and Hey1 (DKO). WT and DKO BMDMs were then untreated or exposed to LPS for 3 hours, and microarray was performed to examine global gene expression profiles to identify Hes1 and Hey1-regulated inflammatory genes
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.
Project description:Bone marrow cells from Baf60a KO or WT mice were differentiated into BMDMs, followed by RNA extraction and sequencing at the University of Michigan AGC.
