Project description:Amyloidogenic peptides are therapeutic in EAE, reducing systemic levels of IL6, TNF, and INFg. The fibrils are bound and endocytosed in peritoneal MFs and B1a cells. Differential gene expression was used to further understand the process. Groups of three female C57BL/6 mice were injected with 10ug of Tau 623-628, Amylin 28-33, LPS, or PBS. 30-40 minutes post injection, the peritoneal cavity was lavaged, CD11b high (MFs) and CD5+CD19+ (B1a lymphocytes) were purified by flow cytometry directly into Trizol. RNA was extracted from the aqueous phase using the Qiagen RNeasy microkits. RNA quality was assessed using the Agilent 2100 bioanalyzer and the RNA 6000 nano reagents kit. One color microarray, Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray, was performed at the human immune monitoring center. All samples were processed and run at the same time.
Project description:Amyloidogenic peptides are therapeutic in EAE, reducing systemic levels of IL6, TNF, and INFg. The fibrils are bound and endocytosed in peritoneal MFs and B1a cells. Differential gene expression was used to further understand the process.
Project description:Gene expression profile of LysMCre/Cre and KLF2∆/∆ primary peritoneal macrophages following 6 hours of LPS treatment. We used microarrays to detail the global program of gene expression following LPS stimulation of LysMCre/Cre and KLF2∆/∆ primary peritoneal macrophages. We identified distinct classes of genes that were altered following LPS stimulation.
Project description:Mouse peritoneal B1a cells were classified into two groups based upon the expression level of PC1. One is PC1 high group and the other is PC1 low. To evaluate gene expression patterns that distinguished PC1 high expressing B1a cells from PC1 low expressing B1a cells, we used Affymetrix GeneChip® Mouse gene 1.0 ST Array.
Project description:Analysis of gene expression in explanted peritoneal macrophages from Aoah -/- and Aoah +/+ mice treated with LPS 21 days prior to harvest. Explanted peritoneal machrophages were challenged with LPS or control (PBS). The study seeks to characterize global gene expression in the state of prolonged LPS tolerance induced in mice lacking the LPS-inactivating enzyme Aoah. Groups of 9 C57BL/6 Aoah+/+ or Aoah-/- mice were given i.p. injections of 10 µg E. coli LPS/mouse. Twenty-one days later (when Aoah-/- mice remain tolerant and Aoah+/+ mice have recovered), peritoneal macrophages were harvested and the yields from three mice were pooled to form 3 samples per group (i.e., three samples of Aoah+/+ and Aoah-/- mice, with each sample comprised of peritoneal macrophages from 3 mice). Next day, cells were challenged with LPS or PBS and whole RNA was isolated 2 hours later and used for microarray experiments.
Project description:We have identified a number of miRNAs that are differentially expressed in LPS treated mouse peritoneal macrophages, as compared to untreated cells Peritoneal macrophages treated with LPS for 0, 4, 12, 24h. RNA was isolated and miRNA array assays were performed by Exiqon (miRCURY™ LNA Array version 10.0)
Project description:Analysis of gene expression in explanted peritoneal macrophages from Aoah -/- and Aoah +/+ mice treated with LPS 21 days prior to harvest. Explanted peritoneal machrophages were challenged with LPS or control (PBS). The study seeks to characterize global gene expression in the state of prolonged LPS tolerance induced in mice lacking the LPS-inactivating enzyme Aoah.
Project description:Gene expression profile of LysMCre/Cre and KLF2?/? primary peritoneal macrophages following 6 hours of LPS treatment. We used microarrays to detail the global program of gene expression following LPS stimulation of LysMCre/Cre and KLF2?/? primary peritoneal macrophages. We identified distinct classes of genes that were altered following LPS stimulation. We injected 3 pairs of LysMCre/Cre (n=3) and KLF2?/? (n=3) mice with 2 ml of thioglycolate broth intraperitoneally. After 72 hours of thioglycolate injection, total cell suspension from peritoneal cavity were collected. These cells were seeded on 100mm tissue culture plates. Cells were allowed to attach for 4 hours and unattached cells were removed by washing with cell culture medium. These cells were placed in humidified incubator for additional 24 hours before LPS treatment. LPS was diluted to final concentration of 100ng/ml in culture medium and this medium was placed on peritoneal macrophages derived from LysM Cre/Cre and KLF2?/? mice for 6hours. Cells were lysed and total RNA was isolated and subjected to hybridization on Affymetrix microarrays.
Project description:To determine the underlying mechanisms by which trametinib affected LPS-induced inflammatory response in macrophages, we performed microarrays to define the global gene expression in murine peritoneal macrophages treating with trametinib or vehicle followed by LPS stimulation.
Project description:Purpose: Previous work has demonstrated that miR-33 is an important regulator of lipid metabolism and atherogenesis. By performing bone marrow transplant experiments into LDLR-/- mice, our work demonstrates that the effects of miR-33 in macrophages play a major role in its ability to reduced atherosclerotic plaque burdon. To have performed extensive additional characterization of the effects of miR-33 deficiency in macrophages icluding RNA-seq analysis of peritoneal macrophages from wildtype, miR-33-/-, LDLR-/-, and miR33-/-/LDLR-/- animals. Methods: Thioglycolate elicited peritoneal macrophages from WT and miR-33-/- mice were harvested by peritoneal lavage. Cells were then plated for 2hr, then washed to remove non-adherant cells. Macrophages were then scraped, pelleted and frozen at -80◦C. Total RNA from WT and miR-33-/- thioglycollate-elicited peritoneal macrophages was extracted and purified using a RNA isolation Kit (Qiagen) followed by DNAse treatment to remove genomic contamination using RNA MinElute Cleanup (Qiagen). The purity and integrity of total RNA sample was verified using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). rRNA was depleted from RNA samples using Ribo-Zero rRNA Removal Kit (Illumina). RNA libraries from WT BMDMs were performed TrueSeq Small RNA Library preparation (Illumina) and were sequenced for 45 cycles on Illumina HiSeq 2000 platformm (1 x 75bp read length). The reads obtained from the sequencer are trimmed for quality using in-house developed scripts. The trimmed reads are aligned to the reference genome using TopHat2. The transcript abundances and differences calculated using cuffdiff. The results were plotted using R and cummeRbund using in-house developed scripts. Results: Our RNA-seq analysis has allowed us to identify genes and pathways that are altered in miR-33 deficient peritoneal macrophages under hyperlipidemic conditions (LDLR-/- vs. miR33-/-/LDLR-/-). Further analysis of gene expression changes that occur between wildtype and LDLR-/- animals has allowed us to identify which of these changes are likely due to differences in lipid loading and which are independent of these effects.
