Transcriptional profiling of Regulatory T cells isolated from mice exposed to Smoke, Morphine and Smoke plus Morphine
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ABSTRACT: Purpose: The goals of this study were to identify gene expression signatures that are unique to Tregs from mice upon treatments with smoke, morphine and smoke plus morphine Methods and results: Wild-type mice were treated with cigarette smoke (SM) for a period of eight weeks, and/or the chronic continuous administration of morphine (M) via mini-pumps for the final four weeks CD4+CD25highCD127low Tregs from spleen were purified by cell sorting to generate mRNA transcription. Transcriptional profiling revealed a unique Treg signatures in SM, M or SM + M relative to control mice. Conclusion: Our study represents the first detailed analysis of splenic Treg transcriptome in mice exposure to smoke plus morphine.
Project description:Critically ill preterm infants experience multiple stressors while hospitalized. Morphine is commonly prescribed to ameliorate their pain and stress. We hypothesized that neonatal stress will have a dose-dependent effect on hippocampal gene expression, and these effects will be altered by morphine treatment. Male C57BL/6 mice were exposed to 5 treatment conditions between postnatal day 5 and 9: 1) Control, 2) mild stress + saline, 3) mild stress + morphine, 4) severe stress + saline and 5) severe stress + morphine. Hippocampal RNA was extracted and analyzed using Affymetrix Mouse Gene 1.0 ST Arrays. Single gene analysis and gene set analysis were used to compare groups with validation by qPCR. Stress resulted in enrichment of genes sets related to fear response, oxygen carrying capacity and NMDA receptor synthesis. Morphine downregulated gene sets related to immune function. Stress plus morphine resulted in enrichment of mitochondrial electron transport gene sets, and down-regulation of gene sets related to brain development and growth. We conclude that neonatal stress alone influences hippocampal gene expression, morphine alters a subset of stress-related changes in gene expression and influences other gene sets. Stress plus morphine show interaction effects not present with either stimulus alone. These changes may alter neurodevelopment. Male mice were exposed to 5 treatment conditions between postnatal day (P)5 and P9 (n=3/group), with birth recorded as P1. Litters were culled to n=7 maximum per dam. Groups included: 1) Untreated controls (CC), 2) mild stress + saline (MSS), 3) mild stress + morphine (MSM), 4) severe stress + saline (SSS) and 5) severe stress + morphine (SSM).
Project description:Critically ill preterm infants experience multiple stressors while hospitalized. Morphine is commonly prescribed to ameliorate their pain and stress. We hypothesized that neonatal stress will have a dose-dependent effect on hippocampal gene expression, and these effects will be altered by morphine treatment. Male C57BL/6 mice were exposed to 5 treatment conditions between postnatal day 5 and 9: 1) Control, 2) mild stress + saline, 3) mild stress + morphine, 4) severe stress + saline and 5) severe stress + morphine. Hippocampal RNA was extracted and analyzed using Affymetrix Mouse Gene 1.0 ST Arrays. Single gene analysis and gene set analysis were used to compare groups with validation by qPCR. Stress resulted in enrichment of genes sets related to fear response, oxygen carrying capacity and NMDA receptor synthesis. Morphine downregulated gene sets related to immune function. Stress plus morphine resulted in enrichment of mitochondrial electron transport gene sets, and down-regulation of gene sets related to brain development and growth. We conclude that neonatal stress alone influences hippocampal gene expression, morphine alters a subset of stress-related changes in gene expression and influences other gene sets. Stress plus morphine show interaction effects not present with either stimulus alone. These changes may alter neurodevelopment.
Project description:Purpose: To identify differentially expressed genes regulated by morphine in mice. Methods: Mice were intrathecally injected with normal saline or morphine (10 ug/10 uL) for 7 days and then spinal cords L4-L5 were collected and subjected to mRNA-sequencing by geneseeq and data analysis. Results: We identified morphine upregulated 42 genes and downregulated 44 genes compared with saline group. KEGG enrichment analysis showed calcium signaling pathway was downregulated by morphine and we noticed that TRPC5 was one of the most significantly downregulated genes by morphine. We confirmed that intrathecal injection of morphine leads to Trpc5 transcriptional suppression via reducing GABA release, contributing to morphine tolerance.
Project description:We extracted RNA of sorted lung SPC/GFP+ EpCAM+ cells and performed microarray analyses. Total RNA was extracted from sorted Ep-CAMhigh/GFPhigh cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The RNA integrity was examined on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA). Biotinylated ss-cDNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA using the GeneChip WT PLUS Reagent Kit User Manual (Affymetrix/Thermo Fisher Scientific). Fragmented and labeled ss-cDNA were hybridized on a GeneChip Clariom S Array (Affymetrix/Thermo Fisher Scientific) (n =3/group). We used microarrays to detail the global gene expression of SPC/GFP+ EpCAM+ cells from the control mice(G37-1_(Clariom_S_Mouse)), 3 week continuous smoke mice (G37-2_(Clariom_S_Mouse)) and 3 weeks intermittent smoke mice(G37-3_(Clariom_S_Mouse)), and identified distinct classes of up-regulated genes during this process.
Project description:We investigated the role of DNMT1 in immune homeostasis by generating mice lacking DNMT1 in Foxp3+ T-regulatory (Treg) cells. These mice showed decreased peripheral Foxp3+ Tregs, complete loss of Foxp3+ Treg suppressive functions in vitro and in vivo, and died from autoimmunity by 3-4 weeks unless they received perinatal transfer of wild-type Tregs that prolonged their survival. Methylation of CpG-sites in the TSDR region of Foxp3 was unaffected by DNMT1 deletion, but microarray revealed more >500 proinflammatory and other genes were upregulated in DNMT1-/- Tregs. CD4-Cre-mediated DNMT1 deletion showed inability of conventional T cells to convert to Foxp3+ Treg under appropriate polarizing conditions. Hence, DNMT1 is absolutely necessary for maintenance of the gene program required for normal Treg development and function. RNA from three independent samples of magnetically separated CD4+CD25+ Treg of fl-DNMT1/Foxp3cre mice, compared to wild type (C57BL6) control
Project description:Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR gene expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine at 2 or 5 mg/kg ip bid (MS5) and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and microRNA expression profiling was performed on hippocampal tissues at P9. Overall, MS2 and MS5 morphine treatment altered expression of a total of 150 mRNAs (>1.5 fold change, P<0.05; 36 up, 114 down), and MS5 affected 63 mRNAs. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. MS5 decreased expression of miR-204-5p, miR-455-3p, miR-448-3p, and miR-574-3p.Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed microRNAs These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR gene expression in stressed neonatal mice. If permanent, morphine–mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.
Project description:Using an oral self-administration paradigm, we have seen morphine increase neural activity in the Paraventricular Nucleus of the Thalamus (PVT). Morphine binds primarily to Mu Opioid Receptors (MORs), which are highly expressed in the PVT. Translating Ribosome Affinity Purification (TRAP)-Sequencing was conducted on PVT neurons that express MORs
Project description:We isolated visceral adipose tissue (VAT) Tregs from Foxp3.YFP-Cre Bmal1WT or Foxp3.YFP-Cre bmal1flox mice fed a normal lean diet or a high-fat diet. VAT Tregs were also sorted after adoptive transfer. We found that Bmal1KO Tregs are more activated in lean mice, after 4 weeks HFD and after adoptive transfer, but loseVAT Treg signature after 16 weeks of high-fat diet feeding.