Murine Microglial Gene Expression after Abdominal Sepsis
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Benjamin Singer
PROVIDER: E-MTAB-4180 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Gut dysbiosis and host genetics are implicated as causative factors in inflammatory bowel disease, yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis, offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically-created blind self-filling (SFL) and self-emptying (SEL) ileal loops. SFL exhibit fecal stasis due to directional peristalsis motility oriented towards away from the loop end, whereas SEL remain empty. In wild type mice, SFL, but not SEL, develop pouch-like microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-/- deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. Germ-free IL10-/- mice conventionalized with wild type SFL, but not SEL, microbiota, develop severe colitis. These data demonstrate an essential role for fecal stasis, gut dysbiosis, and genetic susceptibility and offer insights into human pouchitis and ulcerative colitis. All animal protocols were approved by IACUC at the University of Chicago. All animals were C57Bl/6 mice that were bred and housed under standard 12:12 light/dark conditions at the University of Chicago. Female mice aged 6-8 weeks were fed ad libitum gel diet 76A (Cat# 72-07-5022, Clear H20, Portland, ME) for 5-days prior to surgery to prevent obstruction at the anastomosis. Animals were anesthetized with ketamine/xylazine. Aseptic surgery was performed to resect 2.5cm of ileum 3cm proximal to the ileal-cecal value with anastomosis to the ileum using 8-0 suture (Figure 1a). The abdominal wall was closed with interrupted 4-0 silk suture and skin was closed with staples. Analgesics (betanorphine mg/kg BW) were provided post-operatively. After 5 weeks, mice were humanely euthanized. Intestinal loops were collected for RNA, protein, and histology. Loop, sham ileum, and sham colon contents were collected and snap frozen at −80°C for microbiota analysis. Human biopsies and stool samples were obtained under IRB approval and privacy protocols were followed. Our initial work demonstrated up regulation of TLR4 signaling in the mucosa of self-filling ileal loops. We hypothesized that TLR4 may be in-part responsible for mediating the metaplasia and inflammatory responses observed. Therefore, TLR4 KO mice were used to test this hypothesis and subsequently demonstrated attenuated responses in these parameters.
2015-09-20 | E-GEOD-71997 | biostudies-arrayexpress
Project description:The study was designed in order to identify genes differentially expressed when glucocorticoid signaling is blocked by a glucocorticoid-receptor antagonist (RU486 â?? mifepristone) in the context of brain inflammation induced by bacterial lipopolysaccharide (LPS). LPS is only able to cause murine brain damage in our experimental conditions upon RU486 pre-treatment. Hence, the study may reveal potential candidate genes to mediate neuroprotection or neurotoxicity. Due to the factorial design of the experiment, RU486 main-effect could be dissociated from the effects resultant of RU486/inflammation interaction. In addition, brain dissection was conducted to verify the effects in the brain side ipsilateral or contralateral to the site of intracerebral LPS infusion. Experiment Overall Design: C57Bl/6 mice received an i.p. injection of vehicle (DMSO - 50 microliters) or RU486 (50 mg/kg) and were submitted to surgery 4 h later. The mice receiving intraparenchymal injections were anesthetized and the right caudate putamen was reached, using a small cannula at the coordinates 0.0 mm anteroposterior, -2.0 mm lateral, and -3.0 mm dorsoventral according to a mouse brain atlas. The animals received an infusion of sterile pyrogen-free saline (1 microliter) or LPS (from Eschericia coli; serotype O55:B5; Sigma L2880 - 2.5 micrograms) over 2 min by means of a microinjection 18 pump. Animals were killed 12 h after the intracerebral infusion. The mice were anesthetized under isofluorane and blood was drawn via cardiac puncture before head decapitation. Brains were removed rapidly from the skulls and placed in cold phosphate buffered saline (PBS) solution. A brain region limited at plane anteroposterior +1.5 to -1.5 and dorsoventral -4.0 was dissected, separated in ipsilateral side and quickly immersed in liquid nitrogen. The tissue was stored at -80 oC until RNA extraction was performed. A total of 37 chips (MOE430A â?? Affymetrix, Santa Clara, CA) were used for oligonucleotide array analysis [one chip per biological sample; 8 groups (contralateral dmso/saline, dmso/LPS, RU486/saline, RU486/LPS and ipsilateral dmso/saline, dmso/LPS, RU486/saline, RU486/LPS) with 4 â?? 6 biological replicates each]. Expression values from the CEL files generated from scanning were obtained using RMA algorithm, available at http://www.bioconductor.org. The expression values were also inspected with GeneSpring software (Silicon Genetics). Statistical analysis was performed considering a factorial linear model according to the methods implemented in Limma package (R project packages are available at http://www.cran.r-project.org).
2008-06-14 | E-GEOD-6509 | biostudies-arrayexpress
Project description:Background- Resistant starch is a prebiotic metabolized by the gut bacteria. It has been shown to attenuate chronic kidney disease (CKD) progression in rats. Previous studies employed taxonomic analysis using 16S rRNA sequencing and untargeted metabolomics profiling. Here we expand these studies by metaproteomics, gaining new insight into the host-microbiome interaction. Methods- Differences between cecum contents in CKD rats fed a diet containing resistant starch with those fed a diet containing digestible starch were examined by comparative metaproteomics analysis. Taxonomic information was obtained using unique protein sequences. Our methodology results in quantitative data covering both host and bacterial proteins. Results - 5,834 proteins were quantified, with 947 proteins originating from the host organism. Taxonomic information derived from metaproteomics data surpassed previous 16S RNA analysis, and reached species resolutions for moderately abundant taxonomic groups. In particular, the Ruminococcaceae family becomes well resolved – with butyrate producers and amylolytic species such as R. bromii clearly visible and significantly higher while fibrolytic species such as R. flavefaciens are significantly lower with resistant starch feeding. The observed changes in protein patterns are consistent with fiber-associated improvement in CKD phenotype. Several known host CKD-associated proteins and biomarkers of impaired kidney function were significantly reduced with resistant starch supplementation. Conclusions- Metaproteomics analysis of cecum contents of CKD rats with and without resistant starch supplementation reveals changes within gut microbiota at unprecedented resolution, providing both functional and taxonomic information. Proteins and organisms differentially abundant with RS supplementation point toward a shift from mucin degraders to butyrate producers.
2018-12-18 | PXD008845 | Pride
Project description:NF-kB is a transcriptional factor that consists in homo and heterodimers of the large family of Rel subunits. Among the most important functions for NF-kB, initiation of immunological/inflammatory responses and regulation of cell proliferation/apoptosis which are the major features of severe infections. Although the role of NF-kB is crucial in host defense against pathogens, mice deficient for individual subunits of NF-kB have not been explored in murine models of polymicrobial infection. In this report, we have investigated in vivo the consequences of cRel subunit deficiency in the survival to polymicrobial infection. We have also approached the underlying mechanisms of the host defense by analyzing cytokine production, bacterial clearance and the distribution of innate and adaptive immune cells. Absence of cRel enhances mice mortality to polymicrobial sepsis. The decreased survival of cRel-/- animals upon infection is not related to altered local mechanisms of innate defense such as the peritoneal recruitment of the Gr.1+CD11b+ phagocytic cells and the bacterial clearance. However, cRel deficiency allows to altered systemic cytokine response associated to sustained loss of the lymphoïd subset CD8a+ of spleen dendritic cells, key antigen-presenting cells for the initiation of the adaptive immunity. Genome-wide analysis of the systemic host response to polymicrobial sepsis reveals inflammatory/immune and apoptotic gene signatures associated to cRel subunit. In this study we identified the NF-kB member cRel, as a key factor which plays a critical role in survival to polymicrobial sepsis and also as a regulatory transcription subunit controlling the inflammatory and the adaptive immune responses in severe infection.
2011-10-14 | E-MEXP-2944 | biostudies-arrayexpress
Project description:Expression data from Total RNA extracted from murine spleen. Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture (CLP), followed 6 hours later by an intravenous injection of Mesenchymal Stem Cell (MSC) or saline. Twenty-eight hours after CLP, plasma, bronchoalveolar lavage (BAL) fluid and tissues were collected for analyses. Total RNA was extracted using Trizol (as per manufactures' instruction) followed by clean-up procedure using Qiagen RNA easy Prep (as per manufactures instructions) In the following study we hypothesized that mesenchymal stem cells (MSCs), which have documented immunomodulatory properties, would reduce sepsis-associated inflammation and organ injury in a clinically relevant model of sepsis. To identify the molecular changes associated with decreased inflammation in CLP-injured mice treated with MSCs, we analyzed the gene expression profiles from spleens collected at 28 hours from 4 animals per group: sham/saline, CLP/saline, and CLP/MSCs.
2010-11-22 | E-GEOD-24357 | biostudies-arrayexpress
Project description:Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil Female and male ICR mice (12-14 weeks age) weighing between 25 and 30 grams were anesthetized with a ketamine/xylazine mixture ( i.p.) allowing the subcutaneous implantation of miniosmotic pumps (Alzet, model 2001). The pumps were filled with either verapamil or vehicle (0.02% ascorbic acid). Control animals carried mini-pumps with vehicle and control animals were investigated in parallel with each set of experimental animals. Mini pumps delivered verapamil at 3.6 mg/kg/day for 24 (RNA)-48 (protein) hours. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and the stored at -80oC until studied. Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Left ventricular free wall from female mice was rapidly excised and either snap frozen at -80oC or used immediately for RNA isolation. Three VER treated mice and 3 vehicle treated mice were used to generate RNA for microarray. Total RNA was isolated using the RNAqueous -4PCR kit (Ambion) and quantitated spectrophotometrically at 260nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Microarray data was obtained using the Affymetrix 430 V2 GeneChip (representing 45,101 probe sets), in accordance with the manufacturer’s specifications.
2009-12-31 | E-GEOD-8367 | biostudies-arrayexpress
Project description:STIM1 is a Ca2+ sensor of intracellular Ca2+ stores and essentially activates the Ca2+ entry channels of the plasma membrane. STIM1 is predicted to activate transient receptor potential canonical (TRPC) channels and Orai channels, and has a critical role in promoting the development of cardiac hypertrophy. Gene array experiments were performed to analyze the effects of STIM1 deficiency using total RNA from the left ventricles of WT sham, WT TAC, STIM1KO sham and STIM1KO TAC 4 weeks after the operation.
2014-07-01 | E-GEOD-38733 | biostudies-arrayexpress
Project description:Ischemic stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Buyang Huanwu decoction (BHD), a traditional Chinese medicine prescription, has long been used clinically for neurological recovery after stroke. Its molecular characteristics and related biomarkers for the therapeutic effect in cerebrospinal fluid (CSF) are yet to be explored. In this project, CSF was obtained from acute ischemic stroke induced mice by a middle cerebral ischemic/reperfusion (CI/R) injury. Proteomic and metabolomic analyses of CSF were performed using LC-MS/MS to define the neuroprotective effect of BHD and the related biomarkers.
2019-02-16 | PXD006342 | Pride
Project description:Sprague-Dawley rat retina post-injury and control samples
2007-10-26 | E-GEOD-1001 | biostudies-arrayexpress
Project description:In order to examine the mechanism of TPO on cardiac protection against myocardial infarction damage (MI), we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to delineate the TPO cardioprotective mechanism against infarction. MI and TPO induced gene expressions in rat heart were measured at week 4. Two biological replicates were performed for each treatment group.
2012-12-01 | E-GEOD-22489 | biostudies-arrayexpress