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  • All animal care and experimental procedures adhered to NIH guidelines and were approved by the University of Illinois Institutional Animal Care and Use Committee. Measures were taken to minimize the number of animals used and the pain and suffering of the mice. Microglia and peritoneal macrophages (hereby termed macrophages) were collected from approximately 22 weeks old male C57Bl/6J mice. Mice housing, management, and BCG challenge followed previously described protocols [5,8]. Briefly, mice were housed individually in standard polypropylene cages including corn cob litter. Housing was under a 12:12 h reversed light/dark cycle (lights on 10:00 PM-10:00 AM) with controlled environmental temperature (23°C) and humidity (45%). Mice were offered water and food (Teklad 8640 chow, Harlan Laboratories, Indianapolis, IN, USA) ad libitum and handled daily for one week prior to the trial to ensure adaptation. Mice were acclimated to the light cycle and facility for at least 3 weeks prior to the injection with BCG or saline. All mice were individually handled for a few minutes daily prior to the challenge. The immune challenge involved live attenuated mycobacteria TICE BCG (50 mg wet weight of lyophilized culture containing 1x108 colony forming units or CFU/vial; Organon Teknika Corp. LLC, USA Inc., USA). Each vial’s reconstitution prior to inoculation followed provider instructions using preservative-free saline. No peritoneal mycobacteria infection was reported in mice by day 20 after intraperitoneal infection with BCG; whereas dissemination to other organs, including the spleen, bone marrow, and lung was observed [9]. There are no reports of BCG infection of microglia or brain and the peritoneum is a source of non-infected macrophages rather than macrophages from other tissues that could be infected with BCG. Individual mice were challenged once with either 10 mg/mouse (BCG-challenged group, n = 12) or sterile saline solution (Control group, n = 12) administered via intraperitoneal injection [5]. No mouse became severely ill or died at any time prior to the experimental endpoint. Mice were euthanized by CO2 asphyxiation by trained personnel 7 days after BCG challenge and all efforts were made to minimize suffering. The endpoint was selected based on prior work that demonstrated the recovery from sickness yet persistence of depressive-like symptoms 7 days after challenge [5]. Macrophages were collected from peritoneal tissue using the proven protocols [8,10]. Abdomens were disinfected, skin was retracted, and the peritoneal cavity was flushed with Hank's Balanced Salt Solution (cold harvest medium). The peritoneal fluid was centrifuged and the resulting cell pellet was resuspended and plated. The medium was aspired after 2 h incubation to remove non-adherent cells. The surviving adherent cells constituted the non-thioglycollate elicited peritoneal macrophages and cells from individual mice were stored in Trizol at -80°C until RNA extraction [11]. After peritoneum collection (~5 minutes), mice were perfused, and the brains were excised and minced. Microglia were collected following established protocols [12]. In brief, brains were trypsinized [13], dissociated using cell screens (40 μm), centrifuged, resuspended in 30% Percoll (GE Healthcare, Princeton, NJ), then centrifuged for myelin removal. Brain cells were labeled with anti-CD11b (integrin alpha M antibody) magnetized Miltenyi MicroBeads (Miltenyi Biotec, Germany). Cells were separated in a magnetic field with MS columns (Miltenyi Biotec, Germany). The resulting Cd11b+ fraction collected was centrifuged, resuspended, and stored at -80°C. Flow cytometry validation of cell isolation encompassed cell staining with primary fluorescent antibodies for two primary markers for macrophages and microglia: CD11b and CD45 (protein tyrosine phosphatase, receptor type, C antibody) [14,15]. Fc receptors were blocked by incubation with anti-CD16/CD32 antibody before incubation with eBioscience anti-CD11b and anti-CD45 antibodies (eBioscience Inc., San Diego, CA). Surface receptor expression was identified using a Biosciences LSR II Flow Cytometry Analyzer with BD FACSDiva software (BD Biosciences, San Jose, CA). Antibody gating was determined using isotype-stained controls. Cells were ~93% Cd11b+ and ~91% Cd45+, confirming microglia enrichment. RNA extraction from microglia followed the Tripsin method using a total RNA Kit (Omega Biotek, Norcross, GA) and a DNase step to remove DNA contamination [11]. The Agilent 2100 Bioanalyzer with RNA Pico chip (Agilent Technologies, Palo Alto, CA) was used to assess RNA Integrity Numbers. RNA Integrity Numbers were > 9 in 90% of the samples and > 7 in 100% of the 48 samples. Identification of differential transcript isoform and gene expression: RNA libraries from individual mouse were sequenced using an Illumina HiSeq 2000 (Illumina, San Diego, CA) and 100nt long paired-end reads were obtained. Read quality control was implemented using FastQC [8,16]. Quality control analysis indicated that the Phred nucleotide quality score was > 30 across the length of the reads such that the read sequences were not trimmed. Reads were mapped to the Genome Reference Consortium GRCm38 mouse assembly using Tophat2 (v 2.0.8) [17] with the Illumina iGenomes package (mm10; http://support.illumina.com/sequencing/sequencing_software/igenome.html). Reads were combined into transcripts and differential expression was tested using Cufflinks (v2.1.1) [18]. The specifications beyond the default settings used to obtain transcript abundance levels were: a) Upper Quartile Normalization was used to normalize the median transcript counts across libraries; b) multi-read correction for reads mapping to multiple sites; and c) fragment bias correction [19]. Four comparisons were evaluated: two pairwise contrasts between BGG-challenge groups within cell types, and two pairwise contrasts between cells within BCG-challenge groups. Results from genes with at least 10 mapped reads were considered. Multiple test adjustment used the Benjamini-Hoechberg false discovery rate (FDR) approach [20,21]. Genes exhibiting an FDR-adjusted P-value < 0.05 were considered differentially expressed. Identification of alternative splicing events: Alternative splicing events characterizing differences between groups (i.e., cell types or challenge level) were identified using a quantitative approach [22]. Quantitative characterization considered genes detected in both groups, represented by at least two transcript isoforms, and with at least one transcript isoform differentially expressed between groups (FDR-adjusted P-value < 0.05) between groups in one direction (over- or under-expressed) and the rest of the isoforms not differentially expressed or differentially expressed in the other direction. Identification of functional categories over-represented among gene profiles: Functional analyses of the transcript isoforms differentially expressed between BCG-challenge groups within cell types, between cell types within BCG-challenged groups, and of transcript isoforms expressed exclusively in one cell type were performed using hypergeometric testing and Gene Set Enrichment Analysis (GSEA). These analyses allowed the identification of Gene Ontology (http://www.geneontology.org/) biological processes, molecular functions, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (http://www.genome.jp/org/). The hypergeometric test was implemented in the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov) [23,24]. Gene Ontology (GO) results were reported using the DAVID Functional Annotation Tool (FAT) classes to facilitate interpretation. Category enrichment was measured using Expression Analysis Systematic Explorer (EASE) scores computed based on a one-tailed jackknifed Fisher exact test [25,26]. The GO categories were clustered and the statistical significance of each cluster was assessed using an Enrichment Score (-log10 geometric mean of the cluster members EASE scores [27,28]. Functional annotation clusters were considered significant at Enrichment Score > 2 (comparable to P-value < 0.001) using the Mus musculus genome as background. Gene Set Enrichment Analysis was implemented using the GSEA-P software package [29,30]. This approach offered functional insights complementary to DAVID based on the consideration of the expression profile of all the genes analyzed and annotations to the Molecular Signature Database (MSigDB) [31]. Interpretation of findings and discovery of differences in gene co-expression associated with BCG challenge or cell type were enhanced using network visualization. Networks were visualized using the BisoGenet plug-in [27,28,32] within the Cytoscape environment [24,33]. BisoGenet enabled the visualization of associations between genes detected in the present study using information from the SysBiomics repository. Networks depicting genes as nodes and gene associations as edges and including at most one non-measured gene connecting observed genes were considered. The resulting gene networks were augmented with information on differential gene expression results from the comparison between BCG-challenged and cell type groups obtained in this study. The node size represented the differential expression P-value and the node color denoted over- or under-expression between challenge levels or cell types.
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