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Real-time quantitative PCR analysis of murine splenic chemokines and cytokines [common cytokines assay]

ABSTRACT: C57BL/6 mice were subjected to sham surgery or orthotopic Hepa1-6 hepatic tumor inoculation. Spleen tissues from mice subjected to sham surgery (control mice), mice bearing a 2-week orthotopic hepatoma, and mice bearing a 4-week othotopic hepatoma were collected. Total RNA was extracted from the spleen tissues, and we used the RT² Profiler PCR array-mouse common cytokines (SABioscience) array panels to quantitate gene expression of cytokine genes from the spleens.

ORGANISM(S): Mus musculus

PROVIDER: GSE84247 | GEO | 2016/07/12

SECONDARY ACCESSION(S): PRJNA328475

REPOSITORIES: GEO

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Project description:Purpose: Cachexia is a metabolic phenotype affecting many cancer patients. The immune environment in adipose tissue during the development of cancer associated cachexia has not been characterized. To help address this gap, we isolated visceral adipose tissue from cancer-bearing and sham-operated mice and performed bulk RNA-sequencing. Methods: mRNA expression was evaluated by RNA-sequencing in visceral adipose tissue (VAT) of 10-week old mice which had been subjected to sham surgery (sham) or had received orthotopic pancreatic tumors four weeks prior to harvest (KPC). Paired-end 150 bp single-index sequencing was performed using an Illumina HiSeq system. Transcript quantification was performed with Salmon, using quasi-mapping mode with selective alignment (validateMappings). We set the rangeFactorizationBins to 4 and passed both the seqBias and gcBias flags to correct for sequence-specific bias and fragment-level GC bias, respectively. Transcript quantifications were collapsed to gene-level quantifications using the tximport package and the Ensembl based annotation package EnsDb.Mmusculus.v79. Differential expression analysis was then performed using DESeq2 with fold change shrinkage by ashr. For downstream analysis and visualization, only genes with a mean TPM (transcripts per million) > 2 in VAT from both sham-operated and tumor-bearing mice were considered. Results: We identified over 11,000 genes which were expressed at an average of at least 2 TPM (transcripts per million) in both the sham and KPC adipose tissue samples. Differential expression analysis with DESeq revealed 161 upregulated genes (p < 0.05) and 27 downregulated genes (p < 0.05) in the adipose tissue of cancer bearing mice compared to that of sham operated mice. Gene set enrichment analysis suggested an increase in inflammatory pathways, including type 1 and type 2 interferon pathways, in the adipose tissue of cancer bearing mice. Conclusions: This study represents a comprehensive characterization of adipose tissue in the healthy vs. cachexia setting. The RNA-seq data suggests increased immune infiltration into adipose tissue in the setting of a distant pancreatic tumor. Together with flow cytometry and cytokine abundance analyses, we conclude that reguatory Th2 immunity is maintained in the adipose tissue during this model of cancer associated cachexia.

Project description:Objective – Following destabilization of the medial meniscus (DMM), mice develop experimental osteoarthritis (OA) and associated pain behaviors that are dependent on the stage of disease. We aimed to describe changes in gene expression in knee-innervating dorsal root ganglia (DRG) after surgery, in order to identify molecular pathways associated with three pre-defined pain phenotypes: “post-surgical pain”, “early-stage OA pain”, and “persistent OA pain”. Design – We performed DMM or sham surgery in 10-week old male C57BL/6 mice and harvested L3-L5 DRG 4, 8, and 16 weeks after surgery or from age-matched naïve mice (n=3/group). RNA was extracted and an Affymetrix Mouse Transcriptome Array 1.0 was performed. Three pain phenotypes were defined: “post-surgical pain” (sham and DMM 4-week vs. 14-week old naïve), “early OA pain” (DMM 4-week vs. sham 4-week), and “persistent OA pain” (DMM 8- and 16-week vs. naïve and sham 8- and 16-week). ‘Top hit’ genes were defined as p<0.001. Pathway analysis (Ingenuity Pathway Analysis) was conducted using differentially expressed genes defined as p<0.05. In addition, we performed qPCR for Ngf and immunohistochemistry for F4/80+ macrophages in the DRG. Results – For each phenotype, top hit genes identified a small number of differentially expressed genes, some of which have been previously associated with pain (7/67 for “post-surgical pain”; 2/14 for “early OA pain”; 8/37 for “persistent OA pain”). Overlap between groups was limited, with 8 genes differentially regulated (p<0.05) in all three phenotypes. Pathway analysis showed that in the persistent OA pain phase many of the functions of differentially regulated genes are related to immune cell recruitment and activation. Genes previously linked to OA pain (CX3CL1, CCL2, TLR1, and NGF) were upregulated in this phenotype and contributed to activation of the neuroinflammation canonical pathway. In separate sets of mice, we confirmed that Ngf was elevated in the DRG 8 weeks after DMM (p=0.03), and numbers of F4/80+ macrophages were increased 16 weeks after DMM (p=0.002 vs. Sham). Conclusion- These transcriptomics findings support the idea that distinct molecular pathways discriminate early from persistent OA pain. Pathway analysis suggests neuroimmune interactions in the DRG contribute to initiation and maintenance of pain in OA. We grouped samples based on pain phenotype in the DMM mouse model of osteoarthritis. Group 1: post-surgical pain (DMM and sham +4 week samples); Group 2: post-surgical control (Naïve +4 week samples); Group 3: Early osteoarthritis pain (DMM +4 week samples); Group 4: Early controls (sham+4 and naive+4 week samples); Group 5: Persistent pain (DMM+8 and DMM+16 week samples); Group 6: Persistent controls (sham+8, naive+8, sham+16, and naive +16 samples). We compared Group 1 vs Group 2; Group 3 vs Group 4; and Group 5 vs Group 6 to draw the conclusions presented in our manuscript.

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Real-time quantitative PCR analysis of murine splenic chemokines and cytokines [common cytokines assay]

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