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Difference in characteristics between Endocardially-derived cardiac tissue macrophages (EcTMs) and non-Endocardially-derived cardiac tissue macrophages (non EcTMs)

ABSTRACT: [Purpose] To define the traditional profile of EcTMs, RNA seq analysis were parformed on EcTMs and non EcTMs at postnatal day28. [Method] The workflow consists of first-strand synthesis, template switching, adaptor ligation, cleavage of ribosomal cDNA and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on an Illumina Nextseq500 a single read 75 run. Data quality check was conducted on Illumina SAV. Demultiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program. Reads were aligned to the UCSC mm10 reference genome map using TopHat. DEseq2 was used to identify differentially expressed genes of EcTMs compared to non-EcTMs. 36 upregulated genes in EcTMs compared to non-EcTMs with a padj < 0.05 were subsequently clustered and visualized using R. Gene ontology functional analyses were performed using the database for annotation, visualization, and integrated discovery (DAVID) online tool.[Result] 40 genes were differentially expressed between the two groups: 36 were higher and 4 genes were lower in EcTMs compared to non-EcTMs. Gene Ontology analysis revealed that EcTMs were enriched for genes related to antigen presentation, lysosomes, and phagosomes (H2-Ab1, H2-Aa, H2-Eb1, CD74, Laptm5). [Conclusion] EcTMs would have higher potential for presenting antigen and phagositosis compared to non EcTMs.

ORGANISM(S): Mus musculus

PROVIDER: GSE100495 | GEO | 2019/02/27

REPOSITORIES: GEO

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Project description:To compare tumor associated macrophage (TAM) from naïve and sepsis surviving mice we have employed Agilent microarrays slides with almost 60,000 genes (39,430 mRNA and 16,251 long non coding RNAs). Other experiments we conducted demonstrated TAM accumulation was increased in post-sepsis subjects. For this reason, we asked if TAM from post-sepsis mice could also exhibit a different gene expression profile. Sepsis was induced by cecal and ligation puncture. Naïve mice were used as control group. All animals were treated with ertapenem (20 mg/kg, i.p., 6 hours after surgery, and then each 12 hours for 3 days). B16-F10 melanoma (30,000 cells) were injected subcutaneously at day 14 after sepsis induction. Fourteen days after tumor inoculation, animals were killed and tumors were harvested and digested (collagenase and DNAse). TAM was isolated by a Percoll gradient (70/30) followed by a 1-hour adhesion protocol, reaching a purity of ~75%. For comparative reasons, we assessed TAM from post-sepsis (n = 4), TAM from naïve mice (n = 4), bone marrow derived macrophage from naïve (n = 4) and from post-sepsis (n = 4), M1-polarized macrophage (n = 4) and M2-polarized macrophage (n = 4). We found only minor gene expression differences between TAM from naïve and from post-sepsis mice (61 genes were up-regulated and 98 genes were down-regulated, fold-change > 0.58 or < -0.58, and p < 0.01). We found genes related to leukocyte activation were down-regulated in TAM from post-sepsis mice (e.g. Ccr7, Cd86, H2-Ab1), as well as genes related to antigen processing and presentation of peptide or polysaccharide antigen via MHC class II (H2-DMb1, Cd74, H2-Eb1, H2-Ob). A gene related to M2 polarization was up-regulated (Marco). Also, we found a down-regulation of Nfkbid in post-sepsis-derived TAM. This led us to hypothesize TAM from post-sepsis mice exhibit a more M2-like phenotype, which may in part contribute to post-sepsis tumor expansion. Three independent experiments were conducted for TAM obtaining, each experiment using n = 4 for naïve and n = 4 for post-sepsis. We selected the 4 best within a group of 12 samples, following A260/280 and A260/230 ratios. For bone marrow derived macrophage from naïve and from post-sepsis, and for M1 and M2-polarized macrophage, we conducted two independent experiments using n = 3 per group. The best 4 samples in each group was selected to microarray processing and analysis.

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Difference in characteristics between Endocardially-derived cardiac tissue macrophages (EcTMs) and non-Endocardially-derived cardiac tissue macrophages (non EcTMs)

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