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Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

ABSTRACT: Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

PROVIDER: PRJNA645192 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12187254.fastq.gz	Fastqsanger.gz
	SRR12187255.fastq.gz	Fastqsanger.gz
	SRR12187256.fastq.gz	Fastqsanger.gz
	SRR12187257.fastq.gz	Fastqsanger.gz
	SRR12187258.fastq.gz	Fastqsanger.gz

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Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

Project description:Atlantic salmon (average 550g) were used in this study. The fish were starved for 24 h and were sacrificed with an overdose of MS-222. Then head kidney (HK) and distal intestine (DI) samples were collected. Immune cells from HK and DI were isolated and cultured at 12°C in Leibovitz’s L-15 Medium (L-15). Both HK and DI leukocytes were allowed to adhere on a petri dish with 2 mL L-15+ for 2 days at 12°C. After removing the supernatant containing non-adherent cells, the petri dish with the adherent cells was placed on ice for 10 min, and the cells were detached by washing three times with 1.5 mL ice-cold PBS supplemented with 5 mM EDTA. The cells obtained were centrifuged (500 × g, 5 min, 4°C) and re-suspended in 1 mL L-15+. Then, the cells were counted using a portable cell counter. Total RNA was extracted from adherent cells from HK or DI (< 500,000 cells). To compare the expression of marker genes between adherent cells from HK and DI, 12 libraries were prepared (6 replicates for each). Transcriptomic analysis revealed that adherent intestinal cells (AICs) expressed genes associated with macrophages, T cells, and endothelial cells. In addition, AIC had higher expression of TNF and TGFβ-related genes that can be linked to macrophages and T cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related markers.

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Adherent Intestinal Cells From Atlantic Salmon Show Phagocytic Ability and Express Macrophage-Specific Genes.

Project description:Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of Escherichia coli BioParticlesTM. AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells.

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Project description:The ability of Entamoeba histolytica to phagocytose host cells correlates to observed virulence in vivo. To better understand the mechanism of phagocytosis we used paramagnetic beads coated with host ligand and sorted trophozoites based on phagocytic ability. Gene expression was then measured in both the sorted phagocytic and non-phagocytic populations using a custom Affymetrix chip for E. histolytica. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. PMID 23045476 M280 Streptavidin Dynabeads (Invitrogen) were labeled with 20ug/mL biotinylated Human C1q (Quidel). Entamoeba histolytica (strain HM1) were washed twice with PBS then resuspended with the labeled beads at a 10:1 ratio of beads to trophozoites. Samples were incubated at 37Â°C for 45 minutes, washed twice with agitation to remove adherent beads, then resuspended in MACS buffer. Samples were loaded into magnetic columns (Miltenyi Biotec) and trophozoites were seperated according to manufacturer's protocols. phagocytic vs. non-phagocytic Entamoeba histolytica populations

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Project description:Alzheimer’s disease-induced phagocytic microglia express a specific profile of coding and non-coding RNAs

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Project description:Head kidney (HK) and distal intestine (DI) from Atlantic salmon (average 550g) were collected. Immune cells from DI and HK were extracted and grown at 12°C in Leibovitz’s L-15 Medium (L-15). The isolated DI or HK leukocytes were allowed to adhere on a petri dish with 2 mL L-15+ for 2 days at 12°C. After removing the supernatant containing non-adherent cells, the adherent cells on petri dish were detached by washing three times with 1.5 mL ice-cold PBS supplemented with 5 mM EDTA. The cells were centrifuged (500 × g, 5 min, 4°C) and re-suspended with 2 mL L-15+. Total RNA was extracted from adherent cells from HK or DI (< 500,000 cells). To understand the miRNA expression profiles of adherent intestinal cells (AICs) and to compare them with those of adherent head kidney cells (AKCs), twelve libraries (6 for AIC and 6 for AKC) were prepared. Small RNA-sequencing revealed 37 differentially expressed miRNAs; 20 were up-regulated and 17 were down-regulated. Of them, 12 miRNAs in AIC were found to be involved in regulating macrophage functions.

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