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Paired-end RNA Sequencing on Madin-Darby Canine Kidney (G-MDCK) epithelial cells infected or not with Listeria monocytogenes

ABSTRACT: Purpose: When we infect MDCK epithelial cells we low dosage of Listeria monocytogenes we observe that at late times (24 h) post infection that infected cells pertaining in infection foci get collectively extruded out of the epithelial monolayer forming a 3D mound. The formation of the infection mound results from the competition between two populations: the uninfected cells ("surrounders") that eventually squeeze and extrude the infected cells ("mounders"). Using RNA-Sequencing, we performed gene expression profiling for uninfected MDCK cells, cells infected for 24 h with Listeria monocytogenes at an MOI of 200 (both "mounders" and "surrounders") and cells sorted to infected ("mounders") or uninfected ("surrounders") populations after being infected for 24 h with Listeria monocytogenes. Our goal was to understand how transcriptional regulation leads to the collective extrusion of infected epithelial cells triggered by surrounding uninfected cells. Methods and Results: By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes between all groups. Through pathway enrichemnet analysis we find that compared to cells not exposed to infection "mounders" showed significant downregulation in 31 genes related to tight junctions and ZO-1 was one of the key genes downregulated and to a lesser degree to pathways related to adherens junctions, focal adhesions and regulation of the actin cytoskeleton. "Surrounders" showed significant downregulation to genes related to actin cytoskeleton, to endocytosis to ahderens junctions and to a lesser degree focal adhesions and tight junctions. Especially "surrounders" and to a lesser degree "mounders" showed upregulation on IL-17 and NF-kB and TNF related signaling pathways, which underlines the fact that although "surrounders" are uninfected their distinct mechanical behavior might arise from cytokines released from "mounders" which impact crucially their phenotype. Interestingly "mounders" only showed additionally upregulation on DNA replication, cell cycle related pathways and ferroptosis suggesting that these cells might be undergoing some kind of stress response. Conclusions: Infection of MDCK epithelial cells with Listeria monocytogenes significantly alters the transcriptome of the infected host cells when those are examined 24 h post infection. Both infected and uninfected cells in an infected well show significant transcriptomic changes compared to uninfected cells but also compared to each other. Most importnantly innate immunity related pathways are singificanlty upregulated for un infected cells in an infected well and to a lesser degree for infected cells as compared to cells not exposed to infection.

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE140626 | GEO | 2019/11/19

REPOSITORIES: GEO

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Project description:Purpose: When we infect MDCK epithelial cells we low dosage of Listeria monocytogenes, we observe that at late times (24 h) post-infection the infected cells pertaining in infection foci get collectively extruded out of the epithelial monolayer forming a 3D mound. The formation of the infection mound results from the competition between two populations: the uninfected cells ("surrounders") that eventually squeeze and extrude the infected cells ("mounders") in the infected wells. The extracellular matrix where the host cells adhere appears to impact the formation of infection mounds with more mounding on glassr as opposed to soft 3 kPa substrata, as evidenced by confocal 3D microscopy. Here we built poalyacrylamide hydrogels of varing stiffness (3 kPa soft or 70 kPa stiff) and coated them with collagen I rat tail. We also placed host cells on glass collagen I-coated surfaces. We then infected for 24 h (or not) the host cells with Listeria monocytogenes to infer how matrix stiffness-dependent gene expression of MDCK cells in monolayers changes in uninfected versus infected wells, through RNA-Sequencing. Our goal was to understand how matrix stiffness impacts transcriptional regulation of host cells and whether that could modulate the collective extrusion of infected epithelial cells triggered by surrounding uninfected cells. Methods and Results: By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes between all groups, other than between cells residing on soft versus stiff polyacrylamide hydrogels that behave similarly in terms of gene expression. Through pathway enrichement analysis, we find that cells residing on polyacrylamide hydrogels as opposed to cells residing on glass show higher upregulation of a number of innate immune signaling related pathways (e.g. MAPK, TNF) even when infection is not involved. When comparing infected versus not wells, the relative differences in gene expression for cells residing on glass are higher than for cells residing on hydrogels. However certain specific innate immune signaling pathways that regulate mound formation (https://www.sciencedirect.com/science/article/abs/pii/S1534580721000708), show higher upregulation in infection for cells on hydrogels as opposed to glass (e.g. IL-17, TNF, NF-κΒ; evidenced by pathway enrichement analysis). Finally, through pathway enrichement analysis we find that the relative change based on enrichement score when comparing uninfected versus infected settings for cells residing on glass versus on polyacrylamide hydrogels is lower for the former (i.e. for cells residing on glass) for NF-κB, TNF and IL17. However, with regards to the MAPK pathway the enrichment score of cells from infected versus uninfected wells, is much higher for cells residing on glass as opposed to polyacrylamide hydrogles. Conclusions: Infection of MDCK epithelial cells with Listeria monocytogenes significantly alters the transcriptome of the infected host cells when those are examined 24 h post infection and compared to uninfected cells on all different substrata. Compared to cells residing on glass substrates cells residing on polyacrylamide hydrogels (irrespective of whether the stiffness is low i.e. 3 kPa or high i.e. 70 kPa) show significant differences both when uninfected settings and when infected ones are compared.

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Paired-end RNA Sequencing on Madin-Darby Canine Kidney (G-MDCK) epithelial cells infected or not with Listeria monocytogenes

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