Dataset Information

Single-cell RNA sequencing analysis of HUVEC in monolayer alone or exposed to (bacterially-infected) macrophage-like U937 cells for 8 h.

ABSTRACT: We sought to identify changes in gene expression that occur when endothelial cells (HUVEC) in monolayer interact with either uninfected or Listeria monocytogenes-infected macrophages. To that end we seeded HUVEC in monolayer and either exposed them to nothing, or to uninfected PMA-differentiated macrophage-like U937, or to Listeria monocytogenes-infected PMA-differenitated U937. At 8 hours post U937 exposure (hpe), cells were detached from the wells and cryopreserved for scRNA-seq. scRNa-seq analysis allowed us to distinguish endothelial cells from macrophage like cells and create appropirate clusters. By then analyzing the differentially expressed genes between all conditions for HUVEC and U937 separately, we identified a significant number of differentially regulated genes related to innate immune signaling in both infected U937 exposed HUVEC and to a lesser degree uninfected U937 exposed HUVEC, as compared to control unexposed HUVEC. By perofming pathway enrichment analysis, we also discovered that HUVEC exposed to uninfected U937 showed a higher upregulation in pathways related to mechanotransdcution, like "focal adhesions" and "regulation of the actin cytoskeleton", compared to those exposed to infected U937. This is consistent with the reinforsement of traction and monolayer stresses of HUVEC with uninfected U937 which we observe, a feature that is attenuated significanlty during infection. Finally, when we compare uninfected versus infected U937 cells from co-cultures, we find that the later upregulate cell adhesion molecules, the PI3K signaling pathway and focal adhesions, suggestive of increased cell contractility and adhesion as compared to uninfected U937.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312719 | GEO | 2026/01/27

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 4 of 4

Similar Datasets

Project description:We sought to identify changes in gene expression that occur when endothelial cells in monolayer interact with either uninfected or Listeria monocytogenes-infected macrophages. To that end we seeded HUVEC in monolayer and either exposed them to nothing, or to uninfected PMA-differentiated macrophage-like U937, or to Listeria monocytogenes-infected PMA-differenitated U937. At 8 or 24 hours post U937 exposure (hpe), we washed the samples thoroughly to remove U937 and then collected the mRNA to to perform RNA-Sequencing and gene expression profiling for the six conidtions described. By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes related to innate immune signaling in both (un)infected U937 exposed HUVEC as compared to control unexposed HUVEC, irrespective of the time post-exposure. For example we found significant upregulation of genes related IL-17, NF-kB and TNF related signaling pathways, though to a lesser degree for HUVEC exposed to uninfected U937 as compared to bacterially infected. Interestingly, we found that only HUVEC exposed to uninfected but not infected U937 and only at 8hpe showed significant upregulation of genes related to focal adhesions and regulation of the actin cytoskeleton. We found that consistent with alterations in the dynamics of HUVEC under this condition, which show a significant rise in the traction on monolayer stresses they exert. At later times post-exposure (i.e., 24 hpe) were these changes in gene expression are not present anymore, HUVEC dynamics are also identical irrespective of whether they were exposed or not to (un)infected U937. Thus the RNA sequnecing results at 8 hpe that reveal upregulation of genes related to focal adhesions and the actin cytoskeleton for uninfected U937 exposed HUVEC match with the results of traction force and monolyaer stress microscopy that reveal strong enhancement of these forces over the first 10 hpe and which are absent in the other two conditions or at the later time point of 24 hpe.

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 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.

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.

			Action	DRS
		Other
	GSE312719_DE_analysis_SingleCellRNAseq.xlsx	Xlsx
	GSE312719_RAW.tar	Raw
	filelist.txt	Txt

Dataset Information

Single-cell RNA sequencing analysis of HUVEC in monolayer alone or exposed to (bacterially-infected) macrophage-like U937 cells for 8 h.

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets