Paired-end RNA Sequencing on Madin-Darby Canine Kidney (G-MDCK) epithelial cells infected or not with Listeria monocytogenes and residing on varying stiffness substrates.

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-??; 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-?B, 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. Overall design: Epithelial cell mRNA profiles of cells previously residing in confluency on glass substrates or polyacrylamide hydrogels (of 3 KPa or 70 kPa) coated with 0.25 mg/mL collagen where either (1) uninfected; or (2) infected with low dosage of Listeria monocytogenes (MOI=200) at 24 h post-infection and compared. Sampled were generated in quadruplicate, using Illumina NovaSeq 6000 using the NovaSeq 6000 S4 Reagent Kit with 2x150 read length and 40 million reads per sample.