Paired-end RNA Sequencing on primary human umbilical endothelial cells (HUVEC) exposed to uninfected or bacterially-infected PMA-differentiated macrophage-like U937 for 8 or 24 h

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. Overall design: Primary endothelial cells HUVEC were seeded in monolayer and exposed to: (1) nothing (unexposed HUVEC, UE), (2) uninfected PMA-differentiated U937 macrophage-like cells (uninfected macrophage exposed HUVEC, UI), or (3) Listeria monocytogenes-infected U937 macrophage-like cells (infected macrophage exposed HUVEC, I). After 8 or 24 hours post-exposure (hpe) to U937, the samples were washed to remove U937, and mRNA was collected for RNA sequencing and gene expression profiling across these six conditions. For each condition four samples were analysed, using Illumina NovaSeq 6000 using the NovaSeq 6000 S4 Reagent Kit (300 cycles). Equipment: Illumina with 2x150 read length and 40 million reads per sample.