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

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. Overall design: Primary endothelial cells HUVEC were seeded in monolayer and exposed to: (1) nothing, (2) uninfected PMA-differentiated U937 macrophage-like cells, or (3) Listeria monocytogenes-infected U937 macrophage-like cells. After 8 hours post-exposure (hpe) to U937, samples were gently washed and detached, and then cryopreserved for scRNA-seq . For each condition three samples were analysed using 10x Genomics scRNAseq technology. Equipment: 10X Chromium platform with Chromium Next GEM Single Cell 3' Kit v3.1 following the manufacturer's protocol for library construction. Library sequencing was done with llumina NovaSeq X platform, at 20000 reads per cell per sample. Sequencing read mode applied was: Read 1 28bp; Read 2 90bp; Index Reads 1 and 2 10 bp each.