Analysis of Macrophage Response to Adherent-Invasive E.coli Infection Using RNA Sequencing

In order to understand the role of mucosa-associated E. coli in Crohn pathogenesis, the role of the innate immune system, factors which may contribute to prolonged bacterial survival and therapeutic strategies to target intracellular E. coli replication. The aim of the present study was to assess the cell-specific transcriptional response of cultured RAW 264.7 cells following direct interaction with AIEC strain LF82. To assess the response of those cells in direct interaction with different internalisation of bacterial burden, we used fluorescence-activated cell sorting to specifically isolate cells associated with a recombinant strain of LF82 that constitutively expresses green fluorescent protein. We then applied whole-genome expression microarray technology to assess transcriptional differences between these cell populations.We set out a bulk RNAseq to test how macrophages respond to different pathogenic E.coli loads in a transcriptional status, to decipher the mechanistic underpinnings of this variation in both the host and bacteria. In order to quantitatively characterise outcomes of macrophages within different bacterial loads, FAC sorting was conducted and based on a fluorescent system of GFP-expressing bacteria. There are three possible outcomes: no infection signed as No, low bacterial load as Low and high bacterial load as High. A total of 80,000 cells per sample were collected by FACS. In order to simplify the description of the three groups, the terms Control, No, Low and High will be used instead.