title: Investigation of macrophages response to infection with Crohn's disease associated Adherent-invasive Escherichia coli
Creators: name: Li, Xiang
Publisher: name: University of Glasgow
Year: 2022

description: Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's Disease (CD). CD is believed to be caused by a complex interaction between genetics, microbiome, the immune system and the environment. AIEC are characterised by an ability to survive and replicate intracellularly in macrophages with increased secretion of pro-inflammatory cytokines, which may contribute to further dissemination of the pathogen. Bacterial persistence within cells is thought to lead to immune evasion and chronicity of infections. Understanding the maintenance of AIEC in macrophages may elucidate methods of targeting AIEC colonization as a potential therapeutic intervention strategy for patients with CD. A histopathological hallmark of CD is aggregation of intestinal macrophages, referred to as granulomas, which may result from fusion of monocytes and macrophages giving rise to the formation of multinucleated giant cells (MGCs). However, cell-cell interactions are highly heterogeneous. Therefore, imagebased technologies such as imaging flow cytometry (IFC) and fluorescent microscopy were employed on macrophages during AIEC infection. IFC employs a traditional flow cytometer equipped with a fluorescent camera capable of imaging over different wavelengths. With real high-resolution images of AIECinfected macrophages, images of mono- and bi-nucleated cells as well as polynucleated cells were observed. In addition, the morphologic features and of cell-cell interaction could be monitored. As a result, significantly more multinucleated cells were found in AIEC-infected macrophages when compared to uninfected macrophages. AIEC infection induced an enhanced connection between cells as a result of increased cellular adhesion, phagocytosis, and cell fusion. Therefore, it is possible that the formation of MGCs may result from phagocytosis or cell fusion. The use of IFC for examining host-pathogen interactions is well established, so we applied it to identifying the role of specific host proteins in bacterial infection. Little is known about how macrophage-killing of AIEC is impeded. We used an in vitro infection model to identify macrophage proteins associated with AIEC intracellular replication. Phosphorylated proline-rich tyrosine kinase 2 (p-PYK2) levels were identified as being significantly altered during AIEC infection. The pPyk2 inhibitor PF-431396 significantly decreased intramacrophage replication of AIEC as determined by viable colony count, fluorescence immunostaining and imaging flow cytometry. Meanwhile, Pyk2 inhibition also decreased TNF∝ secretion from AIEC-infected macrophages. Pyk2 has previously been identified as a risk locus in inflammatory bowel diseases through genomewide association studies and is overexpressed in patients with intestinal and colorectal cancer (CRC), the latter a major long-term complication of CD. In this thesis, we have outlined that Pyk2 inhibition could be a potential strategy for CD treatment via controlling intracellular AIEC levels within macrophages. To better understand host-pathogen interactions, it is important to take into account the extreme heterogeneity of the host response after an infection. Infection is a dynamic process and modelling the outcomes of the infectious process has always proved challenging. Using an in vitro infection model, IFC found that less than 50% of RAW 264.7 cells were actually infected by AIEC strain LF82 after 24 hours. In this thesis, using high-throughput RNA-sequencing combined with FACS sorting, infected cells from these heterogeneous populations were sorted into populations based on their intracellular pathogen burden. This allowed a greater understanding of what benefit AIEC bacteria could gain from resisting killing by mucosal macrophages and to what extent the expression of host gene expression affects AIEC intramacrophage replication. Based on this transcriptomic analysis of infected macrophages, I identified specific chemical inhibitors targeting proteins identified as highly expressed in infected cells with heavy bacterial burdens. The ubiquitin E3 ligase Itch, involved in the ubiquitin-proteasome system, was targeted by the drug clomipramine significantly reducing intracellular bacterial burden in macrophages. This finding suggests the possibility that AIEC could manipulate the ubiquitin-proteasome system during infection, allowing AIEC to survive within macrophages. The discovery of potent chemical inhibitors, identified as targeting proteins with a novel role in AIEC infection, will hopefully open up new avenues of research and opportunities for the development of new intervention strategies in infection and CD.