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Title: Phenotypic and functional characterization of macrophages in the normal and inflamed human gut
Author: Ammoscato, Francesca
ISNI:       0000 0004 7962 3816
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2016
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Intestinal macrophages in the lamina propria underlying the gut epithelium play an important role in gut homeostasis. They remove apoptotic cells and cellular debris and phagocytose and destroy invading pathogens and intestinal microbiota crossing the epithelium. Infiltrating macrophages also play an important role in the pathogenesis of inflammatory bowel disease (IBD) by producing pro-inflammatory molecules such as TNF-alpha and metallo-elastase. In this work I developed a new method to isolate macrophages from human lamina propria mononuclear cells (LPMCs) using CD33 magnetic beads. I then carried out functional and phenotypic studies of these cells isolated from the lamina propria of control samples and from patients with either Crohn's disease or ulcerative colitis. The use of additional marker CD14 enabled me to distinguish two populations of macrophages, namely CD33+CD14- LPMCs, the main intestinal macrophage population in the normal colon and CD33+CD14+ LPMCs, predominant in the inflamed mucosa. Intestinal macrophages were also characterized for their expression of CD68, CD206 and CD64. CD33+CD14- LPMCs and CD33+CD14+ LPMCs expressed CD68, the gold standard macrophage marker. CD206 is a macrophage mannose receptor and it is associated with a M2 macrophages phenotype. The numbers of CD206+ cells was higher in CD33+CD14+ cells of control and CD samples and to a lesser extent in CD33+CD14+ cells from UC patients. CD64 is the high affinity Fcgamma type I receptor (FcγRI) used to discriminates macrophages from dendritic cells. The numbers of CD64+ cells were markedly increased in the CD33+CD14+ cells of IBD mucosa and control subjects compared to CD33+CD14- cells. The ability of different macrophage subsets to make TNF- was also studied. The number of CD33+CD14+ TNF-+ LPMCs was significantly increased in IBD patients compared to control subjects. Interestingly, using CD206, I found that the majority of CD33+CD14+CD206+ cells in IBD were TNFα+. Moreover CD33+CD64+ LPMCs produced significantly more TNF- compared to CD33+CD64- LPMCs which probably represent DCs. Since TNF- is first produced as a transmembrane molecule and then cleaved into soluble TNF- by the ADAM17/10 proteases, CD33 LPMCs were cultured with ADAM17/10 inhibitor, GW280264X, which induced a significant increase in transmembrane TNF- and reduced release of soluble TNF-. Moreover reduced production of soluble TNF- and soluble IL-6R where found when biopsies of UC and CD patients were cultured with GW280264X. Finally, gene arrays on CD33 gut macrophages isolated from IBD patients and control subjects showed a dysregulated immune gene profile. IL-24 transcripts and protein were increased in IBD macrophages and when exogenous IL-24 was added to IBD mucosal biopsies cultured ex vivo there was an increase in promoted TNF- release.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Intestinal macrophages ; Irritable Bowel Disease