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Title: Bacteria-macrophage interactions in the pathogenesis of Crohn's disease
Author: Flanagan, Paul Kevin
ISNI:       0000 0004 6495 6431
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly E. coli that replicate inside macrophage phagolysosomes. In this thesis several hypotheses were tested: (i) that CD macrophages might be defective at killing and or responding to Gram-negative bacteria, particularly E. coli; (ii) that killing of phagocytosed bacteria within macrophages might be enhanced by drugs such as hydroxychloroquine (HCQ) (previously shown to raise intravacuolar pH), and vitamin D (previously shown to enhance macrophage function). I assessed CD peripheral blood monocyte-derived macrophages (MDM) for their abilities to kill E. coli, with and without HCQ and vitamin D, and to generate neutrophil chemoattractants. MDM from patients with CD were similar to those from healthy controls (HC) in allowing replication of phagocytosed CD-derived E. coli: HM605 [CD N=10, mean fold replication in 3h: 1.08, (95% confidence interval (CI) 0.39-1.78); HC N=9, 1.50, (95%CI 1.02-1.97); P=0.15] and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD 2.55, 95%CI 2.31-2.80; HC 2.65, 95%CI 2.46-2.85, P=0.42). The only possible exception was reduced bacterial killing in macrophages from the relatively rare patients homozygous for ATG16L1 polymorphisms, reported previously and confirmed in the single example in this series. HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intra-macrophage E .coli replication 3h post-infection, HCQ: 73.9% inhibition (P < 0.001) at 1μg/mL, accompanied by raised intra-phagosomal pH, and 1,25OH2-Vitamin D3: 80.7% inhibition (P < 0.05) at 80nM. HCQ had synergistic effects with doxycycline and ciprofloxacin on killing of phagocytosed E. coli. Thus: CD and HC macrophages generally perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics.
Supervisor: Rhodes, J. M. ; Campbell, B. J. Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral