P055. Relationship of impaired macrophage cytokine release to genetic susceptibility polymorphisms in Crohn's disease
G.W. Sewell1, A.P. Levine1, L. Jostins2, P. Harrison1, P.J. Smith1, A.P. Walker1, S.L. Bloom3, A.W. Segal1, A.M. Smith1
1UCL, Division of Medicine, United Kingdom; 2Wellcome Trust Sanger Institute, United Kingdom; 3University College London Hospital, Department of Gastroenterology, United Kingdom
Background: Mounting evidence suggests that the pathogenesis of CD involves a defective innate immune response to bacteria in the bowel wall. Clearance of bacteria such as E. coli is impaired in CD, as a consequence of delayed neutrophil recruitment. This is associated with diminished pro-inflammatory cytokine secretion by macrophages in response to bacterial stimulation . Genome-wide association studies (GWAS) have identified numerous genetic polymorphisms associated with CD susceptibility . Here, defective release of pro-inflammatory cytokines in response to E. coli is validated, and the relationship to genetic polymorphisms interrogated.
Methods: Monocyte-derived macrophages were cultured from healthy controls (HC) (n = 39) and quiescent CD (n = 43) patients (HarveyBradshaw score <3). Cells were stimulated with heat-killed E. coli (HkEc) for 24 hours. Cytokine secretion was quantified using multiplex cytokine assays. Patients in the macrophage study and overall UCLH cohort (CD n = 356, HC n = 221) were genotyped for 34 CD-associated polymorphisms using Sequenom technology. Weighted genetic risk scores (GRS) were calculated based on the genotyping results, using a logistic regression model and assuming additive genetic effects.
Results: Macrophages from CD patients released significantly reduced levels of TNF (p < 0.01) in response to HkEc, in comparison to HC macrophages. Release of IL‑1β, IL‑10 and IL‑8 did not differ between patients and controls. There was no detectable relationship between the levels of TNF secreted and the genotype for any individual polymorphism (including NOD2, ATG16L1 and IRGM variants). However, there was a weak but significant negative correlation between TNF release and overall GRS in the CD cohort (p < 0.05). As expected, the UCLH cohort of CD patients displayed a significantly higher mean GRS than the HC cohort (p < 0.001).
Conclusions: CD is characterised by both defective macrophage pro-inflammatory cytokine release in response to E. coli and an increased load of GWAS risk alleles. Although the molecular basis of impaired pro-inflammatory secretion remains incompletely understood, an increased burden of GWAS risk alleles is associated with decreased TNF release downstream of HkEc in CD, indicating possible cumulative effects of these variants on macrophage TNF secretion.
1. A. M. Smith et al. (2009), Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease, J. Exp. Med. 18831897, 206(9).
2. A. Franke et al. (2010), Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci, Nat. Genet. 11181125, 42.