Introduction Patients with Crohn’s disease exhibit an attenuated inflammatory response after trauma or Escherichia coli infection and have delayed clearance of subcutaneous bacteria compared to healthy controls. Adherent, invasive E. coli, increased in Crohn’s disease, replicate within macrophages and may have a primary pathogenic role. Crohn’s disease patients’ macrophages may have a primary defect in bacterial killing, allowing survival of AIEC.
Methods We aimed to assess the relative ability of monocyte-derived macrophages from Crohn’s patients to kill intracellular E. coli and Staphylococcus aureus compared to healthy controls. Peripheral blood monocytes were obtained from consenting adults by centrifugation over Lymphoprep™ followed by 2h adherence to plastic Nuncò tissue culture dishes and subsequent differentiation into macrophages by 5d culture (as per Smith et al , J. Exp. Med. 2009, 206: 1883–97). Macrophages were infected with an adherent, invasive E. coli HM605, E. coli K12 or Staph. aureus Oxford strain. Intramacrophage killing was assessed using the gentamicin protection assay. Cytokine release to the culture medium was also determined by sandwich ELISA. Macrophage-mediated chemotaxis of human neutrophils, obtained from healthy controls by centrifugation over Polymorphoprep™, was quantified in Boyden chambers.
Results No significant difference in the relative killing of E.coli K12 (-14 ± 11% vs. -45 ± 9%) and Staph. aureus (-52 ± 4% vs. -63 ± 5%) nor in the relative survival of AIEC HM605 (+50 ± 26% vs. +8 ± 22%) within monocyte-derived macrophages was seen (healthy controls vs. Crohn’s disease respectively; n = 10 each group, ANOVA). TNFα, IL-6 and IL-8 production were not significantly different between the two groups and macrophage mediated neutrophil chemotaxis was equivalent. Smoking status did not affect bacterial survival, with no differences observed in killing between current smokers, ex-smokers and non-smokers.
Conclusion AIEC are ineffectively killed by both Crohn’s disease and healthy macrophages. Macrophages from patients with Crohn’s disease do not appear to have an inherent defect in killing and exhibit equivalent ability to induce neutrophil chemotaxis relative to controls. These data suggest circulating inhibitors of Neutrophil chemotaxis may explain the previously observed defective neutrophil chemotaxis and bacterial clearance in vivo.
Disclosure of Interest P. Flanagan Grant/research support from: Awarded a Shire innovation fund for SpRs, S. Subramanian: None Declared, B. Campbell: None Declared, J. Rhodes Consultant for: A member of advisory boards for Atlantic, Procter and Gamble and Falk, Speaker bureau with: Has received speaking honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble, Schering Plough, Shire and Wyeth, Conflict with: With the University of Liverpool and Provexis UK, holds a patent for use of a soluble fibre preparation as maintenance therapy for Crohn’s disease plus a patent pending for its use in antibiotic-associated diarrhoea.
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