W1799 Defective In Vitro Macrophage Maturation in Crohn's Disease

Abstract

Introduction Whist the cause of Crohn9s disease (CD) remains unclear, an innate defect in the handling of intracellular bacteria appears to be involved. Evidence for this comes from observations of a defect in phagocyte recruitment and the presence of viable E coli within lamina propria (LP) macrophages in these patients. LP macrophages are derived from blood monocytes and develop into pro inflammatory cells under the influence of Th1 cytokine (M1) or anti inflammatory regulatory macrophages (M2) by Th2 cytokines. It is known that macrophage turnover in CD is higher and maturation incomplete compared to those in healthy individuals so these cells are a good candidate for the observed differences. Methods To study in vitro the cytokine-driven differentiation of macrophages from patients with IBD (5 CD, 5 UC) and five healthy controls and follow their behaviour when challenged with CD derived strains of E coli . Monocytes were stimulated for 6 days with INF-γ, IL-4 + IL-13 or IL-10 to generate M1, M2a or M2c macrophage populations, respectively. Specific maturation markers (CD14, CD16, CD33, CD163, CD206, CD209, CCR2 and CCR7) were then measured using flow cytometry. These cells were then challenged with E coli and phagocytic function measured using the gentamicin protection assay. Results Monocytes stimulated with INFγ, from both the control and IBD groups gave a similar M1 type profile. IL-4+IL-13 stimulation gave rise to M2a cells, however the cells isolated from the CD group expressed a higher percentage of CD14 and a greater proportion of these cells expressed low levels of CD163 and CD206. In the CD group, IL-10 gave rise to a population that expressed CD14+, CD16+ and were CD163++, CD206+ and CD209+. When these different cell populations were challenged with E coli the number of recovered viable bacteria at 30 min gave similar numbers of internalised bacteria. However when we recovered bacteria 3 h post infection, significantly higher survival of bacteria in the IL-10, M2c population in the CD Group (M1 cells, 14±9; M2a cells, 23±8; M2c cells, 135±41) was found. No bacteria survived in the controls or UC. Conclusion We have demonstrated a population of IL-10 induced M2c cells that are unable to complete the process of bacterial killing effectively in patients with CD. These CD163+ cells are similar to a subpopulation of lamina propria macrophages that have been shown harbour viable bacteria. This may indicate that a defect in the migration and maturation of monocytes, unable to clear bacteria efficiently, may be the underlying innate defect in CD.