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P069. Anti-TNF induced regulatory macrophages display high levels of autophagy

A.C. Vos1, M. Wildenberg2, A. Levin2, G. van den Brink3, D.W. Hommes4

1Leiden University Medical Center, Gastroenterology-Hepatalogy, Leiden, Netherlands; 2Academic Medical Center, Intestinal and Liver Research, Amsterdam, Netherlands; 3Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands; 4UCLA, Division of Digestive Diseases, Los Angeles, United States

Background: One of the most exciting developments in IBD therapy has been the introduction of anti‑TNF agents. Although originally believed to function through neutralization of soluble TNF, we have previously shown that anti‑TNF agents induce regulatory macrophages which have immune-suppressive and wound healing capacity (Vos et al, Gastroenterology 2010). How these cells exert their effects has not been established thus far. Over the past few years, genome-wide association studies have shown a correlation between Crohn's Disease (CD) and a number of autophagy-related genes, implicating a role for autophagy dysfunction in the pathogenesis. We evaluated the induction of autophagy in anti‑TNF induced macrophages.

Methods: Mixed lymphocyte reactions were established using PBMC from two healthy donors in a 1:1 ratio and treated with anti‑TNF or IgG control. Subsequently, macrophages were isolated from the cultures using CD14-microbeads. Levels of autophagy were determined by enumeration of LC3+ spots and quantification of LC3-I/LC3-II ratios. In addition, expression levels of various autophagy-related genes were studied by gene array.

Results: Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes as indicated by LC3+ spots. In addition, conversion of the autophagosomal protein LC3 was increased in anti‑TNF treated cultures compared to IgG treated cultures. Finally, expression of a number of autophagy related genes including atg5, atg7, atg9 and atg16l2 were upregulated in the induced regulatory macrophages in comparison to inflammatory M1 type macrophages.

Conclusions: Regulatory macrophages induced by anti‑TNF therapy display increased levels of autophagy compared to IgG-induced or inflammatory macrophages. Given the role for impaired autophagy in the pathogenesis of CD, this may be an addition mechanism by which anti‑TNF compounds exert their beneficial effects.