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P014. The inflammasome induces macrophage-mediated disruption of the epithelial barrier

D. Lissner1, M. Schumann1, L.-I. Kredel1, A. Batra1, T. Stroh1, A. Kühl1, B. Siegmund1, 1Charité – Universitätsmedizin Berlin, Gastroenterologie/Infektiologie/Rheumatologie, Berlin, Germany

Background

The inflammasome with its two sharply regulated cytokines Interleukin-1β (IL-1β) and IL-18 is crucial for the maintenance of intestinal homeostasis. In this study, we wanted to examine the relevance of inflammasome-mediated pathways on disruption of the epithelial barrier through pro-inflammatory macrophage subtypes.

Methods

Unpolarized macrophages (M0) were isolated from human peripheral blood and polarized into M1- and M2-macrophages. The cells were stimulated by the addition of lipopolysaccharide (LPS). The influence of these cells types on epithelial integrity was analyzed using resistance measurements on different epithelial cell lines (Caco-2, T-84, HT-29/B6). Cytokine concentration was measured using cytometric bead array. Inhibition of IL-1β and IL-18 was performed using anakinra, a specific IL-1β receptor antagonist (15 µg/ml), or a specific monoclonal IL-18 antibody (1 µg/ml, MBL, Woburn, MA), respectively.

Results

M0-macrophages as well as M1-polarized macrophages caused a massive decrease in epithelial resistance of epithelial cell layers of all subtypes, compared to M2-macrophages. This was further amplified by the addition of LPS. Staining of the epithelial cell layers revealed deregulation of various tight junction proteins (Claudin-1, Claudin-2, Zonula occludens-1) as well as induction of apoptosis in epithelial cells. M0-macrophages predominantly expressed IL-1β and IL-18, which was hardly found in the supernatants of polarized macrophages. M1-macrophages expressed high concentrations of TNF α, whereas the predominant cytokine for M2-macrophages was IL-10. The effect of M0-macrophages on the epithelial cell layer was significantly reduced by inhibition of IL-18, which was not seen for polarized macrophages. The inhibition of IL-1β by anakinra caused a trend in improvement of the epithelial damage caused by M0-macrophages. Again, this was not seen for the polarized macrophages subtypes.

Conclusion

M0- and M1-macrophages are capable of disrupting the epithelial barrier. For M0-macrophages, this is mediated through activation of the inflammasome, as suggested by cytokine inhibition studies.