W1215 MDP Stimulates Memory T-Cell Recruitment Via NOD2 Connecting Bacterial Sensing and Adaptive Immunity

Abstract

BACKGROUND: Macrophage inflammatory protein (Mip)-3α is a CC chemokine attracting memory T lymphocytes and is induced during differentiation of blood monocytes into intestinal macrophages (IMACs) in the intestinal mucosa. It is further released via NFκB activation upon stimulation with proinflammatory cytokines, such as TNF. Bacterial translocation across the epithelial barrier is crucial during the pathogenesis of Crohn's disease (CD). NOD2 is an intracellular sensor of bacterial muramyl dipeptide (MDP) and is able to activate the NF-κB pathway. NOD2 variants have been shown to be the most important susceptibility factor for the development of CD. We have tested the ability of MDP to induce Mip-3α expression and subsequent recruitment of memory T lymphocytes. METHODS: Cells were transfected with vector (pcDNA) to express wtNOD2, SNP13 or empty vector. Cells were stimulated with 0/100/500/1000/2000 ng/ml MDP for 18 hours. Supernatants were collected and Mip-3α was quantified by ELISA and real time-PCR. Functional assays on migration of memory (CD45R0+) T cells were performed in a three dimensional In Vitro differentiation model for IMACs: the multicellular spheroid (MCS) coculture model. Neutralizing antibodies to human Mip-3α (polyclonal, rabbit, acris-antibodies; final concentrations, 15, 1.5, and 0.15 mg/ml) were used for blocking studies. RESULTS: Taqman analysis revealed significantly elevated Mip-3αmRNA expression in HEK293 cells transfected with pcDNA to express NOD2 after stimulation with MDP. Increased Mip-3α secretion was demonstrated by ELISA. MDP induced Mip-3α mRNA and protein secretion in a dose dependent manner. A functional deficit was found after transfection with pcDNA to express SNP13. During the differentiation of monocytes into IMACs Mip-3α was induced in the MCS-In Vitro model during 7 days of coculture. Mip-3α+ IMACs attracted CD45R0+ T cells to migrate into the MCS. CONCLUSION: Mip-3α is induced during differentiation of IMACs. There is evidence for a NOD2 mediated induction of Mip-3α expression followed by subsequent memory T-cell recruitment connecting bacterial sensingwithmemory T-cell functions.