1. DC-SIGN/HLA-DR+ cells increased in coeliac duodenum 1. Immunology Lab, Dept of Paediatrics & Immunology, University of Valladolid, IBGM-CSIC, Spain; 2. Department of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, the Netherlands; 3. Imperial College London, St. Mark’s and Northwick Park Hospital, United Kingdom; 4. Molecular Genetics, Hospital Universitario Rio Hortega, Valladolid, Spain. E-mail: earranz@ped.uva.es Montalvillo E. 1, García-Vallejo J.J. 2, Martínez-Abad B. 1, Vallejo-Diez S. 1,Escudero- Hernández C. 1, Bernardo D 3. Garrote J.A. 1,4, Arranz E. 1 INTRODUCTION: Coeliac Disease (CD) is an intestinal inflammatory disorder induced by the ingestion of dietary gluten which occurs in genetically predisposed individuals. The only treatment is a strict gluten-free diet (GFD), after which a complete remission of the clinical expression is found. DC-SIGN is a C-type lectin and has a high affinity for the ICAM3 molecule. It binds various microorganisms by recognizing high-mannose-containing glycoproteins on their envelopes. Besides functioning as an adhesion molecule, recent study has shown that DC-SIGN can initiate innate immunity by modulating toll-like receptors. DC-SIGN together with other C-type lectins is involved in recognition of tumors by dendritic cells and DC-SIGN is also a potential engineering target for dendritic cell based vaccine. OBJECTIVE: We aimed to determine the DC-SIGN expression by immunofluorescence in duodenal biopsies from coeliac patients and controls. PACIENTS & METHODS: We obtained endoscopic duodenal biopsies from 3 aCD, 3 GFD and 3 HC patients. Tissue was washed three times in PBS before to be embedded in OCT compound. Then, the samples were frozen in liquid nitrogen and stored at -80C for long-term use. After cutting 8μm tissue sections, slides were air dried overnight. We used acetone solution to fix the tissue sections. To rehydrate and to wash them we used PBS. Blocking was performed with 3% BSA in PBS before antibody staining. The staining was done adding rabbit DC-Sign anti-human ([1:1000], labelled with α-rabbit AlexaFluor 488 [1:500]) and mouse HLA-DR anti-human or mouse CD4 anti-human (both [1:500], labelled with α-mouse AlexaFluor 647[1:500]) for 1h at room temperature. We used the corresponding isotypes as controls. Slides were mounted using Vinol mounting medium with DAPI [1:10000] and viewed in a Leica DM6000 microscope. For the analysis, we used Leica Application Suite for Advanced Fluorescence software (Leica Microsystems, 2.3.5 version) CONCLUSSIONS: In aCD CD4 + T cell infiltration occurs in both the lamina propria and the epithelium. The T cells infiltration is accompanied with an increase in both the number of DC-SIGN + cells and the expression level of DC-SIGN on HLA-DR + cells in the lamina propria. This appears to be specific of aCD, as HC do not show CD4 + T cell infiltration and/or increased presence of DC-SIGN +. GFD patients also show a lower CD4 + T cell infiltrate and DC-SIGN expression. This decreased depends on the time elapsed since the start of the gluten- free diet. ACKNOWLEDGEMENTSACKNOWLEDGEMENTS: This project has been partially funded by University of Valladolid (VA016A10-2), Health Institute Carlos III (PI10/01647), Junta de Castilla y Leon (FPI, Montalvillo E.) and University of Valladolid (FPI-UVa; Escudero-Hernández C., Martínez-Abad B.) RESULTS Healthy ControlGluten Free Diet Celiac Disease Active Celiac Disease 20x In active Coeliac Disease CD4 + T cell infiltration occurs in both the lamina propria and the epithelium. As shown in Figure 1, the T cells infiltration is accompanied with an increase in both the number of DC-SIGN + cells and the expression level of DC-SIGN on HLA-DR + cells in the lamina propria. This appears to be specific of active CD, as healthy controls do not show CD4 + T cell infiltration and/or increased presence of DC-SIGN +. Diet- treated CD patients also show a lower CD4 + T cell infiltrate and DC-SIGN expression. This decreased depends on the time elapsed since the start of the gluten-free diet (fig. 1). By Immunofluorescence, DC-SIGN was found to be most highly expressed in the lamina propria cells of aCD patients (Fig. 2). In these patients, we have observed that all the DC-SIGN + cells are HLA-DR + (Fig. 2a). On the contrary, both healthy controls and diet-treated celiac patients show less DC-SIGN/HLA-DR + cells (2b). Also these samples show some HLA-DR + cells that do not express DC-SIGN (2c). 40x c Healthy ControlGluten Free Diet Celiac Disease Active Celiac Disease 20x 40x Figure 1. Immunofluorescence analysis of DC-Sign (green) and CD4 (red) in duodenal biopsies from aCD, GFD and healthy control patients, with a DAPI (blue) nuclear staining Figure 2. Immunofluorescence analysis of DC-Sign (green) and HLA-DR (red) in duodenal biopsies from aCD, GFD and healthy control patients, with a DAPI (blue) nuclear staining. 2c 2a 2c 2b