Increased production of IL-15 protein in adult coeliac disease patients Introduction: Patients with coeliac disease (CD) may have an increased expression of the specific receptor of interleukin (IL)-15 (IL-15Ra) but not in healthy individuals. (Bernardo D. et al. 2008) Furthermore, gluten ingestion in both groups increases the expression of IL-15. Therefore, the study of this cytokine and its receptor could be of great interest in order to obtain a better understanding of CD pathogenesis. Patients and methods: Western blot has been used to determine the expression of IL-15 and IL-15Ra using  actin as a reference. Samples: duodenal biopsies from coeliac patients, CD (n=17: 6 children, 7 adults, 4 non - good responders to gluten-free diet (GFD), one of which is refractory CD patient), non-coeliac pathologic controls, Pc (n=10) and apparently healthy individuals, Hc (n=10). Versadoc and Quantity One sorftwares (BioRad) were used to develop and quantify, respectively. Quantitative PCR using the same samples as western blot was performed for analysis of IL-15Ra, IL-15, STAT3, STAT5A, STAT5B, IL-21 and IFN-g. GADPH was quantified as a reference. We used also flow cytometry to study cell level expression of IL-15 and IL-15Ra, extracellular and intracellular, using duodenal biopsies from coeliac patients (n=5), non-coeliac pathologic controls (n=4) and apparently healthy individuals (n=5) Intraepithelial lympocytes (IELs) and lamina propia lymphocytes (LPLs) were separated using 1M DTT, 1M EDTA and incubated in completed culture medium for 1h at 37ºC. Lamina propia was then isolated using collagenase (2h at 37ºC) Statistical analysis were performed using the non-parametric Mann-Whitney’s test and t-test. Results: Western blot: IL-15Ra analisys shows two bands (approx. molecular weights of 40 and 48KDa) whereas IL- 15 shows five bands: approx. 14KDa (IL-15), 17 KDa (Short signal peptide IL-15) and 20KDa (long signal peptide IL-15), but also 40 KDa and 48KDa bands. (Fig. 1) The appearance of the two high molecular bands in IL-15 and IL-15Ra western blots indicate that both proteins are bound together. The different molecular weight could be due to a different N-glycosilation process inside cells; being the high molecular weight band the one secreted in the cellular membrane. Densitometric analysis: some degree of increase of IL-15 secretion (48KDa band) in adult coeliac patients when compared to healthy controls (p=0.184) and paediatric CD patients (p=0.174) was observed. Surprisingly, levels of secreted IL-15 are significantly decreased in non-responding CD patients (p<0.1) Finally, IL-15Ra western blot did not show any significant variation, though a tendency can be appreciated. (Fig.2) Flow cytometry: A set of IL-15Ra, IL-15, CD45 and CD3 or CD11c antibodies were used to assess the amounts of IL-15 and IL-15Ra proteins in both duodenal intraepithelial and lamina propria lymphocytes and dendritic cells. No significant results were obtained. Compared to western blot, flow cytometry does not provide additional information. Conclusions: IL-15 is tightly regulated at a post-transcriptional level. Because of that, IL-15 should be bore in mind together with its specific receptor IL-15Ra. Here we demonstrate that the IL-15/IL- 15Ra assosiation can be detected by western blot. Furthermore, soluble IL-15Ra (sIL-15Ra) could have been detected (approx. Molecular weight: 42KDa) (Duitman E. et al. 2008) Although IL15 expression is not very reliable because of its strict post-transcriptional regulation, IL-15Ra expression shows the same patter as western blot for pathological controls, children CD and adult CD patients. What is more, different expression patterns in other IL-15-related molecules could be important in different ages and/or stages of the disease (non-good responders to GFD express less STAT5A). On the other hand, GFD non-responding patients do not reach the expected increased levels of IL-15 mRNA expressión or protein as well as IFN-g mRNA when compared to the remainder CD groups. ACKNOWLEDGEMENTS: 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.) Escudero-Hernández C. 1 ; Vallejo-Díez S. 1 ; Martínez-Abad B. 1 ; Montalvillo E. 1 ; Marugán M. 4 ; Fernández-Salazar L. 3 ; Garrote J.A. 2 ; Arranz E Mucosal Immunology Lab, IBGM, University of Valladolid-CSIC, Spain 2. Molecular Genetics, Hospital Universitario Rio Hortega, Valladolid, Spain. 3. Gastroenterology Service, Complejo Hospitalario de León, Spain 4. Paediatrics Service, Hospital Clínico Universitario Valladolid, Spain. Corresponding author : Figure 2 : Graphic representation of densitometric analysis of IL-15Ra (A) and IL-15 (B) western blot and median values for each group. Hc, healthy individuals; Pc, pathological controls; CD, coeliac disease patients; Non-resp. GFD, non-responders to gluten-free diet. Figure 1: Representative IL-15Ra (A) and IL-15 (B) western blots. IL- 15Ra western blot shows two bands (approx. molecular weights of 40 and 48KDa) IL-15 western blot show five bands: approx. 14KDa (IL-15), 17 KDa (Short signal peptide IL-15) and 20KDa (long signal peptide IL-15), but also 40 KDa and 48KDa bands. Healthy controls, Hc; pathologic controls, Pc; coeliac disease, CD. 1.A 1.B Hc Pc Pc Pc child CD adult CD Figure 4: Relative quantification (2 -(  Ct ) method) of IL-15, STAT3, STAT5A and STAT5B expression by qPCR. Figure 5: Relative quantification (2 -(  Ct ) method) of IL-15, IL-21 and IFN-g expression by qPCR. Quantitative PCR: IL-15 expression was compared to STAT3, STAT5A and STAT5B, as molecules included in the JAK/STAT signaling pathway through IL-15Ra activation by IL-15. (Fig.4) While healthy control expression show lower levels in each molecule, pathological controls and CD patients show higher expression of STAT5A and STAT5B. STAT3 is the only molecule where paediatric CD levels are similar to healthy controls. On the other hand, non-responding GFD CD patients show similar levels of mRNA for IL-15, STAT3 and STAT5B, but STAT5A. (Fig.4) IL-21 expression pattern do not display any possible increment. IFN-g was analyzed because of its ability of inducing IL-15, but also as an imflammatory marker. Its relative quantification is consistent with previous studies. (Fig.5) 48KDa 40KDa 48 and 40KDa IL-15 bands 2.A2.B 3 45 Figure 3: Graphic representation of the relative quantification (2 - (  Ct ) method) of IL-15Ra and IL-15 expression respect to healthy controls by qPCR. References:: Bernardo D. et al Clin. Exp. Immunol., 154: “Higher constitutive IL-15Ra expression and lower IL-15 response threshold in coeliac disease patients”. Duitman E.H. et al Mol. Cell. Biol. 48 (15): “How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: Lessons from interleukin-15 (IL-15) / IL-15 receptor alpha”.