NON ABSORBABLE GLUCIDS (PREBIOTICS) DIRECTLY CONTRIBUTE TO THE INTESTINAL IMMUNE DEFENSE INCREASING THE PRODUCTION OF CYTOKINES BY ENTEROCYTES

B Ocón1, M Ortega2, CJ Aranda2, I Romero2, A Anzola1, M Guadix3, A Zarzuelo1, MD Suárez2, F Sánchez de Medina1, O Martínez2. 1University of Granada, Pharmacology 18071, Spain, 2University of Granada, Biochemistry and Molecular Biology ll 18071, Spain, 3University of Granada, Chemical Engineering, Spain

INTRODUCTION : Non absorbable glucids (NAGs) have generally prebiotic effects. They have been widely used for the treatment of chronic intestinal inflammation, showing benefits in colitis animal models. Here we test the hypothesis that these products have the ability to modulate the immune response directly, interacting with the intestinal epithelium irrespective of their well known prebiotic effects. NAGs assayed: fructooligosaccharides (FOS); inulin (INU); galactooligosaccharides (GOS); goat’s milk purified oligosaccharides (GMOS).

AIMS : (1) to evaluate the direct effects of NAGs on the enterocyte mediated immune response

(2) to elucidate the molecular process by which NAGs directly produce an immune response in enterocytes.

METHODS : Intestinal epithelial cell lines were cultured under the following conditions, with or without NAGs; Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS) 10%, 2 mM L-glutamine, 100 U·mL-1 penicillin, 0.1 mg·mL-1 streptomycin and 2.5 mg·mL-1 amphotericin B, except Caco-2/TC7 which were grown under the same medium described above but with FBS 20% instead. IEC18 cell line subsets knocked-down for either TLR4 or MyD88 were obtained following the manufacturer´s protocol (sc-156001-V) for toll-like receptor 4 (TLR4) and (sc-106986-V) for MyD88. Pharmacological inhibitory assays involving AKT, NF-kB or MAPK inhibitors were performed with the presence of those molecules from 1 hour before NAGs addition until samples collection.

RESULTS : NAGs added to the culture medium of intestinal epithelial cells 18 (IEC18 cells) for 24 hours after confluence stimulate the production of growth regulated oncogene alpha (GROα) and monocyte chemotactic protein (MCP1). The addition of a nuclear factor-kappa B (NFκB) inhibitor, but not an AKT inhibitor, caused a sharp inhibition in this stimulatory effect, indicating a major role of NFκB in the NAGs signaling pathway. Mitogen activated protein kinase (MAPK) inhibitors (JNK, p38 and ERK) were also tested and found to exert a small inhibitory effect. Myeloid differentiation primary response gene 88 (MyD88) and TLR4 expression was silenced in IEC18 cells making use of short hairpin (sh) RNA lentiviral particles. Both interventions partially inhibited NAGs effects. Therefore, our results show that NAGs are TLR4 ligands that activate descending signaling pathways in both a MyD88 dependent and independent fashion. Moreover, we pulsed IEC18 cells with lipopolysaccharide (LPS) after an hour of NAGs treatment and found a significant decrease in LPS-elicited cytokine production in those groups which received NAGs (with the exception of FOS) compared to control group, which also suggests that NAGs-triggered epithelial cell response is tightly dependent on TLR4 recognition.

In line with these findings, the response of the epithelial cell lines IEC18, Caco-2, Caco-2/TC7 and HT29 to NAGs correlates with both LPS effect and TLR4 messenger RNA levels. NAGs evoked MCP1 secretion in mouse colonic explants is significantly impaired in TLR4 KO mice. Thus, ex vivo results corroborate those previously observed in epithelial cell lines.

Finally, we have tested NAGs effects on the epithelial cell line (Caco-2) infection caused by either K12 or LF82 GFP-transformed E.Coli strains, analyzed by flow cytometry, an in vitro model developed in our laboratory. Several NAGs caused a sharp decrease in the LF82 dependent epithelial cell invasion rate, reaching well over 50% of inhibition compared to control after 24 h of treatment and 4 hours of bacteria-cells co-culture. This phenomenon might be closely related to the molecular findings described above.

CONCLUSION : NAGs increase the immune response of intestinal epithelial cells stimulating TLR4 and activating NF-κB (and secondarily MAPK) in both a MyD88 dependent and independent fashion.

Note; I´m really sorry but I couldn´t find the way to include the huge amount of data referred to the results I will include in the poster, owing to the word number restriction. I hope that´s not a problem for reviewers and the subsequent publication.