Oral administration of a fusion protein between the B subunit of E. coli LT and a synapsin peptide prevents EAE by inducing Treg cells that modulates the cytokine balance

SCERBO MJ, BIBOLINI MJ, ROTH GA, MONFERRAN GC.

Viña del Mar, Chile

Congreso; 9° Congreso Latiniamericano de Inmunología; 2009

Sociedad latinoamericana de Autoinmunidad

The aim of this work was to characterise the cytokine profile and CD4 T cells populations induced by a protocol of oral tolerance using a fusion  protein (LTBSC) comprising the B subunit of the Heat Labil enterotoxin of E. coli and the C domine of rat Synapsin Ia on the Experimental Autoimmune Encephalomyelitis model. The B subunit of LT (LTB) has been used to direct coupled antigens peptides in order to modulate the immune response against autoantigens. EAE is a demielinating, autoimmune disorder mediated by autoreactive CD4+ against Mielin Basic Protein (PBM) used as a model of human demielinating diseases.       Methods: 43 days old wistar rat were orally fed with 4 doses of 10 ug of LTBSC, an equimolar dose of LTB + SC or vehicle (contro group), the days 10, 8, 6 and 4 previous to the challenge with bovine myelin in complete Freund`s adjuvant, to induce EAE. Mononuclear cells (MNC) were harvest from the mesenteric lymph nodes (MLN), inguinal lymph nodes and spleen previous the challenge to EAE and during the acute face of the disease. The production of  IL-4, IL-10, INF-g and TGF-b were analysed in supernatants of 48 hs  cultures of MNC with 1 ug/ml of ConA. MNC were stain with anti-CD4, anti-CD25 and anti-FoxP3 antibodies without any previous treatment, and the regulatory populations where analysed. Results: The oral treatment with LTBSC generates a decrease of the 50% in the incidence of the disease respect to the control group. This was accompanied by a significant increase the levels of IL-4, IL-10 and TGF-b and a decreased in INF-g in MLN, ILN and spleen the LTBSC group and in a lesser ….in the LTB+SC group, previous to EAE induction. This was accompanied by an increase in the CD4+CD25+FoxP3+ regulatory population. After de EAE challenge the levels of IL-4, IL-10 and TGF-b only remain up regulated in the LTBSC, as well as the triple positive CD4 population. The levels of INF-g in ILN and spleen remain unchanged so we evaluated the ratio INF-g/IL-10 that were significant lower in the LTBSC group respect the vehicle and LTB+SC group indicating a shift towards a regulatory microenvironment in the LTBSC group. The oral therapy with 4 doses of 10 ug of LTBSC reduced the incidence of the EAE by the induction of a regulatory microenvironment characterized by an increase in the levels of anti-inflamatory cytokines (IL-4, IL-10, TGF-b) and the regulatory CD4+CD25+FoxP3+ T cell population.