Anti-inflammatory effect of a fibronectin-binding protein expressing Lactococcus lactis containing a eukaryotic DNA vector coding for interleukin-10 using a murine model of Crohn´s disease

Abstract

Introduction: Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level and its absence is involved in inflammatory bowel diseases (IBD), such as Crohn's disease. However, oral treatment with IL-10 is limited due to its low survival in the gastrointestinal tract (GIT) and systemic treatments lead to undesirable side effects. In this context, the aim of this work was to evaluate the anti-inflammatory effect of the administration of an invasive Lactococcus lactis strain (L. lactis FnBPA+ that expresses the fibronectinbinding protein A of Staphylococcus aureus) capable of delivering a eukaryotic expression vector (pValac) containing the gene coding for IL-10 of Mus musculus, using a Trinitrobenzenesulfonic acid (TNBS) induced Crohn´s disease mouse model, as a new strategy for the prevention and treatment of IBD. Methods and Results: For this purpose, the pValac:IL-10 plasmid was firstly constructed and the expression of IL-10 by cells of the Chinese Hamster Ovary cell line, after transfection with the constructed plasmid, was confirmed by Confocal Microscopy and Flow Cytometry. Conventional BALB/c mice received an intra-rectal inoculation of TNBS to induce intestinal inflammation similar to human Crohn's disease. These mice then either received bacterial supplementation (108 UFC/mouse/day) of L. lactis FnBPA+ (pValac) (Wt-group), L. lactis FnBPA+ (pValac:IL-10) (pValac:IL-10 group) or saline solution (control group). Large intestines were removed, visually inspected for macroscopic evaluation and prepared for histological evaluation and immunohistochemistry using standard methods. The liver was aseptically homogenized and serial dilutions were plated in different growth media to determine microbial translocation. Mice from the pValac:IL-10 group showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), decreased numbers of IL-17 producing cells and lower microbial translocation to liver, compared to mice from the Wt-group or those that did not receive bacterial supplementation (control group). Conclusions: Administration of L. lactis FnBPA+ containing the pValac:IL-10 was effective in the prevention of inflammation in a murine model of Crohn's disease, confirming the potential use of therapeutic plasmids delivered by lactic acid bacteria, such as L. lactis, for the prevention and treatment of a diverse array of diseases.