Evaluation of recombinant Lactococcus lactis strain producing human Pancreatitis-associated Protein I in the treatment of DNBS-induced colitis and 5-Fluoracil-induced mucositis in mice models

Abstract

Inflammatory Bowel Diseases (IBD), including ulcerative colitis (UC) and Crohns disease (CD) are complex intestinal disorders characterized by chronic inflammation of the gastrointestinal tract (GIT). IBD are caused by a deregulation of the mucosal immune system toward the native intestinal microbiota in genetically predisposed individuals, leading to excessive pro-inflammatory immune responses in the GIT. Theincidence of both diseases is increasing in developed countries turning CD and UC a main gastroenterological problem as current treatment drugs are associated with serious side effects. Thus, recent research is focusing on the development of new strategies for the treatment of IBD. Probiotic bacteria, especially the ones belonging to the lactic acid bacteria (LAB) group, were shown to be capable of prevent and treat IBDby restoring the balance of disrupted microbiota and suppressing pro-inflammatory immune responses. In order to increase LAB probiotic effect, molecular cloning and expression of anti-inflammatory molecules are being carried out and LAB recombinant strains are also being evaluated as an alternative treatment for IBD. As the use of these strains, especially the model Lactococcus lactis, showed to be very effective infighting intestinal inflammation, its administration as a therapy for treating other human GIT inflammatory diseases, such as mucositis, are also being evaluated. This disorder is a common side effect of patients undergoing radiotherapy or chemotherapy that strongly affects their quality of life. Like IBD, treatment for mucositis is very limited withfew medicaments and procedures described to contain inflammation. Therefore, given the need to develop alternative treatments for both IBD and mucositis, this study aimed to test the efficacy of either dairy L. lactis NZ9000 or recombinant L. lactis strain expressing Pancreatitis Associated Protein I (PAP) to fight inflammation in mouse models of IBD and mucositis. PAP has been reported as a protein with antimicrobialproperties that plays important roles to keep intestinal homeostasis. Firstly, we constructed and confirmed the expression of human PAP by recombinant L. Lactis. Afterwards, we evaluated the therapeutic effect of this strain in a mice model of dinitrobenzenosulfonic acid (DNBS)-induced colitis. We demonstrated that DNBS injection was able to cause severe acute inflammation in the colon of mice. Moreover,PAP delivery by lactococci protected animals from weight loss, intestinal permeability, and tissue damage. In addition, L. lactis-PAP treatment decreased Th1 (IFN), Th2 (IL- 4, IL-5) and Th17 (IL-17) type-immune responses. It was also observed a higher expression of regulatory TGF- cytokine and increased amount of T regulatory cells intreated mice. The anti-inflammatory effects of both L. lactis-PAP and dairy L. lactis NZ9000 strains were also measured in 5-fluoracil mucositis model. We showed that this model was successfully reproduced in BALB/c mice with an induction of acute inflammation in the small bowel of animals. Administration of L. lactis NZ9000 harboring pSEC vector without the cDNA of PAP was able to prevent histological damage, reduce eosinophils infiltrate and IgA secretion in the ileum of mice. On theother hand, L. lactis expressing PAP preserved mucosal architecture and improved Paneth cells activity. Taking together, our results demonstrate that L. lactis, expressing PAP peptide is a promising strategy to treat IBD. Moreover, L. lactis NZ9000 strain, derived from dairy L. lactis MG1363 used extensively for cheese production, surprisingly presented anti-inflammatory effects in mice injected with 5-FU.