Avaliação do efeito anti-inflamatório de linhagens de Lactococcus lactis pEXU:gdf11 em um modelo murino de mucosite intestinal induzida por 5-fluorouracil

Abstract

Lactic Acid Bacteria (LAB) are the main representative species of the probiotic microorganisms and their use as Genetic Modified Organisms (GMO) has been demonstrated through several studies showing promising approaches, especially for the treatment of inflammatory diseases of the Gastrointestinal Tract (GIT). The LAB model Lactococcus lactis displays a myriad of recombinant genetic tools, such as heterologous expression systems and shuttle vectors, therefore presenting great advantages on the development of novel therapies based on the delivery of molecular targets in situ, especially to the mucosa. Moreover, some strains present probiotic properties and are Generally Recognized as Safe (GRAS). Mucositis is an inflammatory process of the GIT induced by chemotherapeutic agent 5-fluorouracil (5-FU) as a side effect often reported in chemotherapy patients. The small bowel is heavily affected by 5- FU, whose main mechanism of action occurs through blocking DNA and RNA synthesis, hence, inhibiting cell proliferation and leading to apoptosis of both malignant and healthy cells. Besides cell death, intestinal mucositis is characterized by a pro-inflammatory response that leads to generation of Reactive Oxygen Species (ROS) and activation of pro-inflammatory cytokines, that, in turn, will exacerbate mucosal damage and dysfunction of the gut microbiota. Currently, there is no cure available for mucositis, although novel alternative therapeutic and preventive approaches have been glimpsed. In the search for recombinant molecular targets with anti-inflammatory potential, the GDF11 protein has been reported by previous pre-clinical studies as a promising candidate for the treatment of other inflammatory conditions such as rheumatoid arthritis, psoriasis and Ulcerative Colitis. In this context, this work aimed at investigating the anti-inflammatory effects of GDF11 protein-coded DNA molecules delivered by L. lactis strains as in a murine intestinal mucositis model induced by 5-FU. To achieve that, the coding sequence of GDF11 was cloned to eukaryotic expression vector pEXU and transformed into strains L. lactis subps. lactis NCDO 2118 and L. lactis subsp. cremoris MG1363. Then, BALB/c male mice (n=7) received recombinant strains via gavage in a daily rationale during 13 days. On day 10, mice were given an intraperitoneal injection of 5-FU and 72 hours later, animals were euthanized and the ileum was sampled to perform histological analysis, evaluation of inflammatory infiltrate and evaluation of expression of inflammatory markers. Besides, body weight and food and water intake were daily assessed. Administration of recombinant strains of L. lactis pEXU:gdf11 showed no effect on food and water intake as well as no prevention of body weight loss on animals that received 5-FU. However, mice receiving L. lactis NCDO 2118 pEXU:gdf11 showed significant attenuation of histopathological damage specially in reduction of inflammatory infiltrate, despite no effect was seen on villus and crypt depth shortening. Gene expression analysis revealed the administration of both recombinant lactococci strains decreased the inflammatory response via Nlrp3 and NfkB down-regulation, while no decrease was observed in Tnf expression levels. Moreover, an increase of Il10 levels was also observed on the same groups. Therefore, this study concludes that GDF11 delivered by L. lactis is able to down-regulate mucositis immune responses. However, only the recombinant strain L. lactis NCDO 2118 pEXU:gdf11 was capable of attenuating intestinal damage induced by 5-FU. Hence, this recombinant strain could be a potential therapeutic alternative for intestinal mucositis.