Inflammatory response, antioxidant and gut microbiota alteration in a murine model of inflammation challenged with Salmonella enterica serovar Typhimurium consuming milk kefir
Authors:   , Pereira Mariana de Fátima Albuquerque 
Published: August 2023

The use of probiotics has been suggested as a possible bacterial prophylaxis against Salmonella infections because some probiotic strains have protective effects. The combination of several mechanisms is described in the literature, such as the production of antibacterial substances against Salmonella and the increase of the host immune response against infection in the intestinal mucosa. Among the microorganisms used in the production of probiotic fermented beverages, the kefir culture stands out. Kefir is a fermented beverage composed of a microbial community that lives in a symbiotic association. Studies report that the use of milk kefir has anti-inflammatory, antimicrobial, antioxidant activities, in addition to increasing the concentration of short-chain fatty acids (SCFAs), altering the microbiota, among other benefits to intestinal health. An unbalanced composition of the intestinal microbiota, known as dysbiosis, is involved not only in intestinal diseases, but also in pathologies of other organs, such as the brain. Then, through the production of hormones, immunological factors and metabolites, the intestinal microbiota can modulate the intestinal environment and interfere with the behavior and function of the host's central nervous system. However, there are still gaps regarding the role of kefir in the mechanisms of action in acute infections and safety. Thus, the general objective of our work is to investigate the effect of milk kefir consumption on the immune system, antioxidant and intestinal microbiota composition of wild-type C57BL-6 and IL-10 knockout mice on health and oral infection by Salmonella enterica serovar Typhimurium. In manuscript 1, the objective was to review data from studies, from the last 10 years in different databases, with murine models on the role of kefir against inflammation and the main response mechanisms involved in this process. In total, 23 studies were included in this review. The experimental design of manuscript 2 was carried out with C57BL6 mice (n=20) subdivided into groups that received 0.1mL of water or 0.1mL (10% w/v) of kefir. The kefir underwent maturation for 48 hours, and was then administered via orogastric route, to the animals for 4 weeks. In the manuscript 3 experiment, C57BL-6 wild type (WT) (n=40) and C57BL-6 IL-10 -/- (KO) (n = 40) mice were subdivided into eight experimental groups treated or not with kefir. In the first 15 days, the water groups received filtered water (0.1 mL) while the kefir groups received milk kefir (0.1 mL, 10% w/v) via orogastric route. Then, two groups of each strain received a single dose (0.1 mL) of S. Typhimurium inoculum (ATCC 14028, dose: 10 6 CFU/mL). According to the revised data (Manuscript 1), kefir was shown to act reducing inflammation, initially by alternating between innate, Th1 and Th2 responses, reducing pro- inflammatory cytokines while increasing anti-inflammatory ones. In addition, it also acts in the mediation of immunomodulatory and protective effects through the numerous metabolites and organic acids produced and secreted by kefir in the intestinal microbiota, and has also been shown to act as an antihypertensive, antioxidant, antitumor and hypocholesterolemic and hypoglycemic action, factors that contribute to reducing inflammation. In manuscript 2, the aim was to analyze the microbiota profile of milk kefir and its effect on metabolism, oxidative stress, and the microbiota-gut-brain axis in a healthy murine model. Milk kefir drink exhibited high antioxidant potency compared to milk and in vivo increased antioxidant enzymes. The composition of the microbiota of the drink and the fecal microbiota were different, but composed mainly of SCFA-producing bacteria (Comamonas in the drink; Lachnospiraceae and Lachnoclostridium in mice). In metabolism, kefir improves liver function, reduces triglycerides and uric acid. The increase in cerebral and fecal SCFAs and the antioxidant effect found are associated with the change in the intestinal microbiota caused by kefir, which indicates that kefir positively influences the gut-microbiota-brain axis and contributes to the preservation of intestinal and brain health. In manuscript 3, our objective was to evaluate the role of IL-10 in inflammation and gut microbiome in mice consuming milk kefir and orally challenged with Salmonella enterica serovar Typhimurium. Kefir was observed to prevent systemic infections only in IL-10 -/- mice, which were able to survive, regulate cytokines, and control colonic inflammation. The abundance in Lachnospiraceae and Roseburia and the higher production of SCFA in the pre-infection showed that kefir has a role in intestinal health and protection, colonizing and offering competition for nutrients with the pathogen, in addition to acting in the regulation of Salmonella infectivity only in the lack of IL-10. These results demonstrate the role of IL-10 in the prognosis of salmonellosis and how milk kefir can be used in acute infections. Keywords: Milk kefir. Probiotic. Fermented milk. C57BL-6 mice. Salmonella enterica serovar Typhimurium. Inflammation. Anti-inflammatory pathways. Immune system. Bacterial diversity. Short-chain fatty acids. Microbiota-gut-brain axis. Fecal microbio- ta. Kefir microbiota.