Novel probiotic evidence of lactobacilli on immunomodulation and regulation of satiety hormones release in intestinal cells

Highlights

• Lactobacillus strains survive gut conditions in simulated digestion in vitro model.

• Studied Lactobacillus strains are able to adhere on Caco-2 cell model.

• Strains aren't cytotoxic on Caco-2 cells and modulate secretion of interleukins.

• Studied Lactobacillus strains modulate secretion of CCK and GLP-1 on STC-1 cells.

Abstract

Lactobacilli strains with probiotic traits were tested for their ability to survive to the digestion process modelled during this study. These strains managed to sustain the harsh conditions of the gastric and duodenal phases and showed good adhesion capacities to human Caco-2 cell line. These probiotic microorganisms have survived during these steps, exposed to low pH, high concentration of bile salts and enzymes occurring in the digestion and virtually reached the duodenal compartment in sufficient amount with limited population loss. These lactobacilli strains appeared to be non-cytotoxic after contact with Caco-2 cells for 24 h. Importantly, some of these strains showed immunomodulatory effect, lowering the pro-inflammatory cytokine IL-8 and promoting secretion of the anti-inflammatory IL-10. Besides, Lactobacillus gasseri CMUL34 and Lactobacillus acidophilus CMUL67 strains were able to modulate secretion and expression of two intestinal hormones: the Glucagon-Like Peptide 1 (GLP-1) and the cholecystokinin (CCK) in STC-1 cells.

Introduction

Gut microbiota is a major constituent of the morpho-functional system including intestinal epithelium and mucosal immune system (Scarpellini et al., 2010). This complex system is continually on a dynamic balance and is responsible of the host local intestinal integrity (Scarpellini et al., 2010). Probiotics are live microorganisms that when consumed in adequate amounts, produce beneficial effects on both digestive and systemic levels. Probiotics are overall safe, acid and bile tolerant, able to adhere and colonize the intestinal tract (Sanders et al., 2010). However, the term “probiotic” should be dedicated to live microbes for which a health benefit has been shown in controlled studies (Gaurner et al., 2008). The most commonly used microorganisms as probiotics are lactic acid bacteria (LAB), including Lactobacillus, Enterococcus, Streptococcus and Bifidobacterium species. Nevertheless, further microbial species including Escherichia coli Nissle, Bacillus cereus and Saccharomyces cerevisiae are also recognized as probiotics (Fijan, 2014). Indeed, lactobacilli colonize the gut and vagina of mammalians. They are widely described as beneficial microorganisms because of their aptitudes to relieve irritable bowel syndrome, reduce diarrhoea, promote immunity and restore the balance of gut microflora (Akoglu et al, 2015, Aragon et al, 2010, Galdeano, Perdigón, 2006, Mitsuoka, 1996, Orel, Kamhi-Trop, 2014, Van Niel et al, 2002). Moreover, lactobacilli play an important role in the protection of the host against harmful microorganisms and also strengthen the immune system (Soccol et al., 2010). Some species were described to improve food digestibility and reduce metabolic disorders (Kumar et al., 2012). LAB and particularly lactobacilli were used, since centuries, for food preservation and food fermentation as beneficial agents. LAB are able to modify physicochemical properties of food by fermentation, enhancing their taste and their digestibility and to prevent contamination of potential pathogens by lowering the pH (Gaurner et al., 2008). The genus Lactobacillus is commonly found in the upper gastrointestinal tract of mammals. To express their beneficial effects in the host, probiotic strains must be able to survive the passage through the digestive tract. Tolerance to high concentration of bile salts is an important parameter to microbial survival in the intestinal tract. So far, research has mainly focused on strains sensitivity toward low pH, proteolytic enzymes and bile salts (Sanders et al., 2010). In several reports, human enterocyte-like Caco-2 cells have been used for in vitro studies to elucidate the mechanisms of cellular adhesion of nonpathogenic lactobacilli (Chauvière et al, 1992, Ferreira et al, 2011). The ability of lactobacilli to adhere to mucosal surfaces of the intestine, and the subsequent long or short-term colonization have been the most commonly encountered criteria for the selection of probiotic strains.

Another recent probiotic lactobacilli interest is their action on gastrointestinal hormones involved in food intake regulation. Among these hormones, two are the focus of different investigations: the glucagon like peptide-1 (GLP-1) and the cholecystokinin (CCK) (de Graaf, Blom, Smeets, Stafleu, & Hendriks, 2004). These hormones were described to be involved in the control of appetite as satiation signals secreted by the intestine in response to meal stimulation (Mars et al, 2012, Moran, Dailey, 2011, Walsh, 1994). GLP-1 is produced by enteroendocrine L cells, stimulates glucose-induced insulin secretion and inhibits glucagon secretion. This hormone has two active forms, GLP-1 (7–36) amide and GLP-1 (7–37), product of a posttranslational processing of a proglucagon precursor in the mammalian intestine (Oh, Lee, Ko, Choi, & Kim, 2003). CCK is produced in the gut by enteroendocrine I cells and is a member of a peptide hormone family, characterized by the same carboxyl-terminal pentapeptide sequence (-Gly-Trp-Met-Asp-Phe-NH₂). This hormone is widely distributed throughout the central nervous system and the digestive tract (Oikonomou, Buchfelder, & Adams, 2008). The STC-1 plurihormonal cell line is derived from an endocrine tumour developed in the small intestine of a double transgenic mouse (Rindi et al., 1990). The STC-1, murine endocrine cells, are considered a suitable model for the in vitro study of gastrointestinal hormones release (Cordier-Bussat et al, 1997, Cordier-Bussat et al, 1998, Sufian et al, 2007). These endocrine cells are able to secrete both GLP-1 and CCK hormones (Cordier-Bussat et al, 1997, Cordier-Bussat et al, 1998, Cudennec et al, 2012, Geraedts et al, 2011).

This study provides further probiotics traits to recently proven beneficial lactobacilli isolated from vaginal origin of Lebanese women (Al Kassaa, Hamze, Hober, Chihib, & Drider, 2014). The resistance of these strains to the harsh conditions of the digestion process was evaluated throughout the buccal, gastric and duodenal phases simulated in vitro. Then the adhesion capacities of these lactobacilli strains were studied and their cytotoxicity toward Caco-2 cells was determined. Immunomodulatory effect of these strains, particularly on IL-6, IL-8 and IL-10 secretion, was also considered. Finally we look for the impact of these lactobacilli strains on the secretion and expression of CCK and GLP-1.