In vitro screening of probiotics and synbiotics according to anti-inflammatory and anti-proliferative effects

https://doi.org/10.1016/j.ijfoodmicro.2010.09.007Get rights and content

Abstract

There is emerging evidence of the efficiency of probiotic, prebiotic and synbiotic treatments in inflammatory bowel diseases (IBDs) and one of their long-term complications, colorectal cancer (CRC). In this study, various strains of probiotic lactic acid bacteria, prebiotic glucooligosaccharides (GOS) or a synbiotic combination of the two were screened for anti-inflammatory and anti-proliferative effects in different in vitro models in the context of such diseases. To mimic IBD response to Gram negative bacteria, HT-29 cells were sensitised to inflammatory response to lipopolysaccharide (LPS) by IFNγ which increased expression of TLR4, the LPS biosensor, and were then treated by probiotics, prebiotics and synbiotics. Secreted IL-8 and activated NF-κB were monitored as inflammation biomarkers. A selection of active strains were then subjected to a second inflammatory cell culture model consisting of inflammatory activated transgenic Caco-2 cells transfected by a reporter gene under the control of NF-κB inducible promoter. Quantification of reporter gene expression allowed us to demonstrate some probiotic inhibitory properties or to confirm such characteristics in two different models. Proliferation of cancerous HT-29 cells was monitored by XTT assay. Only three probiotic strains induced a proliferation decrease, but with a lack of reproducibility. Binary or ternary probiotic associations, complemented or not by prebiotic GOS, significantly decreased proliferation, especially with a synbiotic association of Bifidobacterium breve, Lactococcus lactis and oligoalternan, a GOS. This combination was selected for the following experiments. We showed the involvement of both bacterial and carbohydrate compounds of this synbiotic in the observed effect by dose range tests. We demonstrated that this decrease in proliferation may be due to an induction of a differentiated phenotype, as shown by the up-regulation of intestinal alkaline phosphatase, a biomarker of differentiation, monitored by real-time RT-PCR in HT-29 cells treated by the selected synbiotics. Thus, this study demonstrates the ability of probiotics to exert anti-inflammatory effects and shows some anti-proliferative characteristics for a specific synbiotics. These products should be further evaluated in animal models to confirm the in vitro results.

Introduction

Inflammatory bowel diseases (IBDs) are mainly represented by ulcerative colitis and Crohn's disease and have had a high and increasing prevalence in western countries during recent decades (Karlinger et al., 2000, Lakatos, 2006). These pathologies are characterized by an excessive and prolonged inflammatory response (Laroux et al., 2001) and, despite their unknown aetiology, genetic and environmental risk factors seem to be involved in IBD pathogenesis (Bosani et al., 2009). Some genetic alterations in IBDs concern microbial Pattern Recognition Receptors (PRRs), such as NODs or TLRs, which influence NF-κB activation and lead to a deregulated inflammation of the intestinal mucosa (Yamamoto-Furusho, 2007). Environmental risk factors are related to smoking, appendectomy or the diet of western countries (Loftus, 2004). The key role of microbiota imbalance has been highlighted in IBDs in several studies, with a loss of diversity and an increase in Gram negative bacteria (Baumgart et al., 2007, Gradel et al., 2009, Wohlgemuth et al., 2009). Lipopolysaccharide (LPS) of Gram negative bacteria is recognised by PRRs and triggers an inflammatory response through the NF-κB pathway and the subsequent overexpression of pro-inflammatory cytokines like IL-8 (Riedel et al., 2006). The major involvement of gut microbiota imbalance in IBD pathogenesis suggests that modulating this part of the intestinal ecosystem could help to prevent such diseases. Probiotics, prebiotics and synbiotics are new concepts recently tried in IBD prevention and/or treatment. Some probiotics, which are defined as “live microorganisms which beneficially affect the health of the host” (FAO/WHO, 2002), belonging to lactic acid bacteria and especially the genera Bifidobacterium and Lactobacillus, have recently been demonstrated to have therapeutic properties in IBD (Isaacs and Herfarth, 2008, Martins et al., 2009, Vanderpool et al., 2008). Probiotics could also prevent IBD crises by inhibiting pathogenic bacteria (Servin, 2004) or modulating intestinal mucosa immunity (Delcenserie et al., 2008). The revised definition of a prebiotic is “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.” (Roberfroid, 2007). Most of the prebiotics studied have been carbohydrate polymers and they have been shown to be effective in attenuating inflammation. Prebiotics exert their anti-inflammatory effects through different pathways such as fermentation into short chain fatty acids (SCFA) by colic microflora (Lara-Villoslada et al., 2006) or the specific stimulation of beneficial gut microflora (Hoentjen et al., 2005). This observation has led to the development of the concept of synbiotics as a synergistic combination of probiotics and prebiotics (Roberfroid, 1998). They have been demonstrated to be efficient in preventing IBD symptoms and improving patients' quality of life in clinical trials (Fujimori et al., 2009, Furrie et al., 2005).

Chronic inflammation occurring in IBDs induces persistent damage along the digestive tract and plays a role in the long-term development of colorectal cancer (CRC) (McConnell and Yang, 2009). As for classical chronic inflammatory symptoms, some data suggest that probiotics could be useful to prevent such pathologies. Lactobacillus salivarius UCC118 was shown to reduce both inflammatory response and gastrointestinal tumour development in IL-10 knockout mice (O'Mahony et al., 2001). Lactobacillus peptidoglycan is able to induce Th1 response and thus activate peritoneal macrophages which exert effects against cancer CT26 cell development in mice (Sun et al., 2005). Prebiotics have also been evaluated for their antitumorigenic effects. Germinated barley foodstuff treatments showed some significant protective effects against chemically induced CRC in rat models (Kanauchi et al., 2008). Furthermore, resistant starch induced apoptosis of cancerous cells in another CRC rat model (Bauer-Marinovic et al., 2006). These results could be explained by the selective induction of growth of beneficial bacteria by prebotiotics and their fermentation leading to antitumorigenic compounds. These results obtained with probiotics and prebiotics against CRC led to the assaying of synbiotic anticancer properties in an attempt to increase the effects one of these compounds in comparison with when the components were used alone. In a CRC rat model, Bifidobacterium longum and inulin showed a synergistic reduction of colon carcinogenesis (Rowland et al., 1998) and B. lactis and resistant starch induced cancerous cell apoptosis (Le Leu et al., 2005).

These results suggest that probiotics, prebiotics and synbiotics could be useful in IBD and/or CRC prevention or treatments. In the present study, we first aimed to evaluate the anti-inflammatory characteristics of products in in vitro models by monitoring inflammation biomarkers. For a second aim, we tested them for their anti-proliferative properties on cancer cell lines. All assays were conducted with probiotics and/or with glucooligosaccharides (GOS), which are prebiotic carbohydrates and had been previously demonstrated to support growth of some of the tested probiotic strains (Grimoud et al., 2010)*. These compounds were tested in an attempt to provide probiotics, prebiotics or synbiotics efficient for inhibiting the mentioned phenomenon in vitro as a first step to selecting them for in vivo studies.

Section snippets

Probiotic strains and growth conditions

The probiotic lactic acid bacteria tested are listed in Table 1. Bifidobacterium bifidum 02, 20 and Bifidobacterium pseudocatenulatum 14 had previously been described newborn faeces clinical isolates (Vaugien et al., 2002) and were chosen on the basis of their high adherence to human colonic epithelial cells and their immunomodulatory activities on macrophage cell lines (Vaugien et al., 2005). Lactobacillus farciminis CIP 103136 was obtained from Institut Pasteur Collection (Paris, France) and

NF-κB and IL-8 quantification in HT-29 cells

None of the tested strains or GOS alone induced secretion of IL-8 or NF-κB activation when they were co-cultured with HT-29 cells without any activation (data not shown). Table 3 shows the variation values of both secreted IL-8 and activated NF-κB in inflammatory stimulated HT-29 cells treated by probiotics in comparison with the untreated control. By considering the parameters monitored, different groups can be distinguished. Three of the five bifidobacteria (B. bifidum LMI 02 and LMI 20, B.

Discussion

In the present study, we first aimed to select some potential anti-inflammatory probiotics in vitro. None of our tested strains or prebiotic GOS had any intrinsic pro-inflammatory characteristics, which suggests safe use for a possible human application. Through our first cell culture model, we demonstrated the anti-inflammatory properties of 11 strains, belonging to the different genera tested, in HT-29 cells during a pro-inflammatory treatment in a strain-dependent manner. 5 of these strains

Acknowledgements

This work was supported by grant from Région Midi-Pyrénées. The authors would like to thank Céline Courtin for her technical assistance during her internship.

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