Modulation of the cytokine profile in Caco-2 cells by faecal lactobacilli and bifidobacteria from individuals with distinct dietary habits
Introduction
The intestinal epithelium lies at the interface between the microbiota and the gut-associated lymphoid tissue (GALT). In addition to the barrier function of enterocytes, they actively defend against pathogens and limit penetration of commensal microbes into underlying tissues. Specifically, enterocytes play an important role in the intestinal immune system as they regulate mucosal dendritic cell (DC) specialisation. Among other unidentified mediators, enterocytes release thymic stromal lymphopoietin (TSLP) which blocks interleukin (IL)-12 production by DCs in response to bacteria and drives Th2-polarising cells, inhibiting the inflammatory potential of DCs [1]. Interestingly, the majority of enterocytes isolated from patients with Crohn’s disease (CD) were shown not to express TSLP failing to control the DC pro-inflammatory response [1]. On the other hand, the gut microbiota is directly involved in the development and modulation of the intestinal immune system. In particular, lactobacilli and bifidobacteria are considered key players because they constitute essential members of the normal intestinal microbiota in animals and humans, particularly bifidobacteria in infants [2], [3]. Changes in diet, use of antibiotics and intestinal colonisation by helminths can modify intestinal microbial communities [4], [5] Furthermore, alterations in intestinal microbiota have been reportedly documented in a growing list of diseases, such as inflammatory bowel disease [6] and celiac disease [7]. The role of diet, in particular the impact of dietary macronutrients (carbohydrates, protein and fats) in microbial ecology, is significant. Very recently, the gut microbiota and metabolome in 153 Italian individuals recruited from different regions in Italy, who followed omnivore, ovo-lacto-vegetarian or vegan diets, were analysed. Results showed that a high-level of consumption of plant foodstuffs was associated with beneficial microbiome-related metabolomic profiles in subjects consuming a Western diet [8]. Interestingly, the subsequent analysis of their faecal microbiota indicated that the samples clustered differently, according to the recruitment site, highlighting a greater impact of geographical location than type of diet [9]. In the present work, we further analysed the same populations by addressing the immune mechanisms by which lactobacilli and bifidobacteria from these individuals may influence the enterocyte response. Accordingly, we evaluated, for the first time, a large microbiological screening of lactobacilli and bifidobacteria isolated from individuals undergoing omnivorous, vegan and ovo-lacto-vegetarian diets to determine the role of diet in modulating in vitro immune markers of Caco-2 cells. Our data revealed peculiar modulatory activities of selected bacteria on inducible cytokines produced by enterocytes undergoing a stimulatory signal, which were found to be dependent on dietary habit for lactobacilli. Furthermore, the geographical area also influenced the pro-inflammatory activity of lactobacilli in Caco-2 cells.
Section snippets
Participant recruitment and faecal sample collection
Healthy adult volunteers (n = 155) who followed an omnivorous (n = 55), ovo-lacto-vegetarian (n = 53) or vegan (n = 47) diet were recruited from 4 four Italian centres (Bari, Bologna, Parma and Turin) [8], [9] (https://clinicaltrials.gov; ClinicalTrials.gov Identifier: NCT02118857; MRMOVVD), as indicated in Table 1. The exclusion criteria were dietary regimen followed for less than 1 year, age under 18 or over 60 years, regular consumption of drugs, regular supplementation with prebiotics or probiotics,
Plate counts and DGGE analysis
We used cell morphology and cell motility of colonies grown on selective media as general principle for genus confirmation [13]. The average numbers of lactobacilli and bifidobacteria found in the different dietary groups were then calculated and are reported in Table 2. In agreement with previous observations [9], counts were found lower in the vegan group than in the other two groups for both examined genera (P < 0.05). To further characterize the cultivated microbial populations, randomly
Discussion
In this study, we compared faecal populations of lactobacilli (155 samples) and bifidobacteria (110 samples) isolated from three dietary cohorts (omnivores, ovo-lacto-vegetarians and vegans). We found that both genera down-regulated cytokine expression in Caco-2 cells undergoing a pro-inflammatory stimulus; however, only lactobacilli activity was influenced both by the dietary habit and by the geographical origin. A major challenge for understanding the microbiota-host interactions is the
Conflict of interest
The authors have no financial or personal conflict of interests.
Acknowledgments
This study was supported by the Italian Ministry of University and Research (MIUR) PRIN 2010–2011 program (grant number 2010WZ2NJN; http://www.diet4microgut.it). The funders had no role in the design, analysis or writing of this article.
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