Elsevier

Cytokine

Volume 90, February 2017, Pages 80-87
Cytokine

Modulation of the cytokine profile in Caco-2 cells by faecal lactobacilli and bifidobacteria from individuals with distinct dietary habits

https://doi.org/10.1016/j.cyto.2016.11.007Get rights and content

Highlights

  • Lactobacilli and bifidobacteria from three dietary human cohorts are examined.

  • Both genera show anti-inflammatory properties on enterocytes.

  • Enterocyte immunomodulation of lactobacilli is associated with a specific dietary habit.

  • The geographical area has an impact on the immune activity of lactobacilli.

Abstract

Enterocytes are actively involved in the defense against pathogens and they limit penetration of commensal microbes into tissues. They also have an important role in gut immunity as enterocytes confer mucosal dendritic cell specialisation. On the other hand, the microbiota is directly involved in the development and modulation of the intestinal immune system. Particularly, lactobacilli and bifidobacteria play a primary role in shaping the immune response. We further explored this issue by evaluating whether functional differences in Caco-2 cells could characterise faecal populations of lactobacilli (155 samples) and bifidobacteria (110 samples) isolated from three dietary cohorts (omnivores, ovo-lacto-vegetarians and vegans) recruited at four Italian centres (Turin, Parma, Bologna and Bari). According to our findings, tested bacteria were unable to modulate expression of IL-8, IL-10, TGF-β or thymic stromal lymphopoietin (TSLP) cytokines in unstimulated Caco-2 cells. Conversely, in phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) stimulated Caco-2 cells, lactobacilli from the omnivorous group and all bifidobacteria significantly down-regulated IL-8. Notably, both genera also lowered the TSLP expression in stimulated Caco-2 cells, regardless of the diet regimen. By further examining these data on the basis of geographical origin, we found that lactobacilli from the vegetarian group recruited in Bari, significantly up-regulated this cytokine. In conclusion, we highlighted a peculiar immune-modulatory activity profile for lactobacilli on enterocytes undergoing a stimulatory signal, which was associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the inflammatory potential of members of the Lactobacillus genus.

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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