Numerous studies have shown that resistance to oxidative stress is crucial to stay healthy and to reduce the adverse effects of aging. Accordingly, nutritional interventions using antioxidant food-grade compounds or food products are currently an interesting option to help improve health and quality of life in the elderly. Live lactic acid bacteria (LAB) administered in food, such as probiotics, may be good antioxidant candidates. Nevertheless, information about LAB-induced oxidative stress protection is scarce. To identify and characterize new potential antioxidant probiotic strains, we have developed a new functional screening method using the nematode Caenorhabditis elegans as host. C. elegans were fed on different LAB strains (78 in total) and nematode viability was assessed after oxidative stress (3 mM and 5 mM H2O2). One strain, identified as Lactobacillus rhamnosus CNCM I-3690, protected worms by increasing their viability by 30% and, also, increased average worm lifespan by 20%. Moreover, transcriptomic analysis of C. elegans fed with this strain showed that increased lifespan is correlated with differential expression of the DAF-16/insulin-like pathway, which is highly conserved in humans. This strain also had a clear anti-inflammatory profile when co-cultured with HT-29 cells, stimulated by pro-inflammatory cytokines, and co-culture systems with HT-29 cells and DC in the presence of LPS. Finally, this Lactobacillus strain reduced inflammation in a murine model of colitis. This work suggests that C. elegans is a fast, predictive and convenient screening tool to identify new potential antioxidant probiotic strains for subsequent use in humans.

Citation: Grompone G, Martorell P, Llopis S, González N, Genovés S, Mulet AP, et al. (2012) Anti-Inflammatory Lactobacillus rhamnosus CNCM I-3690 Strain Protects against Oxidative Stress and Increases Lifespan in Caenorhabditis elegans. PLoS ONE 7(12): e52493. https://doi.org/10.1371/journal.pone.0052493

Editor: Paul D. Cotter, Teagasc Food Research Centre, Ireland

Received: October 11, 2012; Accepted: November 19, 2012; Published: December 26, 2012

Copyright: © 2012 Grompone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Funders of this work were: ANII (Agencia Nacional de Investigacion e Innovacion, URUGUAY): PE_ALI_1_1702 and Danone Research. ANII had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Danone Research had no role in study design, data collection and analysis, but checked the manuscript before publication.

Competing interests: The authors have read the journal's policy and have the following conflicts: GG is Danone Research employee as well as other authors. Work was also performed by Biopolis S.L. employees. Part of this work has been submitted for a patent application. This does not alter the authors′ adherence to all the PLOS ONE policies on sharing data and materials

Aerobic metabolism leads to the production of harmful byproducts. Organisms can only stay healthy by reducing natural by-products of oxygen metabolism, such as reactive oxygen species (ROS), which are mainly produced by mitochondria [1] and damage proteins, lipids and DNA on accumulating in cells [2], [3], [4], [5]. Oxidative stress plays an important role in the rate of aging processes, often referred to as the Mitochondrial Free Radical Theory of Aging [6], [7], [1]. This process is also a key factor in aging-associated degenerative diseases such as certain types of cancer, diabetes and heart failure, among others [4], [7]. Oxidative stress also plays an obvious role in the pathogenesis of a number of gastrointestinal diseases, including: gastric and duodenal ulcer disease, pancreatitis, IBD; gastric, esophageal, and colon cancers [8], [9]. Although the causal role of ROS in aging remains controversial, recent reports suggest that ROS mediate a stress response to age-related damage, rather than being the underlying cause of aging [10]. Moreover, ROS are shown to have protective effects in model organisms such as Saccharcomyces cerevisae, Caenorhabditis elegans and Drosophila melanogaster [11].

The search for new nutritional antioxidant compounds able to counterbalance the effects of oxidative stress poses an exciting research challenge. There is increasing pre-clinical and clinical evidence that nutritional interventions, using food products or food-grade compounds, can protect against oxidative stress [12], [13], [14], [15]. Probiotics, defined by FAO/WHO in 2001 as live micro-organisms which, when administered in adequate amounts, confer a health benefit to the host [16], can be good candidates for providing such antioxidant effects. While various methodologies have been developed to identify natural LAB strains with intrinsic antioxidant properties [17], [18], there are insufficient methods available for the fast and reliable massive screening of bacterial culture collections in vivo using complex multicellular organisms. Indeed, only a few natural antioxidant lactic acid bacteria (LAB) have been characterized in animals so far [19]. In systems mimicking colon fermentation, Lactobacillus paracasei Fn032, Lactobacillus rhamnosus GG and Lactobacillus spp Fn 001 have been shown to prevent hydroxyl radical production [20]. Moreover, it has been shown that orally-administered live recombinant LAB (Lactococcus lactis or Lactobacillus plantarum strains) producing bacterial SOD can improve TNBS-induced colitis in rats [21], [22] and mice (Foligné et al., unpublished data).

However, laboratory animals cannot be used in the first steps of screening large sets of strains and, thus, there is the need for more convenient and predictable screening tools for natural antioxidant LAB. In this respect, the use of less evolved animals may provide an attractive alternative [23], [24].

To identify new natural LAB strains with antioxidant effect, we have developed a completely new and highly predictive method that uses Caenorhabditis elegans as an in vivo screening model. Caenorhabditis elegans is an extremely powerful and well-studied biological system, which has been used as a model to study aging and related diseases [25], [10], [26]. This nematode is a good biological model for genetic studies [27] since many of its pathways are conserved in humans. Oxidative damage and its effects on aging have been studied in a C. elegans model system using a nematode mutant strain exhibiting hyper-resistance to oxidative stress, as compared to its parental strain [28]. Moreover, C. elegans proved an appropriate model to screen potential anti-Salmonella infection bacteria [29] and antibacterial compounds [30]. Furthermore, C. elegans longevity is known to be related to the insulin pathway and to orthologous genes in humans, involved in the insulin-like growth factor and diabetes [31], [32]. So far, little is known about the potential use of C. elegans as a screening tool for probiotic bacteria inducing resistance against oxidative stress or improving longevity.

Here, we have used the nematode Caenorhabditis elegans as a new preclinical model to carry out preliminary antioxidant screenings to identify potential probiotic strains and to provide insights into the mechanisms by which these strains lower oxidative stress. This animal model has enabled us to identify a new strain of Lactobacillus rhamnosus, designated CNCM I-3690, which exerted a strong antioxidant effect and extended nematode lifespan through the insulin-like pathway DAF-2/DAF-16. Furthermore, since inflammation can be associated with generation of ROS leading to oxidative stress [33], we observed that CNCM I-3690 strain might have the ability to protect against oxidative stress through an anti-inflammatory effect. These findings suggest that C. elegans can be a good predictive screening tool for new potential probiotic strains.

We included 78 bacterial strains from a Danone Research collection in the in vivo antioxidant screening methodology, using the model organism C. elegans, adapted from a previously reported protocol performed to measure the antioxidant activity of plant extracts [12]. This collection is composed by 62 Lactobacillus strains belonging to acidophilus, bulgaricus, casei, paracasei, plantarum and rhamnosus species, 9 Streptococcus thermophilus isolates and 6 Bifidobacterium strains belonging to the animalis, breve and longum species. See Table S1 for genera and species specifications of each strain used in this study. The strains belonging to Bifidobacterium, Lactobacillus and Streptococcus genera were grown in MRS with cysteine, MRS and Elliker media, respectively. As the bioassay of the in vivo antioxidant activity is to be carried out with samples of live cells of different LAB, cells must be recovered in the logarithmic phase growth. After growth curve analysis, the optimal time for cell recovery was established to be after 15 h of incubation at OD600 = 1, 1.5 and 1.7 for Streptococcus, Lactobacillus and Bifidobacterium, respectively. The different LAB cultures were added to the mixture at a final concentration of 4×106 cells/mL. See Supplementary Material S1 for growth curves of representative strains for each genus and detailed protocol.

In addition, we analyzed the sensitivity of bacterial strains to kanamycin, an antibiotic used to inhibit Escherichia coli growth. A concentration of 30 µg/mL was sufficient to inhibit E. coli OP50.

Experiments were carried out with a C. elegans mutant strain BA17 fem-1(hc17) (Caenorhabditis Genetics Center at the University of Minnesota, USA) which is infertile at 25°C. BA17 worms were synchronized by isolating eggs from gravid adults at 20°C, hatching the eggs overnight in M9 buffer and isolating L1-stage worms in the wells of a microtiter plate. The worms were grown without shaking for three days at 25°C and 80–85% relative humidity. After this incubation period, adult worms were subjected to oxidative stress with H2O2 (3 mM and 5 mM) or without H2O2 (no stress control). Two controls were used during this experiment: wells with Escherichia coli instead of LAB as the control of bacterial feeding and wells with E. coli and 3 mM or 5 mM H2O2 as the control for oxidative stress. Worms were incubated in these conditions for 5 h. In terms of scoring for antioxidant capacity, we considered paralyzed worms to be dead (stressed).

In these experiments, we used the C. elegans wild type strain N2 (Caenorhabditis Genetics Center at the University of Minnesota, USA) to validate the potential antioxidant activity of a group of selected bacterial strains. For this purpose, worms were grown in NG medium (Nematode Growth medium: Agar 17.5 g/l, Sodium Chloride 3.0 g/l, Peptone 2.5 g/l, Cholesterol 0.005 g/l) on agar plates with a lawn of E.coli OP50 (control media) and NG with a lawn of each LAB. Worms were incubated at 20°C for 5 days, and then transferred to a medium with 3 mM of H2O2. After 5 h of incubation, worm viability was scored.

To measure the lifespan of C. elegans, synchronized worms of the wild-type strain (N2) and the mutant strains LG333 (skn-1), GR1307 (daf-16) and CB1370 (daf-2) were grown at 20°C until they reached the young adult stage. All mutant strains of C. elegans were obtained from the Caenorhabditis Genetics Center at the University of Minnesota (USA). Worms were then transferred to NGM agar plates covered with lawns of E. coli OP50 or the corresponding LAB (CNCM I-3690 or CNCM I-4317). The plates were incubated at 20°C and the numbers of live and dead worms were scored every two days. Parents were moved periodically to new plates to separate them from their progeny. A worm was considered as dead if it failed to respond to a platinum wire. Three independent assays were carried out with each strain.

Gene expression in C. elegans wild-type strain (N2) was analyzed in worm populations fed with E. coli OP50 (control condition) or the corresponding LAB (Lactobacillus rhamnosus CNCM I-3690 or Lactobacillus rhamnosus CNCM I-4317). Three days feeding period was analyzed (young-adult worms). Synchronized populations were obtained from embryos isolated from gravid adults in the different feeding conditions. After feeding period (3 days), samples of worms were collected with M9 buffer, washed three times and collected in eppendorf tubes for worm disruption by sonication (3 pulses at 10 W, 20 s/pulse). Total RNA isolation was performed with RNAasy Kit (Qiagen, Barcelona, Spain). RNA samples were processed for hybridization using the GeneChip® C. elegans Genome Array of Affymetrix (UCIM, University of Valencia). These chips contain oligonucleotide probesets designed to asses over 22500 transcripts from the C. elegans genome. Four biological replicates were examined per condition using Bioinformatics (CIPF, Valencia, Spain). Raw data obtained from Affymetrix arrays were background corrected using RMA methodology [34]. Signal intensity was standardized across arrays via quantile normalizaton algorithm. Differential gene expression assessment between control and treated conditions was carried out using limma moderated t-statistics. The p-values obtained for each gene were adjusted with multiple testing p-value correction procedures [35]. Finally, gene set analysis was carried out for each comparison using logistic regression models ner [36].

Data analysis was accomplished in “R” (R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org) mainly through packages in the “Bioconductor” suite [37]. Normalization was performed using rma (robust multi-array average expression measure) in “affy” [38] and differential expression was assayed via “limma” [39]. Genes were considered differentially expressed when the multiple testing adjusted P-value