Lactobacillus paracasei CBA L74 metabolic products and fermented milk for infant formula have anti-inflammatory activity on dendritic cells in vitro and protective effects against colitis and an enteric pathogen in vivo

Abstract

The rapid expansion of commercially available fermented food products raises important safety issues particularly when infant food is concerned. In many cases, the activity of the microorganisms used for fermentation as well as what will be the immunological outcome of fermented food intake is not known. In this manuscript we used complex in vitro, ex-vivo and in vivo systems to study the immunomodulatory properties of probiotic-fermented products (culture supernatant and fermented milk without live bacteria to be used in infant formula). We found in vitro and ex-vivo that fermented products of Lactobacillus paracasei CBA L74 act via the inhibition of proinflammatory cytokine release leaving anti-inflammatory cytokines either unaffected or even increased in response to Salmonella typhimurium. These activities are not dependent on the inactivated bacteria but to metabolic products released during the fermentation process. We also show that our in vitro systems are predictive of an in vivo efficacy by the fermented products. Indeed CBA L74 fermented products (both culture medium and fermented milk) could protect against colitis and against an enteric pathogen infection (Salmonella typhimurium). Hence we found that fermented products can act via the inhibition of immune cell inflammation and can protect the host from pathobionts and enteric pathogens. These results open new perspectives in infant nutrition and suggest that L. paracasei CBA L74 fermented formula can provide immune benefits to formula-fed infants, without carrying live bacteria that may be potentially dangerous to an immature infant immune system.

Conflict of interest statement

Competing Interests: Francesca Fasano and Andrea Budelli are employees of Heinz. The rest of the authors have declared that they have no conflict of interest. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Lactobacillus paracaseii CBA L74 supernatant has anti-inflammatory properties on MoDCs.

MoDCs were infected with either Salmonella SL1344 (MOI 10∶1) or Lactobacillus CBA L74 (MOI 10∶1) for 1 hour in medium without antibiotics or in the presence of 2% LB, MRS, FB62 supernatant (Sn FB62) or CBA L74 supernatant in MRS (Sn CBA L74). After washing and 24 h incubations in medium with antibiotics cytokine abundance was evaluated. A. Concentrations of IL-12p70 and IL-10 as determined by ELISA and CBA assays respectively. B. The % of response relative to FB62 infected MoDCs is shown after grouping six experiments together. C. Anti-inflammatory index calculated as ratio of IL-10/IL12p70 concentrations. * p<0,05; *** p<0,001.

Figure 2. Lactobacillus paracaseii CBA L74 supernatant ameliorates DSS colitis when administered intraperitoneally.

Mice (n = 5) were administered on a daily basis for 14 consecutive days (days −2 to 11) with carbonate or live CBA L74 (109 CFU) intragastrically (ig) or with MRS or CBA L74 supernatant (SN CBA L74) intraperitoneally (ip). Acute colitis was induced by dissolving 3% DSS in drinking water for 9 days (days 0–9). Panels from left to right show weight curves of single mice, mean weight loss ad Kaplan-Meier survival for A. carbonate and live CBA L74 ig treated groups B. MRS and CBA L74 supernatant (SN CBA L74) ip treated groups. C. Histological parameters for carbonate, MRS ip and SN ip treated groups. D. Representative histologies for carbonate, MRS ip and SN ip treated groups. Arrows indicate ulcers, arrowheads lymphoid follicles and asterisks inflammation. Bar size  = 500 µm, magnification 50x. * p<0,05; ** p<0,01; *** p<0,001.

Figure 3. Fermented milk preparation has anti-inflammatory properties on MoDCs.

MoDCs were infected with Salmonella FB62 (MOI 10:1) for 1 hour in medium without antibiotics or in different infant formulas: non-fermented milk (NFM) and fermented milk (FM) obtained through fermentation of Lactobacillus CBA L74 alone (CBA L74), fermentation in the presence S. thermophilus TH3 (S. th) or double fermentation with CBA L74 and S. thermophilus TH3 (CBA L74+S. th). After washing and 24 h incubations in medium with antibiotics cytokine abundance was evaluated. Panels show % of response of IL-12p70 and IL-10 relative to MoDCs infected with SL1334 in medium without antibiotics. *** p<0,001.

Figure 4. Oral administration of fermented milk preparation protects against colitis.

Mice (n = 5) were orally administered on a daily basis for 14 consecutive days (days −2 to 11) with vehicle (water), non-fermented milk (NFM) or fermented milk (FM). Acute colitis was induced by dissolving 3% DSS in drinking water for 9 days (days 0–9). A, Single mouse body weight curves, Kaplan-Meier survival and colon length for vehicle, NFM and FM. B. Body weight curves (mean ± SEM) for two pooled experiments. * p

Figure 5. Oral administration of CBA L74 fermented milk preparation protects against colitis.

Mice (n = 5) were orally administered on a daily basis for 14 consecutive days (days −2 to 11) with non-fermented milk (NFM) or milk fermented with only CBA L74 (FM). Acute colitis was induced by dissolving 3% DSS in drinking water for 9 days (days 0–9). A. Single mouse body weight curves, Kaplan-Meier survival curve and colon length for NFM and FM. B. Histological parameters. C. Representative histologies for NFM and FM. Arrows indicate ulcers, arrowheads lymphoid follicles and asterisks inflammation. Bar size  = 500 µm, magnification 50x. * p

Figure 6. CBA L74 fermented milk reduces inflammatory mediators expression.

At the end of the experiment described in Figure 5 RNA was extracted from colons and expression levels of Tbet, Gata3, Inf-γ, Il-17a, Il-6, Il-22, Il-33, Il-1β, Kc, Ccl2, Cox2, iNos, Ahr, Indo, Zo-1, Reg3β, Reg3γ were assessed by RT-qPCR. Expression levels are normalized to the housekeeping mRNA Ralp32. Dots represent individual mice measurements, lines represent the average value.

Figure 7. Fermented milk is protective against Salmonella infection in ex vivo and in vivo infection models.

Murine colonic tissue was incubated with NFM of FM for 1 h at 37°C. Then tissue was either infected or not with Salmonella FB62 (102 CFUs) for 2 hours. Tissue was then incubated with gentamicin for 24 h in oxygen chamber. A. Cytokine concentration culture media expressed relative to non-infected samples (NI). B. Representative histologies for NFM and FM treated samples infected with FB62, magnification 40×. C. Mice (n = 20) were fed with modified diet containing 5% of non-fermented milk (NFM) or fermented milk (FM). After 10 days mice were orally administered with 106 CFUs of Salmonella FB62. Survival was monitored daily. Graph represents Kaplan-Meier Survival Probability Estimate and statistical significance was calculated with two-way ANOVA. * p<0,05; ** p<0,01.