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. 2012 Jun;78(12):4209-16.
doi: 10.1128/AEM.00325-12. Epub 2012 Apr 13.

In vitro functional and immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3 association that are relevant to the development of a pharyngeal probiotic product

Valentina Taverniti  1 , Mario Minuzzo, Stefania Arioli, Ilkka Junttila, Sanna Hämäläinen, Hannu Turpeinen, Diego Mora, Matti Karp, Marko Pesu, Simone Guglielmetti
Affiliations

In vitro functional and immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3 association that are relevant to the development of a pharyngeal probiotic product

Valentina Taverniti et al. Appl Environ Microbiol. 2012 Jun.

Abstract

The use of proper bacterial strains as probiotics for the pharyngeal mucosa is a potential prophylactic strategy for upper respiratory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promising pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by inducing potentially protective effects. In particular, we found that the strains MIMLh5 and ST3 activate U937 human macrophages by significantly inducing the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Nonetheless, the induction of the anti-inflammatory interleukin-10 (IL-10) by MIMLh5 or ST3 was never lower than that of TNF-α, suggesting that these bacteria can potentially exert a regulatory rather than a proinflammatory effect. We also found that the strains MIMLh5 and ST3 induce cyclooxygenase 2 (COX-2) expression and demonstrated that toll-like receptor 2 (TLR-2) participates in the recognition of the strains MIMLh5 and ST3 by U937 cells. Finally, we observed that these microorganisms grow efficiently when cocultured in milk, suggesting that the preparation of a milk-based fermented product containing both MIMLh5 and ST3 can be a practical solution for the administration of these bacteria. In conclusion, we propose the combined use of L. helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyngeal mucosa.

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Figures

Fig 1
Fig 1
Probiotic properties of Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 on a FaDu human pharyngeal cell layer. (A) Bacterial adhesion as observed with Giemsa staining under a light microscope; FaDu cell nuclei appear in red. (B) Antagonistic exclusion activity against bioluminescent Streptococcus pyogenes C11lucFF; the control is a cell layer treated only with PBS before incubation with S. pyogenes. (C) Effect of bacteria on FaDu cells stably transfected with an NF-κB/luciferase reporter vector. The control is a FaDu cell layer incubated without bacterial cells. MOI, multiplicity of infection (bacterial cells per FaDu cell). IL-1β was used as the positive control for NF-κB activation. Data in histograms are the means (± standard deviations) from at least three independent experiments conducted in triplicate. Bacterial and FaDu cell luciferase activities are expressed as relative luminescence units (RLU). Asterisks indicate statistically significant differences compared to the control (P < 0.001).
Fig 2
Fig 2
Transcription analysis of cytokine genes in U937 cells stimulated with Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 after 4 h of incubation with bacterial strains used alone or in association at two different MOIs. Lipopolysaccharide (LPS) was used as the positive control at a concentration of 1 μg ml−1. The values are the means (± standard deviations) for a result representative of three independent experiments, expressed as the fold change in induction relative to the result for the control (U937 unstimulated cells), which was set at a value of 1. Asterisks indicate statistically significant differences compared to results for the corresponding control (*, P < 0.05). (A) Expression levels of IL-10 and TNF-α. (B) Expression levels of COX-2. (C) Expression levels of IL-10 and TNF-α in the presence of a neutralizing antibody against TLR-2 (anti-TLR2). Anti-TLR2 was added to U937 cells 1 h before stimulation with bacteria. Immunoglobulin-A2 isotype (IgA2) was used as the control for nonspecific blocking activity.
Fig 3
Fig 3
Growth curves of Streptococcus salivarius ST3. (A) Growth in M17 medium supplemented with 2% of 8 different carbon sources. (B) Growth in M17 medium supplemented with 6 different concentrations of Actilight fructooligosaccharides (FOS). The curves are representatives of two experiments carried out in sextuplicate.
Fig 4
Fig 4
Growth of Lactobacillus helveticus MIMLh5 and Streprococcus thermophilus ST3 (single culture and coculture) at 42°C in skim milk supplemented with 2% glucose or FOS. (A) Bacterial plate counts of coculture; T0, immediately after 1% inoculum; T3 and T6, after 3 and 6 h, respectively. (B) Acidification curves.
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