A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response

Abstract

The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-κB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α), in a dose-dependent manner. After stimulation of cells with IL-1β, active NF-κB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-α and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties.

FIG. 1.

Bacterial adhesion to FaDu epithelial cell layer. (A) Adhesion indexes (AdI; number of bacteria/100 FaDu cells). Adhesion values are the means for two independent experiments conducted in duplicate. The variation between the replicates was less than 15%. (B) Cell layers observed after Giemsa staining, using light microscopy. Bar, 8 μm. N, FaDu cell nucleus, indicated once for each layer.

FIG. 2.

Antagonistic exclusion activities of dairy and probiotic bacteria against bioluminescent Streptococcus pyogenes C11^Luc on FaDu hypopharyngeal carcinoma cells (A) and HaCaT keratinocytes (B). Data are reported as percentages of variation in light emission relative to the cell layer treated with PBS buffer only before incubation with S. pyogenes. Numerical results are given as arithmetic means ± standard deviations. Each sample was processed in triplicate in at least two independent experiments. Statistically significant differences compared to strain K12 were determined by unpaired Student's t test. **, P < 0.001; *, P < 0.05.

FIG. 3.

Antagonistic exclusion activity against bioluminescent Streptococcus pyogenes C11^Luc on FaDu hypopharyngeal carcinoma cells. (A) Light emission curves registered after addition of d-luciferin. RLU, relative luminescence units. (B) Percentage variation in light emission relative to the cell layer treated with PBS buffer only before incubation with S. pyogenes. Results are given as arithmetic means ± standard deviations calculated for three replicates. (C) Agar plate counts of S. pyogenes cells that remained adhered after treatment of the FaDu cell layer with MIMLh5, K12, or PBS (control).

FIG. 4.

Histograms showing selected cytokines whose secretion changed after overnight incubation of the FaDu cell layer with bacterial cells (2 × 10⁸ cells ml⁻¹), as determined using Bio-Plex assay. The values are the means for two independent experiments conducted in duplicate. The error bars indicate standard deviations.

FIG. 5.

Modulation of light emission expressed by FaDu cells stably transfected with an NF-κB/luciferase reporter vector and incubated with selected bacterial strains, at baseline (A) or stimulated with 2 ng ml⁻¹ of IL-1β (B). Luciferase activity is expressed as the percent change in light emission (relative light units [RLU]), assuming the control without IL-1β to be 100%. Control, FaDu cell layers incubated without bacterial cells. Footnote a, bacterial strains employed at an MOI of 1,000 each (number of bacterial cells per FaDu cell). The values are the means (+ standard deviations) for at least three independent experiments conducted in duplicate. Asterisks indicate statistically significant differences compared to the corresponding control (**, P < 0.001; *, P < 0.05).

FIG. 6.

Production of TNF-α and IL-2 by BMDCs after 24 h of stimulation with the indicated bacteria. MOI, multiplicity of infection (number of bacterial cells per BMDC); LPS, lipopolysaccharide from Escherichia coli (positive control); MIMLh5, Lactobacillus helveticus MIMLh5; La-5, Lactobacillus acidophilus La-5; NF4, Bacillus coagulans NF4 (negative control).