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.2013 Feb;79(4):1221-31.
doi: 10.1128/AEM.03056-12. Epub 2012 Dec 7.

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity

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S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity

Valentina Taverniti et al. Appl Environ Microbiol.2013 Feb.

Abstract

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

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Figures

Fig 1
Fig 1
Biochemical analyses of L. helveticus MIMLh5 S-layer protein. (A) SDS-PAGE profile of MIMLh5 cells before (lanes 1 to 3) and after (lanes 3 to 6) treatment with LiCl solutions; (B and C) SDS-PAGE profile with Coomassie blue staining (B) and silver staining (C) of purified S-layer protein; 50 ng to 3 μg of protein was loaded per well on the gel; (D) HPLC profile of purified S-layer protein (AU210, absorbance units at 210 nm); (E) ESI-MS spectrum of the S-layer protein and reconstructed mass spectrum, indicating an average mass value of 43,853 Da.
Fig 2
Fig 2
Effect of Lactobacillus acidophilus NCFM, Lactobacillus helveticus MIMLh5, and the purified S-layer protein (Slay) of MIMLh5 on human epithelial colorectal Caco-2 cells stably transfected with an NF-κB/luciferase reporter vector at baseline (A) or stimulated with 2 ng/ml of IL-1β (B). A recombinant Caco-2 cell layer was incubated with EMEM only (A) or with the addition of IL-1β (B). Bacterial strains were used at an MOI of 1,000 (bacterial cells per Caco-2 cell). S-layer protein was tested at two different concentrations (100 and 10 μg/ml). Data in the histograms are the means (+ standard deviations) from at least three independent experiments conducted in triplicate. Data are reported as percent variation of light emission (relative luminescence units [RLU]), assuming that the value for the corresponding control was 100%. Asterisks indicate statistically significant differences: ***,P < 0.001; **,P < 0.01; *,P < 0.05.
Fig 3
Fig 3
Quantitative analysis of cytokine gene expression in U937 human macrophages after 4 h of stimulation. Expression profiles of TNF-α, IL-10, and COX-2 are indicated as the FOI relative to the induction level of the control, which was set at a value of 1. Presented data are the means (+ standard deviations) for a result representative of three independent experiments. Asterisks indicate statistically significant differences: **,P < 0.01; *,P < 0.05. (A) U937 cells stimulated with L. helveticus MIMLh5 (MOIs, 1,000 and 100) and its S-layer protein (10 μg/ml); control, unstimulated U937 cells; MIMLh5 w/o Slay, MIMLh5 cells after LiCl extraction of the S-layer protein; MIMLh5 w/o Slay + Slay, S-layer-depleted MIMLh5 cells supplemented with 10 μg/ml of the purified S-layer protein; LPS was used as a positive control at a concentration of 1 μg/ml. (B) Cytokine expression profiles in the presence of neutralizing antibodies against TLR2 (a-TLR2) and TLR4 (a-TLR4); IgA2 was used as a control for nonspecific blocking activity; IgA2 and anti-TLRs were added at a concentration of 5 μg/ml on U937 cells 1 h before stimulation with the S-layer protein; anti-TLR2/4, anti-TLR2 and anti-TLR4 simultaneously added at a concentration of 2.5 μg/ml each; control, U937 cells incubated with IgA2. (C) Cytokine expression profiles in U937 cells stimulated with L. helveticus MIMLh5 S-layer protein and LPS; LPS and S-layer protein were added at concentrations of 1 μg/ml and 10 μg/ml, respectively, both when used alone and in association; control, unstimulated U937 cells.
Fig 4
Fig 4
Quantitative analysis of cytokine gene expression in murine BMDMs after 4 h stimulation with L. helveticus MIMLh5 and its S-layer protein. Expression levels of TNF-α, IL-10, and COX-2 are indicated as the FOI relative to the induction level of the control (unstimulated BMDMs), which was set at a value of 1. LPS was used as a positive control at a concentration of 1 μg/ml. S-layer protein was tested at a concentration of 10 μg/ml. MIMLh5 was used at MOIs of 1,000 and 100. MIMLh5 w/o Slay, MIMLh5 cells after removal of the S-layer protein by LiCl extraction. Presented data are the means of measurements (+ standard deviations) for a result representative of three independent experiments. Asterisks indicate statistically significant differences compared to results for the corresponding control: *,P < 0.05.
Fig 5
Fig 5
Quantitative analysis of cytokine gene expression in murine macrophages isolated from the peritoneal cavity after 4 h stimulation with L. helveticus MIMLh5 and its S-layer protein. Expression levels of TNF-α, IL-10, and COX-2 are shown as the FOI relative to the induction level of the control (unstimulated peritoneal macrophages), which was set at a value of 1. LPS was used as a positive control at a concentration of 1 μg/ml. S-layer protein was tested at concentrations of 10 and 0.1 μg/ml. MIMLh5 was used at MOIs of 1,000 and 100. Presented data are the means of measurements (+ standard deviations) for a result representative of three independent experiments.
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