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. 2008 Dec 9;105(49):19474-9.
doi: 10.1073/pnas.0810305105. Epub 2008 Dec 1.

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Sergey R Konstantinov  1 , Hauke Smidt, Willem M de Vos, Sven C M Bruijns, Satwinder Kaur Singh, Florence Valence, Daniel Molle, Sylvie Lortal, Eric Altermann, Todd R Klaenhammer, Yvette van Kooyk
Affiliations

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Sergey R Konstantinov et al. Proc Natl Acad Sci U S A. .

Abstract

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
L. acidophilus NCFM induces concentration-dependent iDC cytokine responses. iDCs were incubated with L. acidophilus NCFM, LPS (10 ng/mL), or no supplement for 2 days at 37 °C. DCs supernatants were harvested after 48 h and analyzed for IL-10 and IL-12p70. Experiments were repeated 3 times, and the values are the average ± SD.
Fig. 2.
Fig. 2.
Differential binding of DC-SIGN-Fc to L. acidophilus NCFM (SlpA+) and NCK1377-CI (SlpB+). By using the ELISA system, the DC-SIGN-Fc-binding specificity was determined in the presence of EGTA and blocking antibody to DC-SIGN (AZN-D1) and DCIR-Fc. L. acidophilus NCFM indicates L. acidophilus NCK1377-CI. All results are representative for 3 independent experiments (average ± SD); *, P < 0.05.
Fig. 3.
Fig. 3.
Cellular DC-SIGN is a receptor for L. acidophilus NCFM. CHO cells transfected with DC-SIGN and mock (CHO cells alone) (5 × 104 cells) were treated with 5 × 106 FITC-labeled L. acidophilus NCFM (SlpA+) or SlpA knockout NCK1377CI (SlpB+) for 45 min at 37 °C followed by FACScan analysis. The DC-SIGN-binding specificity was determined by measuring of the bacterial attachment (percentage positive CHO or CHO–DC-SIGN cells) in the presence of blocking antibody against DC-SIGN, D1 (AZN-D1), and EGTA. The error bars represent standard deviation of data from 3 independent experiments; *, P < 0.05.
Fig. 4.
Fig. 4.
Purified S layer protein ligates to DC-SIGN. After the SlpA purification (A), the protein was assayed for its binds to DC-SIGN-Fc by ELISA (B). The data are expressed as the mean ± SD of 3 experiments. (C) Western blotting results for DC-SIGN-Fc and DCIR-Fc binding to different concentrations of SlpA.
Fig. 5.
Fig. 5.
L. acidophilus NCFM SlpA mediates binding to DC-SIGN on iDCs. (A and B) Microscopic analysis of DCs interacting with fluorescently labeled L. acidophilus NCFM (A) and SlpA-knockout mutant, NCK1377-CI (B). (Scale bars, 5 μm.) (C and D) FACScan analysis of L. acidophilus NCFM or NCK1377-CI binding to iDCs when iDCs (5 × 104 cells) were treated with 5 × 106 FITC-labeled L. acidophilus NCFM (SlpA+) (C) or SlpA-knockout NCK1377-CI (SlpB+) (D) for 45 min at 37 °C. (E and F) Blocking antibodies to DC-SIGN (AZN-D1, c = 20 μg mL−1) were used when iDCs were treated with L. acidophilus NCFM (E) or NCK1377-CI (F). A representative experiment of 4 is shown.
Fig. 6.
Fig. 6.
L. acidophilus NCFM (SlpA+) and NCK1377-CI (SlpB+) elicit differential iDC cytokine responses. The production of the antiinflammatory cytokines IL-10 and IL-6, and the proinflammatory cytokines IL-12p70, TNF-α, and IL-1β was analyzed by ELISA at different ratios (DCs:NCFM or DCs:NCK1377-CI) or after the iDCs interactions with a purified SlpA (1 μg/mL) with and without LPS (10 ng/mL); −, cytokines produced by iDCs left untreated in those experiments; *, significant difference (P < 0.05) for the cytokines induced at the respective DC:NCFM or DC:NCK1377-CI ratios.
Fig. 7.
Fig. 7.
L. acidophilus NCFM induces Th2 polarization via C-type DC-SIGN. DCs were maturated with L. acidophilus NCFM and the SlpA-knockout, NCK1377CI-(SlpB+) at a ratio of 100:1 (bacteria:DCs), or SlpA+LPS, in the presence or absence of anti-DC-SIGN Ab (aDC-SIGN). The percentage of IL-4- and INFγ-producing T cells was analyzed upon restimulation of the cultures. The data are the result of 4 independent experiments. *, significant difference (P < 0.05) for the cytokines induced after L. acidophilus NCFM stimulation.
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References

    1. Altermann E, et al. Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM. Proc Natl Acad Sci USA. 2005;102:3906–3912. - PMC - PubMed
    1. Sanders ME, Klaenhammer TR. Invited review: The scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic. J Dairy Sci. 2001;84:319–331. - PubMed
    1. van Vliet SJ, Dunnen JD, Gringhuis SI, Geijtenbeek TBH, van Kooyk Y. Innate signaling and regulation of dendritic cell immunity. Curr Opin Immunol. 2007;19:435–440. - PubMed
    1. Smits HH, et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol. 2005;115:1260–1267. - PubMed
    1. Mohamadzadeh M, et al. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci USA. 2005;102:2880–2885. - PMC - PubMed

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