L. plantarum WCFS1 enhances Treg frequencies by activating DCs even in absence of sampling of bacteria in the Peyer Patches

Abstract

Probiotics such as L. plantarum WCFS1 can modulate immune responses in healthy subjects but how this occurs is still largely unknown. Immune-sampling in the Peyer Patches has been suggested to be one of the mechanisms. Here we studied the systemic and intestinal immune effects in combination with a trafficking study through the intestine of a well-established immunomodulating probiotic, i.e. L. plantarum WCFS1. We demonstrate that not more than 2-3 bacteria were sampled and in many animals not any bacterium could be found in the PP. Despite this, L. plantarum was associated with a strong increase in infiltration of regulatory CD103⁺ DCs and generation of regulatory T cells in the spleen. Also, a reduced splenic T helper cell cytokine response was observed after ex vivo restimulation. L. plantarum enhanced Treg cells and attenuated the T helper 2 response in healthy mice. We demonstrate that, in healthy mice, immune sampling is a rare phenomenon and not required for immunomodulation. Also in absence of any sampling immune activation was found illustrating that host-microbe interaction on the Peyer Patches was enough to induce immunomodulation of DCs and T-cells.

Figure 1

Lymphocytes were gated in the forward side scatter plot and the frequency of CD3⁺ T cells was determined. Within the T cell population, the frequency of CD8⁺ T cells and CD4⁺ T cells was determined. Within both the CD4 and CD8 T cell population the isotype controls for CD69, CD25, or the cytokines, were used to set the gate to 99% negative cells. This gate was then copied to the sample stained for CD69, CD25, or cytokines and the frequency of positive cells was determined. Further, within the CD4 T cell population the FoxP3 isotype control was used to set the gate to 99% negative cells. This gate was copied to the sample stained for FoxP3 and the frequency of positive cells was determined.

Figure 2

L. plantarum does induce dendritic cell activation in the Peyer Patches even without sampling of the bacteria. Frequency of dendritic cell subsets in the spleen (N = 6) following oral treatment with medium (white bars), or L. plantarum WCFS1 (black bars). Dendritic cells were gated based on the expression of CD11c and MHC II (A). DCs were gated in the forward side scatter plot, based on size and granularity, and the frequency of MHC II⁺ CD11c⁺ cells was determined. CD19⁺ B-cells were excluded from analysis. Within the DC populations CD80, CD86, or CD103 isotype controls were used to set the gate to 99% negative cells. This gate was copied to the sample stained for CD80, CD86, and CD103 and the frequency of positive cells was determined (A). Dendritic cell frequencies are depicted as the frequency of CD103⁺ cells within the CD11c⁺ MHC II⁺ cell compartment in the spleen (B). Activated dendritic cells are depicted as the frequency of CD80⁺ cells within the CD11c⁺ MHC II⁺ cell compartment in the spleen (C), or the frequency of CD86⁺ cells within the CD11c⁺ MHC II⁺ cell compartment in the spleen (D). Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t-test. *Represents P-values < 0.05.

Figure 3

Bioluminescent L plantarum trafficking through the GI tract of healthy mice. Monitoring of intestinal transit of L. plantarum by bioluminescence imaging in whole animals. L. plantarum-CBRluc (1–2 × 10⁸ CFU) was inoculated intragastrically into mice, and the bioluminescent signal was measured transcutaneously in whole animals at different time points postfeeding. (A) The intensity of the transcutaneous photon emission is represented as a pseudocolor image. Two representative mice are shown. (B) Transit of L. plantarum in the digestive tract of mice. Mice were fed once with 1–2 × 10⁸ CFU of L. plantarum-CBRluc. One representative image of one mouse is shown after 10, 45, 60, 75, 90, and 120 min. d-Luciferin was given intra-gastrically 1 h before administration of bacteria, and the bioluminescence signal was measured 3 h after inoculation of the substrate. (C) The digestive tract of the mouse was then dissected after sacrifice, and the bioluminescent signal was quantified on intact organs. A representative mouse is shown. No signal was detected.

Figure 4

Uptake of the bacteria in the PPs. L. plantarum-CBRluc (1–2 × 10⁸ CFU) was inoculated intra-gastrically into mice. After 5 days, mice were sacrificed and PPs were excised. All PPs were completely sliced and stained. PPs were frozen in precooled iso-propane, sectioned at 5 mm, and processed for immuno-histochemical staining. As primary antibody, we applied Anti-Firefly Luciferase antibody ab181640 (Abcam) in a 1:250 dilution. As second antibody, we applied FITC-conjugated rabbit anti-goat antibody (1:100, Dako) and as third biotinylated swine anti-rabbit antibody (1:100, Dako). Representative image showing that in only two of the eight animals we observed remnants or intact bacteria in the PPs.

Figure 5

L. plantarum WCFS1 induced increased frequencies of regulatory T cells in the spleen while had no effect on the frequency of pro-inflammatory activated T cells. Regulatory T cell frequencies in the spleen (N = 6) following oral treatment with medium (white bars), or L. plantarum WCFS1 (black bars). Regulatory T cell frequencies are depicted as the frequency of CD25⁺FoxP3⁺ cells within the CD4 T cell compartment, CD69⁺ cells within the CD4 T cell compartment (C&D), and CD69⁺ cells within the CD8 T cell compartment. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t- test. *Represents P-values < 0.05.

Figure 6

L. plantarum WCFS1 treatment had an attenuating effect on Th2 responses. Polarized CD4 T cell frequencies in the spleen (N = 6) following oral treatment with medium (white bars), or L. plantarum WCFS1 (black bars). Polarized CD4 T cell frequencies are depicted as the frequency of IFNγ⁺ cells within the CD4 T cell compartment, IL5⁺ cells within the CD4 T cell compartment, IL10⁺ cells within the CD4 T cell compartment, and IL17⁺ cells within the CD4 T cell compartment. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t-test. *Represents P-values < 0.05.

Figure 7

L. plantarum had a more pronounced effect on CD8+ T cells. A 2-fold increase in frequencies of IFN-γ producing cells was observed in the CD8+ T cells as well as an increase in IL10 producing CD8+ T cells. Polarized CD8 T cell frequencies in the spleen (N = 6) following oral treatment with medium (white bars), or L. plantarum WCFS1 (black bars). Polarized CD8 T cell frequencies are depicted as the frequency of IFNγ⁺ cells within the CD8 T cell compartment, IL5⁺ cells within the CD8 T cell compartment, IL10⁺ cells within the CD8 T cell compartment, and IL17⁺ cells within the CD8 T cell compartment. Results are depicted as the mean ± standard error of the mean (SEM). Statistical significance was calculated using the Students t-test. *Represents P-values < 0.05, **Represents P-values < 0.01.