Cytokine production by CD4⁺ T lymphocytes stimulated with <i>Bifidobacterium</i> treated DCs.

<p>DCs maturated with LPS, <i>B. bifidum</i> LMG13195, L22, A8 and IF10/10 or <i>B. breve</i> BM12/11 were cocultured with allogeneic naïve CD4⁺CD45RA⁺ T cells at T-cell:DC ratio of 1∶10. The concentration of IL-10, IL-17, IFNγ and TNFα was quantified by multiplex assay. Bars represent the mean and standard deviation of four independent experiments performed with different blood donors.</p

Cytokine production by human PBMC stimulated with different bifidobacteria strains.

<p>PBMCs were cultured with RPMI alone, or in the presence of LPS (<i>E. coli</i>) or 21 UV-killed bifidobacteria strains at bacteria:cell ratio of 5∶1. After four days of culture, supernatants were collected to quantify the amounts of IL-10, IL-17, IFNγ and TNFα by a multiplex assay (Flex set CBA). Histogram shows mean and standard deviation obtained in four independent experiments performed with different blood donors.</p

Induction of functional Treg cells by <i>Bifidobacterium</i> strains.

<p>(A) CD4⁺CD45RA⁺ T cells were co-cultured with DCs stimulated with different bifidobacterial strains in a 1∶10 ratio. After 12 days of culture determination of Treg cells was assessed by extra/intra-cellular staining by flow cytometry. Different gates for CD45RO⁺ cells with low/intermediate (blue dots) and high (red dots) expression of CD25 were defined, allowing the quantification of CD25^high FOXP3^highCD127^low lymphocytes. (B) PHA-activated effector cells (allogenic PBMCs) were cultured in the presence of cells obtained after 7-day stimulation of PBMCs with LPS or <i>Bifidobacterium</i> spp. After 4 days of culture, cells were pulsed with [³H]thymidine and incorporation was measured 16 h later. Histogram shows mean and standard deviation of the cpm in stimulated triplicates obtained in 4 independent experiments performed with different blood donors. (C) Analysis of CD4/CD25 expression in the same cultures.</p

Structure, purification and location of the ST peptide.

<p><b>a</b>) Domain structure of the protein D1, where the ST peptide is encoded (S/T domain); <b>b</b>) Western blot performed with a specific horseradish peroxidase-conjugated anti His₅ antibody, showing the anomalous migration of the purified His-tagged ST peptide (marked with an arrow); <b>c</b>) Western blot using the polyclonal anti-STp serum as primary antibody; specific immunoreactive bands are labelled with arrows; -: complete medium (negative control), HC#: protein extracts obtained from culture supernatants of healthy colonic biopsies, F#: Some culture supernatants were 0.2 µm filtered prior to protein extraction, D# and E#: protein extracts from culture supernatants of human epidermal and dermal layer cultures respectively.</p

STp induced i) regulatory cytokines in blood enriched DC and ii) stimulated T-cells, which acquired a skin homing profile.

<p><b>a</b>) Intracellular ongoing cytokine production of IL-12(p40/p70), IL-10, IL-6 and TGFβ in blood DC of healthy controls after 24 hours stimulation with STp (10 µg/ml, 1 µg/ml and 0.1 µg/ml) or LPS (100 ng/ml) compared to a basal culture. Closed histograms represent the percentage of positive cells assessed by intracellular cytokine staining and SED normalized subtraction from antibody stained cells cultured in the absence of monensin. That approach accurately quantifies the ongoing cytokine production of DC in a time window of 4 hours (monensin incubation) irrespectively of the initial cytokine amounts within the cells. <b>b</b>) Stimulatory capacity of DC was assayed in a mixed leukocyte reaction (MLR). T-cells were identified in the forward (Fw) and side (Sd scatter) and subsequent CD3 identification of dividing T-cells as CD3+ and CFSE^low. <b>c</b>) Dose response proliferation of T-cells upon 5 days stimulation with different doses of allogeneic DC (0%, 1%, 2% and 3%) previously pulsed with different doses of STp or LPS compared to untreated DC (basal). Results show the mean±SEM of three independent experiments. <b>d</b>) Imprinted homing profile of stimulated T-cells (CFSE^low) was determined regarding their surface expression and intensity ratio (IR) for the gut-homing integrin β7 and the skin-homing CLA molecules compared to resting T-cells cultured in the absence of DC. <b>e</b>) Acquired cytokine profile of such cells was determined as intracellular cytokine content of both IL-10 and IFNγ. Closed histograms represent the percentage of positive cells after subtraction from respective isotypes. All displayed histograms are representative of three independent experiments performed with similar results.</p

STp primes human intestinal DC towards a regulatory phenotype.

<p><b>a</b>) DC were identified in total colonic lamina propria mononuclear cells from healthy controls by flow cytometry according to the Forward and Side scatters and the subsequent HLA-DR/lineage cocktail dot plot. DC were defined as HLA-DR⁺ and lineage^– (CD3, CD14, CD16, CD19 and CD34). <b>b</b>) Ongoing intracellular IL-10 and IL-12(p40/p70) cytokine production (closed histograms) was determined in colonic DC cultured with and without STp (10 µg/ml). Pooled data of 8 independent experiments are shown in panel <b>c</b>). <b>d</b>) Stimulatory capacity of such intestinal DC was determined upon 5 days culture in the presence of allogeneic CFSE-labelled T-cells as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036262#pone-0036262-g002" target="_blank">Figure 2c</a>. Results show the mean±SEM of 8 independent experiments. <b>e</b>) Imprinted homing profile (gut-homing: β7; skin-homing: CLA) and intracellular cytokine content (IL-10 and IFNγ) of stimulated T-cells (CFSE^low) was compared to resting T-cells cultured in the absence of intestinal DC. Pooled data of eight independent experiments is shown in panel <b>f</b>). Closed histograms represent the percentage of positive cells after subtraction from respective isotypes. Lines and bars represent mean±SEM.</p

Clustering of Cas1 into distinct phylogenetic groups.

<p>Cas1 protein sequences were aligned using the MUSCLE algorithm and used to generate a UPMGA tree to show the divergence of different CRISPR-Cas systems. The system type and sub-type is noted on the right. The (*) indicates <i>B</i>. <i>moukalabense</i> which contained an “Undetermined” CRISPR-Cas system.</p

CRISPR repeat-spacer array size distribution.

<p>The graph shows the variability in size of the repeat-spacer arrays using number of spacers in each array, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133661#pone.0133661.t001" target="_blank">Table 1</a>. The error bars show the range of the locus size.</p

Occurrence of CRISPR-Cas systems in bifidobacteria.

<p>Occurrence of CRISPR-Cas systems in bifidobacteria.</p

CRISPR-Cas locus architecture.

<p>One representative for each unique CRISPR subtypes represents the locus architecture of <i>cas</i> genes, CRISPR repeats, spacers and other system-specific components (e.g. tracrRNA). The signature gene for each subtype is colored in red (<i>cas3</i> or <i>cas9</i> for Type I and II, respectively). The universal <i>cas1</i> and <i>cas2</i> genes are colored in blue. Accessory genes are grey. The tracrRNA for Type II systems is shown in yellow. The direction of the arrows indicates directionality of the coding sequences. The repeat-spacer array only shows the CRISPR repeats (black rectangles). Each operon is shown at a scale of 11,000 base pairs. Long repeat-spacer arrays were shortened for simplicity indicated by a double line break. Numbers under the arrays indicate the first and last spacer location, showing the size of the array.</p