<i>In vivo</i> influence of <i>E</i>. <i>faecalis</i> CECT7121-pulsed DCs, and IFNγ production by spleen cells.

<p>C57BL/6 BM-DCs were pulsed with heat-killed <i>E</i>. <i>faecalis</i> CECT7121 (Ef∅) for 18 h, and then adoptively transferred to naïve C57BL/6 mice. After 7 days, spleen cells were <i>ex vivo</i> stimulated with Ef∅ (72 h) and cytokines were analyzed in culture supernatants by ELISA (A, IFNγ; B, IL-13; <b>C</b>, IL-10). Cytokine levels are expressed as mean pg/mL ± SEM. Comparisons were performed between DC and Ef-DC groups. ns: non-significant, * <i>p</i><0.05 (Unpaired Student’s <i>t</i> test). <i>E</i>. <i>faecalis</i> CECT7121-primed BM-DCs adoptively transferred to naïve mice induce the specific-secretion of IFNγ by spleen cells without production of IL-13 or IL-10.</p

IFNγ production after <i>in vivo</i> immunization with <i>E</i>. <i>faecalis</i> CECT7121.

<p>Supernatants from spleen (A and B) and MLNs (C and D) cell cultures were analyzed by direct ELISA to determine the levels of IFNγ. Results were expressed as mean pg/mL ± SEM from 2 independent experiments, and comparisons were performed between PBS and EF groups. ns: non-significant, *<i>p</i><0.05, **<i>p</i><0.01 (Mann-Whitney U-test). The inoculation of <i>E</i>. <i>faecalis</i> CECT7121 induces a rapid IFNγ production at local (MLNs) and systemic (spleen) levels.</p

CD4⁺ and CD8α⁺ cells in spleen and MLNs after probiotic immunization.

<p>Sub-populations of T lymphocytes in the spleen and MLNs of control and probiotic-treated animals were determined by flow cytometry. Results are from 2 independent experiments. ^nsnon-significant (One-way ANOVA plus Dunnett’s Multiple Comparison tests).</p><p>CD4⁺ and CD8α⁺ cells in spleen and MLNs after probiotic immunization.</p

Total number of spleen and MLN cells after i.g. treatment with <i>E</i>. <i>faecalis</i> CECT7121.

<p>Total cell numbers in the spleen and MLNs of PBS and <i>E</i>. <i>faecalis</i> CECT7121-treated BALB/c mice were determined by counting in Neubauer’s haemocytometer. Results are from 2 independent experiments.</p><p>Total number of spleen and MLN cells after i.g. treatment with <i>E</i>. <i>faecalis</i> CECT7121.</p

BM-DCs stimulation with <i>E</i>. <i>faecalis</i> CECT7121 cell walls and soluble lysate.

<p>BM-DCs were <i>in vitro</i> stimulated with <i>E</i>. <i>faecalis</i> CECT7121 cell walls (μg) and soluble lysate (μg of total protein), and IL-6, TNFα, IL-12, and IL-10 were measured in culture supernatants after 24 h (A, B, C, and D, respectively). Heat-killed <i>E</i>. <i>faecalis</i> CECT7121 (MOIs 1:1 and 100:1) was employed as a positive control. Cytokine levels were expressed as mean pg/mL ± SEM. ns: non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (One-way ANOVA and Dunnett’s Multiple Comparison tests after ‘sqrt(IL-12)’ or ‘Log(TNFα)’ transformations). <i>E</i>. <i>faecalis</i> CECT7121 cell walls and soluble lysate show different behavior. Both stimuli are able to induce IL-12 and IL-6, but the soluble lysates induce low TNFα and fail to induce IL-10 production.</p

Evaluation of regulatory cells in the spleen of probiotic-immunized animals.

<p>Spleen and MLNs cells were stained employing specific antibodies to determine CD4⁺FoxP3⁺ Tregs by flow cytometry (A). CD11b⁺Ly6C^intGr-1^high gMDSCs and CD11b⁺Ly6C^highGr-1^int mMDSCs were also determined by flow cytometry in the spleen of PBS and immunized mice (B). Results were expressed as mean cpm ± SEM from 2 independent experiments, and comparisons were performed between PBS and probiotic-treated mice. ns: non-significant (One-way ANOVA and Dunnett’s Multiple Comparison tests). The inoculation of <i>E</i>. <i>faecalis</i> CECT7121 does not induce Tregs accumulation neither in spleens nor in MLNs, but causes a non-significant accumulation of gMDSCs in the spleen of immunized mice.</p

Spleen immune cell populations after <i>in vivo</i> treatment with <i>E</i>. <i>faecalis</i> CECT7121.

<p>Cellular populations in the spleen of control and immunized mice were determined by flow cytometry. Results are from 2 independent experiments. ^nsnon-significant (One-way ANOVA plus Dunnett’s Multiple Comparison tests).</p><p>Spleen immune cell populations after <i>in vivo</i> treatment with <i>E</i>. <i>faecalis</i> CECT7121.</p

<i>In vitro</i> stimulation of genetically modified BM-DCs by <i>E</i>. <i>faecalis</i> CECT7121.

<p>BM-DCs generated from MyD88^-/-, hSIGN, MR^-/-, hSIGN × MyD88^-/-, and hSIGN × MR^-/- mice were <i>in vitro</i> stimulated with heat-killed <i>E</i>. <i>faecalis</i> CECT7121 (Ef∅) for 24 h. MHC-II, CD80, and CD40 expression levels were analyzed by flow cytometry (A), and IL-6, TNFα, IL-12, and IL-10 were determined by ELISA (B). Flow cytometry results are representative from 2 independent experiments. Cytokine levels are expressed as mean pg/mL ± SEM. ns: non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (Kruskal-Wallis ANOVA and Dunn’s Multiple Comparison tests). Absence of MyD88 abrogates BM-DC maturation induced by <i>E</i>. <i>faecalis</i> CECT7121. Modifications in the expression of lectins such as human DC-SIGN or mannose receptor do not seem to influence DC activation by <i>E</i>. <i>faecalis</i> CECT7121.</p