Upon priming with BLG, cord blood derived CD4⁺CD25⁺ T cells become highly suppressive.

<p>(<b>A</b>) Cord blood-derived mononuclear cells were co-cultured for 6 days with the antigen BLG. At day six CD4⁺CD25⁺ T cells were MACS-sorted and an inhibition assay was performed with the CD4⁺CD25⁻ (frozen on day 0, without prior antigen exposure) and stimulated with α-CD3 or BLG. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Results are representative for 6–9 independent experiments. Wilcoxon signed rank test was applied. P-values of less than 0.05 were considered significant (*); p<0.005 (**); p<0.001 (***). (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, RUNX 1, and RUNX 3) after allergen exposure. Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p

Comparable Treg-related transcription factor and marker expression in CB and PB on day 0.

<p>(<b>A</b>) Cord and peripheral blood derived mononuclear cells were analyzed via flow cytometry on day 0 without prior stimulation. Surface and intracellular staining was performed and one out of three representative experiments is shown. (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, TGF-β, RUNX 1, RUNX 3, and IL-10). Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p

CD103⁺RALDH⁺ dendritic cells are elevated in the LP following <i>B. infantis</i> feeding.

<p>Flow cytometric assessment of LP and MLN revealed that <i>B. infantis</i> feeding is associated with increased CD103⁺RALDH⁺ dendritic cells within the LP (n = 7), compared to the control group (n = 8), analysed using unpaired student t-tests (<b>a</b>). Citral blocked the increase in LP CD103⁺RALDH⁺ dendritic cells (n = 7). (<b>b</b>) Multispectral flow cytometry imaging identified CD103⁺ dendritic cells that efficiently bind CFSE-labelled <i>B. infantis</i>. (<b>c</b>) Flow cytometric analysis of CD11c⁺MHCII⁺CD103⁺ dendritic cells from the mucosa demonstrated that approximately 38% of CD103⁺ dendritic cells bound <i>B. infantis</i>. Isolated mucosal CD11c⁺ dendritic cells upregulate mRNA for RALDH enzymes following <i>in vitro</i> incubation with <i>B. infantis</i> (<b>d</b>), while the increase in gene expression is specific to CD103⁺ dendritic cells (<b>e</b>).</p

<i>B. infantis</i> induction of Foxp3⁺ lymphocytes is not RALDH-dependent.

<p>(<b>a</b>) Representative flow cytometric dot-plots are illustrated for CD4 and Foxp3 populations within MLN and LP. (<b>b</b>) The increase in CD4⁺Foxp3⁺ T lymphocytes following <i>B. infantis</i> feeding in the LP (n = 6) is not reversed by citral treatment (n = 5). (<b>c</b>) CD4⁺IL-10⁺ T lymphocytes increased within MLN (n = 8), but not the LP (n = 7). LP statistical significance was estimated using the non-parametric Mann-Whitney test, while MLN statistics were determined using the parametric unpaired student t-test.</p

DSS-induced colitis is reduced by <i>B. infantis</i> feeding.

<p>Four murine study groups were examined – Untreated (no DSS and no <i>B. infantis</i>), Control (DSS alone), <i>B. infantis</i> (DSS and <i>B. infantis</i>), citral (DSS and citral) and <i>B. infantis</i> & citral (DSS, <i>B. infantis</i> and citral). (<b>a</b>) The histopathology inflammatory score was significantly reduced by <i>B. infantis</i> feeding (n = 5), but not when citral was co-administered (n = 8). Citral administered with DSS did not increase inflammation in the colon in comparison to DSS alone (n = 7). (<b>b</b>) Representative slides of the murine gut are illustrated. (<b>c</b>) Colonic myeloperoxidase (MPO) levels were reduced in <i>B. infantis</i>-fed mice, which was not observed with <i>B. infantis</i> and citral treatment. Statistical significance was determined using non-parametric Mann-Whitney tests.</p

<i>B. infantis</i> suppresses the expansion of a pro-inflammatory dendritic cell phenotype within the LP.

<p>Untreated (n = 8), DSS colitis (n = 7), DSS and <i>B. infantis</i> treated (n = 6) and DSS, <i>B. infantis</i> and citral treated animals (n = 7) were compared for dendritic cell subsets within the LP. (<b>a</b>) The proportion of CD103⁺ dendritic cells within the LP was increased for all groups with DSS-induced colitis. (<b>b</b>) However, the CD103⁺ cells expressing RALDH was significantly reduced in the inflamed LP, which was normalized by <i>B. infantis</i> feeding. (<b>c</b>) The CD11c⁺CD103⁻RALDH⁺ cell numbers were reduced in the control DSS group and <i>B. infantis</i> did not reverse this suppression. (<b>d</b>) The increase in CD103⁺ dendritic cells in the inflamed LP primarily consists of CD11b⁺CD103⁺ dendritic cells, while <i>B. infantis</i> feeding was associated with an increase in CD11b⁻CD103⁺ dendritic cells. Statistical significance for (a), (b) and (c) was estimated using the non-parametric Mann-Whitney test, while (d) statistics were determined using the parametric unpaired student t-test.</p

CB-derived CD4⁺CD25⁺ T cells are deficient with regard to their regulatory function harboring a subset with limited net suppressive effects.

<p>(<b>A</b>) Specificity of proliferation to BLG and BLG+LPS is displayed via CFSE labeling experiments. (<b>B</b>) Inhibition experiments were performed using and increasing amount of MACS-sorted CD4⁺CD25⁺ T cells to the CD4⁺CD25⁻ fraction in presence of irradiated APCs. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Graphs indicate the means of 5–8 independent experiments with means +/− SEM. Wilcoxon sign rank test was applied. P-values of less than 0.05 were considered significant. (<b>C</b>) CBMCs were separated via FACS on day 0 according to their CD25 and CD127 expression. Four subgroups (CD4⁺CD25^highCD127^low (F1), CD4⁺CD25^intermediateCD127^low (F2), CD4⁺CD25^intermediateCD127^high (F3), and CD4⁺CD25⁻ T cells (CD25⁻)) were obtained. An inhibition assay was performed with the first three fractions using the CD4⁺CD25⁻ T cells as effector cells. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ subgroup. Graphs indicate the means of 4 independent experiments and SEM. (<b>D</b>) Real-time PCR analysis was performed to analyze the FOXP3, TGF-β, and IL-10 expression for further examination of the four fractions. Data represents three independent experiments showing the normalized expression of means +/− SEM.</p

<i>B. infantis</i> alters lymphocyte phenotypes within the LP.

<p>(<b>a</b>) IL-17⁺ lymphocytes were significantly reduced and IFN-gamma⁺ cells were substantially reduced within the LP of <i>B. infantis</i>-fed mice (n = 8), compared to the control group (n = 6), an effect that was blocked by citral (n = 5). No effect was observed for IL-4⁺ lymphocytes. (<b>b</b>) Isolated LP was cultured <i>in vitro</i> and cytokine secretion measured after 24 hours. <i>B. infantis</i> feeding reduced the <i>in vitro</i> secretion of the T_H17-polarising cytokines IL-1β and IL-6, which was partially reversed by citral. (<b>c</b>) <i>B. infantis</i> feeding was associated with a decrease in the proportion of LP lymphocytes expressing the gut homing receptors α4β7 and CCR9. Citral did not reverse the decrease in α4β7⁺ lymphocytes and had a minor influence on CCR9⁺ lymphocytes. Statisical significance was estimated using unpaired student t-tests.</p

<i>B.</i><i>infantis</i> is sampled by dendritic cells within PP and LP.

<p>(<b>a</b>) Flow cytometric analysis of CD11c⁺MHCII⁺ dendritic cells within the PP and LP at 0 or 2 hours following gavage of CFSE-labelled <i>B. infantis</i>, revealed that a subpopulation of dendritic cells at both sites become CFSE⁺. (<b>b</b>) Visualization by multispectral flow cytometry imaging confirmed the presence of CFSE-labelled bacteria on PP dendritic cells at 2 hours.</p

The interaction of IL-33 with Th1, Th2 and Th17 responses in chronic rhinosinusitis.

<p>IL-33 may act as an “alarmin” that is released by HSECS via a Th1 dependent mechanism. IL-33 itself induces IL-13 and IL-5 and thereby contributes to the limitation of both Th17- and Th1-related arms of the inflammation.</p