Effects of JCM5805 on mPIV1 infection.

<p>A. Experimental procedure of mPIV1 infection. Mice in the control and JCM5805 groups were fed diet with or without 1 mg / mouse / day of JCM5808 during the study period (day -14 to 15). Mice were intranasally infected with mPIV1 on day 0. On 3 days post-mPIV1 infection, six mice were sacrificed from each group for lung histopathology. Thereafter survival rate, body weight and clinical scores were investigated with remained control mice n = 12, and JCM5805 mice n = 13. B. Survival rate of mice infected with mPIV1. The control (circle) and JCM5805 (square) groups consisted of 12 and 13 mice, respectively. The survival of each animal was monitored daily. <i>P</i><0.001 (Log-Rank test). C. Body weight of mice infected with mPIV1. The control (circle) and JCM5805 (square) groups consisted of 12 and 13 mice, respectively. The body weight of each surviving animal was measured daily. The body weight values are shown as mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01 (Student’s t test). The data shown is representative of two independent experiments.</p

Activation of pDCs in intestine by JCM5805 administration.

<p>Healthy C57BL / 6J mice were divided into control and JCM5805 groups (n = 4 in each group), and mice in the JCM5805 group were orally administered JCM5808 daily for 2 weeks. A. Low density cells prepared from PP of each group were analyzed by FACS. Expression level of cell surface activation marker was evaluated for MHC class II as median fluorescence intensities (M.F.I.) in left panel. Ratio of pDCs to total population was shown in right panel. pDCs was defined as “CD3⁻ Siglec-H⁺ CD11c⁺ in total population”. Short line represents the mean values. *<i>P</i><0.05 (Student’s t test). B, Total mRNA was extracted from PP pDCs from mice in the control (open columns) and JCM5805 groups (dot columns) (n = 8 in each group). <i>Ifnα</i> and <i>Ifnβ</i> gene expressions were measured by qRT-PCR and normalized to <i>Gapdh</i> gene expression. Data are shown as mean ± SD. *<i>P</i><0.05 (Student’s t test). These data are representative of three independent experiments. Each data are mean ± SD.</p

Lung histopathology of mPIV1-infected mice.

<p>A. Representative hematoxylin and eosin (H & E)-stained sections of lung tissues from control and JCM5805 group mice (6 mice per group). Lung tissues were prepared from mice 3 days after infection. Scale bars, 300 μm. B. Histological scoring of lung tissues from mPIV1-infected mice belong to control (open columns) and JCM5805 (dot columns) group. Sections were scored at four levels as follows: 0, no symptoms; 1, low pathogenicity; 2, medium pathogenicity; 3, high pathogenicity. The mean ± SD of the tissues in each group is shown. *<i>P</i><0.05, **<i>P</i><0.01 (Mann-Whitney U test). The data shown is representative of two independent experiments.</p

Morphology of pDC stimulated by LABs and incorporation into pDC.

<p>A. <i>L. lactis</i> JCM5805, JCM20101 and <i>L. rhamnosus</i> ATCC53103 were added to pDC for 48 hrs, respectively. Cells were placed onto slide grass by cytospin and stained by Diff-Quick. B. FITC-stained <i>L. lactis</i> JCM5805, JCM20101 and <i>L. rhamnosus</i> ATCC53103 were added to BM-derived pDC, and were subsequently incubated for 24 hrs. Cells were stained with PE-Cy5.5 labeled anti-B220 antibody as pDC marker, and imaged by fluorescence microscopy.</p

Effect of oral administration of <i>L. lactis</i> JCM5805 to healthy C57BL/6 mice.

<p>Healthy C57BL/6 mice were divided into two groups (n = 8 each). Control group (ctrl) were fed a normal diet, and <i>L. lactis</i> group (JCM5805) were fed a diet containing 1 mg of JCM5805 per day. Two weeks later, low density cells fractions prepared from SPN (A) or MLN (B) derived each group were analyzed for MHCII and CD86 on pDC or mDC. Representative data from three independent experiments are shown.</p

Role of TLR signaling in IFN-α production elicited by LAB.

<p>Flt-3L induced BM-derived DC were prepared from wild-type (WT), TLR2^-/- and TLR4^-/- (left panel), or WT, TLR7^-/-, TLR9^-/- and MyD88^-/- (right panel). Cells were cultured in the presence of 0.1 µM of CpG-A or 10 µg/ml of LAB for subsequent 48 hrs. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures. *, p<0.05 for comparison to wild-type.</p

LAB-induced cytokines production in purified pDC, mDC and mixed cultures.

<p>A. pDC and mDC were sorted from Flt-3L induced BM-derived DC. 10 µg/ml of <i>L. lactis</i> JCM5805, JCM20101 and <i>L. rhamnosus</i> ATCC53103 were added to pDC, mDC or pDC and mDC mixed at same ratio (pDC+mDC) for 48 hrs. Concentrations of IFN-α, IL-12 and TNF-α were determined by ELISA. *, p<0.05 for comparison to pDC alone (IFN-α) or to mDC alone (IL-12 and TNF-α). B. Effect of inhibition of direct pDC-mDC contact by transwell system. “pDC+mDC” indicates same number (1×10⁵ cells each) of pDC and mDC cultured together. “pDC/mDC“ indicates pDC and LABs added to the upper chamber, and mDC added to the lower chamber. “mDC/pDC” indicates mDC and LABs added to the upper chamber, and pDC added to the lower chamber. *, p<0.05 for comparison to pDC+mDC. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures.</p

CD4⁺CD25⁺FoxP3⁺Treg and IFN-γ producing CD4⁺T cell generation by <i>L. lactis</i> JCM5805-treated pDC.

<p>MLR reactions using naïve CD4⁺T cell prepared from BALB/c and purified C57BL/6 derived-pDC stimulated by <i>L. lactis</i> JCM5805 (10 µg/ml) or <i>L. rhamnosus</i> ATCC53103 (10 µg/ml) or CpG-A (0.1 µM) was performed. After 7 days, cells were collected and stained for induced Treg cells (Data was gated for CD3⁺CD4⁺ cells, and graphed for CD25 and FoxP3 expression) or IFN-γ producing CD4^+?T cells (Data was gated for CD3⁺ cells, and graphed for CD4 and IFN-γ expression). A representative dataset from three independent experiments with singlet culture is shown.</p

Comparison of DC activation by <i>L. lactis</i> and <i>L. rhamnosus</i>.

<p>Flt-3L induced BM-derived DC were treated with 10 µg/ml of stimulatory (<i>L. lactis</i> JCM5805 or JCM20101) or non-stimulatory (<i>L. rhamnosus</i> ATCC53103) LAB strains; 0.1 µM of CpG-A was included as a positive control. After 48 hrs, cells were evaluated for expression level of cell-surface markers by flow cytometry. A. Gated on pDC. B. Gated on mDC. Numbers in graph indicates Median Fluorescent Intensity (MFI). All test conditions were significantly elevated (p<0.05) compared to medium control, except for ICOS-L for mDC (B). *, p<0.05 for comparison to medium control; #, p<0.05 for comparison to <i>L. rhamnosus</i> ATCC53103. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures.</p

Production of IFNs by Flt-3L induced DC in response to <i>L. lactis</i> and <i>L. rhamnosus</i>.

<p>A. pDC and mDC mixed Flt-3L BM DC were stimulated with 10 µg/ml of <i>L. lactis</i> JCM5805, JCM20101 and <i>L. rhamnosus </i>ATCC53103 for 48 hrs. 1 µg/ml of Pam₃CSK₄ (TLR2L), 5 ng/ml of LPS (TLR4L), 0.1 µM of CpG-A (TLR9L) were used as positive controls. Culture supernatants were assayed for IFN-α, IFN-β, IFN-γ and IFN-λ by ELISA. B. pDC and mDC mixed Flt-3L BM DC were stimulated with heat-killed or live JCM5805 strain. 10⁶ or 10⁸ cells of JCM5805 were added to culture, respectively. Culture supernatants were assayed for IFN-α. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures. *, p<0.05 for comparison to medium control. n.s  =  no significant difference.</p