GC/MS analysis of fractionated peaks and DHE.

<p>GC/MS was performed using an Agilent Technologies Network GC/MS system (7890 GC plus a 5975 mass detector) with a HP-5MS capillary column. (A) GC/MS chromatogram of the fractionated peak as FrC. (B) GC/MS chromatogram of dehydroergosterol as a standard. (C) Mass spectrum of peak 1. (D) Mass spectrum of peak 2.</p

Neurotoxity assay of microglial culture supernatant treated with DHE.

<p>(A) Quantification of H₂O₂-induced apoptosis and cell-death in Neuro-2A cells. Left; caspase-3/7, Right; YoPro-3. (B) Representative phase contrast images of Neuro-2A cells. Left, image of untreated cells; Middle, image of cells treated with 5 ng/ml LPS only; Right, image of cells treated with 5 ng/ml LPS and 5 μM DHE. (C) Quantification of LPS-induced apoptosis and cell-death in Neuro-2A cells. Left; caspase-3/7, Right; YoPro-3.</p

Characteristic of microglial cells treated with DHE using a flow cytometer.

<p>(A) Dot plot of microglia to gate CD11b-positive and CD206-positive cells treated either with or without DHE. (B) Ratio (%) of CD11b-positive and CD206-positive cells to CD11b-single positive cells. (C-F) Median fluorescent intensity of I-A/I-E, CD86, CD80 and CD68 in CD11b-positive cells. (G-J) Median fluorescent intensity of I-A/I-E, CD86, CD80 and CD68 in CD11b-positive and CD206-positive cells. K-N. Ratio (%) of TNF-α, IL-1β, MIP-1αin IL-12p40/p70-positive to CD11b-positive cells, respectively. O-R. Ratio (%) of TNF-α, IL-1β, MIP-1α and IL-12p40/p70-positive in CD11b-positive and CD206-positive cells, respectively.</p

The effect of solvent extracts and fractionated samples on the anti-inflammatory activity of microglial cells.

<p>(A) Freeze dried camembert cheese was treated with either <i>n</i>-hexane, chloroform, chloroform/methanol mixture (1:1 by volume) or methanol. Each extract was evaporated to dryness before dissolving the residue in a suitable buffer. These solutions were then used in the microglial TNF-α production assay. Evaluation of the fractionation and cytokine assay was repeated twice. (B) Methanol extract of camembert cheese was fractionated into Fr1-Fr12 using a C30 column. (C) Effect of the pretreatment of Fr1-Fr12 on production of TNF-α by microglial cells. (D) Major peaks with a retention time of 40.0 min to 55.0 min were re-fractionated as FrA-FrD. (E) Effect of the pretreatment of FrA-FrD on the production of TNF-α by microglial cells.</p

The effect of dairy products on the anti-inflammatory activity of microglial cells.

<p>(A) Isolated microglia were stimulated with 5 ng/ml LPS and 0.5 ng/ml of IFN-γ after pretreatment with an extract of each dairy product, and then TNF-α in the supernatant was quantified. Evaluation of the extraction and cytokine assay was repeated twice. (B) Effect of pretreatment with an extract of dairy product (with or without <i>P</i>. <i>candidum</i> fermentation) on the production of microglial TNF-α.</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

Effect of LAB-derived nucleic acids and combination with TLRLs on IFN-α production.

<p>A. DNA was extracted from <i>L. lactis</i> JCM5805, JCM20101 and <i>L. rhamnosus</i> ATCC53103. Each sample was added at the concentrations of 1, 5, and 10 µg/ml, respectively, to Flt-3L induced BM-derived DC. B. TLR2Ls (1 µg/ml Pam₃CSK₄, 10 µg/ml LTA), TLR3L (10 µg/ml Poly(I:C)) and TLR4L (5 ng/ml LPS) in combination with 0.1 µM of CpG-A and IFN-α production were measured. *, p<0.05 for comparison to CpG-A alone. C. Effect of LTA on IFN-α production induced by LAB-derived DNA. 10 µg/ml of LTA was mixed with 10 µg/ml of DNA prepared from <i>L. lactis</i> JCM5805, JCM20101 or <i>L. rhamnosus</i> ATCC53103 and added to Flt-3L induced BM-derived DC. *, p<0.05 for comparison to LAB-derived DNA alone. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures.</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

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