Spherical Lactic Acid Bacteria Activate Plasmacytoid Dendritic Cells Immunomodulatory Function via TLR9-Dependent Crosstalk with Myeloid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are a specialized sensor of viral and bacterial nucleic acids and a major producer of IFN-α that promotes host defense by priming both innate and acquired immune responses. Although synthetic Toll-like receptor (TLR) ligands, pathogenic bacteria and viruses activate pDC, there is limited investigation of non-pathogenic microbiota that are in wide industrial dietary use, such as lactic acid bacteria (LAB). In this study, we screened for LAB strains, which induce pDC activation and IFN-α production using murine bone marrow (BM)-derived Flt-3L induced dendritic cell culture. Microbial strains with such activity on pDC were absent in a diversity of bacillary strains, but were observed in certain spherical species (Lactococcus, Leuconostoc, Streptococcus and Pediococcus), which was correlated with their capacity for uptake by pDC. Detailed study of Lactococcus lactis subsp. lactis JCM5805 and JCM20101 revealed that the major type I and type III interferons were induced (IFN-α, -β, and λ). IFN-α induction was TLR9 and MyD88-dependent; a slight impairment was also observed in TLR4-/- cells. While these responses occurred with purified pDC, IFN-α production was synergistic upon co-culture with myeloid dendritic cells (mDC), an interaction that required direct mDC-pDC contact. L. lactis strains also stimulated expression of immunoregulatory receptors on pDC (ICOS-L and PD-L1), and accordingly augmented pDC induction of CD4+CD25+FoxP3+ Treg compared to the Lactobacillus strain. Oral administration of L. lactis JCM5805 induced significant activation of pDC resident in the intestinal draining mesenteric lymph nodes, but not in a remote lymphoid site (spleen). Taken together, certain non-pathogenic spherical LAB in wide dietary use has potent and diverse immunomodulatory effects on pDC potentially relevant to anti-viral immunity and chronic inflammatory disease

Modulation of Innate Immunity by lignin-Carbohydrate, a Novel TLR4 Ligand, Results in Augmentation of Mucosal IgA and Systemic IgG Production

Previous study revealed that a specific lignin-carbohydrate preparation, named as lignin-rich enzyme lignin (LREL) derived from plant husk, is a novel toll-like receptor 4 ligand and shows a potent immune-stimulatory activity against dendritic cells (DCs) in vitro. In this report, we investigated immune-stimulatory activity of LREL in vivo. Single intraperitoneal (i.p.) or oral treatment of LREL elicited activation of systemic and mucosal DCs, which were accompanied by significant elevation of cell surface activation markers and ratio of IL-12p40 producing cells. In addition, LREL-fed mice showed not only mucosal DCs activation but also significant increase of IFN-γ+ CD4+ T cells in mesenteric lymph node (MLN), respectively. We further examined the effect of LREL oral immunization in combination with ovalbumin (OVA) on the activation of acquired immune system. In LREL administered group, total mucosal IgA concentration was significantly increased, while antigen-specific immunoglobulin A (IgA) concentration was not changed between groups. On the other hand, both total and antigen-specific IgG concentrations in plasma were significantly increased in the LREL administered group. Taken together, oral treatment of LREL is able to affect mucosal and systemic antibodies induction and might be useful for effective immune-stimulatory functional foods and mucosal vaccine adjuvant

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

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

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

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

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