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Cited by 133Immune Response and InflammationProbiotics and Fermented FoodsImmunotherapy and Immune Responses

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

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Jounai, Kenta, Ikado, Kumiko, Sugimura, Tetsu, Ano, Yasuhisa, Braun, Jonathan, Fujiwara, Daisuke·2012·PLoS ONE
DOI 10.1371/journal.pone.0032588PMC PMC3323594
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Abstract

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.

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Figure 1 Comparison of DC activation by L. lactis and L. rhamnosus. Flt-3L induced BM-derived DC were treated with 10 µg/ml of stimulatory (L. lactis JCM5805 or JCM20101) or non-stimulatory (L. rhamnosus 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 L. rhamnosus ATCC53103. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures.
Figure 1 Comparison of DC activation by L. lactis and L. rhamnosus. Flt-3L induced BM-derived DC were treated with 10 µg/ml of stimulatory (L. lactis JCM5805 or JCM20101) or non-stimulatory (L. rhamnosus 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 L. rhamnosus ATCC53103. Representative data from three independent experiments are shown. Each experiment was done with triplicate cultures; data are mean ± SD for triplicate cultures.CC BY · PubMed Central
Figure 2 Production of IFNs by Flt-3L induced DC in response to L. lactis and L. rhamnosus. A. pDC and mDC mixed Flt-3L BM DC were stimulated with 10 µg/ml of L. lactis JCM5805, JCM20101 and L. rhamnosus 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.
Figure 2 Production of IFNs by Flt-3L induced DC in response to L. lactis and L. rhamnosus. A. pDC and mDC mixed Flt-3L BM DC were stimulated with 10 µg/ml of L. lactis JCM5805, JCM20101 and L. rhamnosus 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.CC BY · PubMed Central
Figure 3 LAB-induced cytokines production in purified pDC, mDC and mixed cultures. A. pDC and mDC were sorted from Flt-3L induced BM-derived DC. 10 µg/ml of L. lactis JCM5805, JCM20101 and L. rhamnosus 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.
Figure 3 LAB-induced cytokines production in purified pDC, mDC and mixed cultures. A. pDC and mDC were sorted from Flt-3L induced BM-derived DC. 10 µg/ml of L. lactis JCM5805, JCM20101 and L. rhamnosus 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.CC BY · PubMed Central
Figure 4 Morphology of pDC stimulated by LABs and incorporation into pDC. A. L. lactis JCM5805, JCM20101 and L. rhamnosus 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 L. lactis JCM5805, JCM20101 and L. rhamnosus 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.
Figure 4 Morphology of pDC stimulated by LABs and incorporation into pDC. A. L. lactis JCM5805, JCM20101 and L. rhamnosus 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 L. lactis JCM5805, JCM20101 and L. rhamnosus 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.CC BY · PubMed Central
Figure 5 Role of TLR signaling in IFN-α production elicited by LAB. 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.
Figure 5 Role of TLR signaling in IFN-α production elicited by LAB. 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.CC BY · PubMed Central
Figure 6 Effect of LAB-derived nucleic acids and combination with TLRLs on IFN-α production. A. DNA was extracted from L. lactis JCM5805, JCM20101 and L. rhamnosus 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 L. lactis JCM5805, JCM20101 or L. rhamnosus 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.
Figure 6 Effect of LAB-derived nucleic acids and combination with TLRLs on IFN-α production. A. DNA was extracted from L. lactis JCM5805, JCM20101 and L. rhamnosus 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 L. lactis JCM5805, JCM20101 or L. rhamnosus 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.CC BY · PubMed Central
Figure 7 CD4+CD25+FoxP3+Treg and IFN-γ producing CD4+T cell generation by L. lactis JCM5805-treated pDC. MLR reactions using naïve CD4+T cell prepared from BALB/c and purified C57BL/6 derived-pDC stimulated by L. lactis JCM5805 (10 µg/ml) or L. rhamnosus 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.
Figure 7 CD4+CD25+FoxP3+Treg and IFN-γ producing CD4+T cell generation by L. lactis JCM5805-treated pDC. MLR reactions using naïve CD4+T cell prepared from BALB/c and purified C57BL/6 derived-pDC stimulated by L. lactis JCM5805 (10 µg/ml) or L. rhamnosus 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.CC BY · PubMed Central
Figure 8 Effect of oral administration of L. lactis JCM5805 to healthy C57BL/6 mice. Healthy C57BL/6 mice were divided into two groups (n = 8 each). Control group (ctrl) were fed a normal diet, and L. lactis 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.
Figure 8 Effect of oral administration of L. lactis JCM5805 to healthy C57BL/6 mice. Healthy C57BL/6 mice were divided into two groups (n = 8 each). Control group (ctrl) were fed a normal diet, and L. lactis 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.CC BY · PubMed Central

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  1. H Kitazawa, 1994, Expression of mRNA encoding IFN α in macrophages stimulated with Lactobacillus gasseri., FEMS Microbiol Lett, 120, 315

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Metadata

Authors
Jounai, K., Ikado, K., Sugimura, T., Ano, Y., Braun, J., & Fujiwara, D.
Affiliations
  • Kirin (Japan) (JP)
  • University of California, Los Angeles (US)
Corresponding author
Fujiwara, Daisuke
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-04-10
Volume
7
Issue
4
Pages
e32588
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0032588
PMC
PMC3323594
OpenAlex
W2062007417
Cited by
133
References
54
Citations by year
2026: 9 · 2025: 10 · 2024: 12 · 2023: 15 · 2022: 11 · 2021: 11 · 2020: 7 · 2019: 7 · 2018: 12 · 2017: 9 · 2016: 8 · 2015: 10 · 2014: 5 · 2013: 6 · 2012: 1
FWCI
3.07
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health (1ul1rr033176-01, UL1TR000124)
  • National Institute of Allergy and Infectious Diseases (R01AI078885)
  • National Center for Research Resources (UL1-RR-033176)
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
lactic acid bacteria, plasmacytoid dendritic cells, myeloid dendritic cells, TLR9, MyD88, interferon-alpha, Lactococcus lactis, type I interferon, interferon lambda, TLR4, ICOSL, PD-L1, regulatory T cells, oral administration, mesenteric lymph nodes, immunomodulation, probiotics, antiviral immunity
MeSH terms
Animals, Bone Marrow, Cell Communication, Cells, Cultured, Dendritic Cells, Female, Immunologic Factors, Leuconostoc, Mice, Inbred C57BL, Pediococcus, Streptococcus, Lactococcus lactis, Interferon-alpha, Coculture Techniques, Lactic Acid, Myeloid Cells, T-Lymphocytes, Regulatory, Toll-Like Receptor 9, Mice, Myeloid Differentiation Factor 88

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