1. Volume 21, Issue 5, Pages 1215-1226 (October 2017)
Commensal Lactobacillus Controls Immune Tolerance during Acute Liver Injury in Mice 
Nobuhiro Nakamoto, Takeru Amiya, Ryo Aoki, Nobuhito Taniki, Yuzo Koda, Kentaro Miyamoto, Toshiaki Teratani, Takahiro Suzuki, Sayako Chiba, Po-Sung Chu, Atsushi Hayashi, Akihiro Yamaguchi, Shunsuke Shiba, Rei Miyake, Tadashi Katayama, Wataru Suda, Yohei Mikami, Nobuhiko Kamada, Hirotoshi Ebinuma, Hidetsugu Saito, Masahira Hattori, Takanori Kanai 
Cell Reports 
Volume 21, Issue 5, Pages (October 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1215-1226DOI: (10.1016/j.celrep.2017.10.022)
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3. Figure 1
Repetitive Con A Injection Induces Immune Tolerance in the Liver
(A) Study design. Male C57BL/6 mice were injected with PBS or given a single injection of Con A or two injections either 1 or 7 days apart and analyzed 12 hr after the final injection.
(B) Representative photomicrographs of H&E-stained sections of the liver. The area comprising necrotic hepatocytes, which is encircled by dashed lines, was larger in Con A (−1D)-Con A mice, whereas the histological findings were mild in Con A (−7D)-Con A mice. Data are representative of each group. Scale bars, 500 μm.
(C) Serum ALT level. Data show mean ± SEM (n = 6/group). Data are representative of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
See also Figure S1.
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4. Figure 2
CD11bintCD11chigh cDCs Accumulate in the Late Phase of Con A-Induced Liver Inflammation
(A) Study design. Each group of mice was sacrificed and analyzed either before (day 0) or 1 day or 7 days after a single Con A injection, respectively.
(B) Representative CD11b and CD11c staining on whole liver mononuclear cells of mice at the indicated time point. Mononuclear cells were gated on a forward scatter (FSC)/side scatter (SSC) plot, followed by exclusion of dead cells using 7-amino-actinomycin D (7-AAD).
(C and D) Mean percentages of three cell subsets (pDCs, cDCs, and macrophages) in whole liver mononuclear cells (C) and absolute cell numbers of the three subsets (D). Blue bars, before Con A injection (day 0); orange bars, 1 day following Con A injection; green bars, 7 days following Con A injection. Data show mean ± SEM (n = 4/group). Data are representative of three independent experiments. ∗∗p < 0.01, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
(E) Phenotypic characterization of isotype control (top), day 1 macrophages (center), and day 7 cDCs (bottom) of the liver. Shown is surface expression of CD80, CD86, MHC class II, CD8a, CCR9, and CX3CR1 on the indicated APC subsets. Red dashed lines indicate the cutoff value determined by the isotype control.
See also Figure S2.
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5. Figure 3 D7-cDCs Produce IL-10 and TGF-β in a TLR9-Dependent Manner
(A) Whole liver mononuclear cells (2 × 104 cells/well) derived from mice at the indicated time point following Con A injection were stimulated with TLR ligands for 24 hr in vitro. The supernatants were collected and measured by cytometric bead array (CBA). TNF, IFN-γ, IL-10, and TGF-β concentrations are shown in the graph. Blue bars, no stimulation; orange bars, FSL1 stimulation; green bars, LPS stimulation; purple bars, ODN1826 stimulation. Data show mean ± SEM (n = 4/group). Significant changes compared with the day 0 value in each TLR ligand stimulation were calculated by ANOVA with Tukey’s multiple comparisons correction. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p <
(B) IL-10 (top) and TGF-β (bottom) concentration. Sorted liver APCs (2 × 104 cells/well) derived from mice 7 days after Con A injection were stimulated with TLR9 ligand (ODN1826) in vitro. The supernatants were collected and measured by CBA. Data show mean ± SEM (n = 3/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
(C) Study design. PBS- or Con A-pre-injected mice were either treated with control or IL-10R-neutralizing antibody (25 μg/g mouse, clone 1B1.3a, BioLegend) 6 hr prior to the second Con A injection on day 7 and sacrificed 12 hr following the final Con A injection.
(D) Serum ALT levels. Data show mean ± SEM (n = 5/group). Data are representative of two independent experiments. ∗∗p < 0.01, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
See also Figures S3 and S4.
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6. Figure 4 Adoptive Transfer of Day 7 cDCs Attenuates Liver Inflammation
(A) Experimental design. Macrophages and cDCs were sorted from the livers of Ly5.2+ mice 1 day or 7 days after Con A administration. Mice were divided into three groups as follows: Ly5.1+ mice intravenously (i.v.) injected with PBS (Con A group), Ly5.1+ mice intravenously transferred with day 1 macrophages (CoNa+D1-macrophage group), Ly5.1+ mice intraveously transferred with day 7 cDCs (CoNa+D1-cDC group). All mice were immediately injected with Con A following the cell transfer.
(B) Representative photomicrographs of H&E-stained sections of the liver. Data are representative of each group. Scale bars, 500 μm.
(C) Serum ALT levels. Data show mean ± SEM (n = 5/group). Data are representative of two independent experiments. ∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
(D) Representative CD11b and CD11c staining on liver mononuclear cells of the indicated mice.
(E) Absolute cell numbers of macrophages (left) and of cDCs in the liver. Data show mean ± SEM (n = 5/group). Data are representative of two independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
(F) Relative abundance of host (CD45.1+, white bars) or donor (CD45.2+, black bars) origin in liver macrophages (left) and cDCs (right) derived from the indicated mice. Each bar indicates the mean value in each group of mice (n = 5).
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7. Figure 5
Con A-Induced Hepatitis Results in Structural Changes in Gut Microbiota
(A) Principal coordinate analysis (PCoA) based on the weighted UniFrac analysis of bacterial community structures (green, day 0; orange, day 1; blue, day 4; purple, day 7). The two components of the weighted PCoA plot explained 42.8% and 27.1% of the variance. Dissimilarities between two groups were evaluated by permutational multivariate analysis of variance (PERMANOVA). ∗p < 0.05.
(B) The composition of gut microbiota at the phylum level on collected feces derived from mice at the indicated time point following Con A injection. Each bar indicates the mean value in each group of mice (n = 4).
(C) Serial change in the composition of gut microbiota (genus) on collected feces derived from mice at the indicated time point following a single Con A administration. For genus-level assignment of 16S reads (16S rRNA gene V1-V2 region) mapped to the known FL-16S sequences and unmapped OTUs, a 94% sequence identity threshold was applied. Green bars, before Con A injection (day 0); orange bars, day 1 after Con A injection; blue bars, day 4 after Con A injection; purple bars, day 7 following Con A injection. Dominant bacteria are shown in the graph.
(D) Differentially abundant taxa at the genus level. Linear discrimination analysis effect size (LEfSe) analysis showing genera that were significantly different (linear discriminant analysis [LDA] score > 3).
(E) Serial changes in the composition of OTU that were assigned as genus Lactobacillus on collected feces derived from mouse feces at the indicated time point following a single Con A administration. The vertical axis represents the relative abundance (percent) of each OTU in the microbiota of day 0 (green bars), day 1 (orange bars), day 4 (blue bars), and day 7 fecal samples (purple bars) subjects.
(C and E) Data show mean ± SEM (n = 4/group). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction. The significant point compared to the day 0 value is shown.
See also Figure S5.
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8. Figure 6
Gut Colonization by Lactobacillus Attenuates Acute Liver Injury
(A) Study design. Gut-sterilized mice treated with broad-spectrum antibiotics for 3 weeks were either left untreated (recovery group) or inoculated with an isolated L. johnsonii strain (L. johnsonii group) every other day for 2 weeks, followed by Con A injection. All mice were sacrificed and analyzed 12 hr after the final Con A injection.
(B) The amount of L. johnsonii in cecal contents of recovery and L. johnsonii-inoculated mice, assessed by 16S qPCR. Data show mean ± SEM (n = 5/group). ∗∗p < 0.01 by Student’s t test.
(C) Representative photomicrographs of H&E-stained sections of the liver. Scale bars, 500 μm.
(D) Serum ALT levels. Data show mean ± SEM (n = 6/group).
(E) Representative CD11b and CD11c staining on liver mononuclear cells derived from the indicated mice.
(F) Absolute cell numbers of macrophages (left) and cDCs (right) in the livers of the indicated mice following Con A administration. Data show mean ± SEM (n = 6/group).
(D and F) Data are representative of two independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < by Student’s t test.
(G) Correlation between the ratio of the number of macrophages to cDCs and serum ALT levels in recovery and L. johnsonii mice (n = 12). Correlations were evaluated by Spearman rank correlation test.
(H) Absolute cell numbers of cDCs in the liver of gut-sterilized mice inoculated with either L. johnsonii, Lactococcus lactis JCM5805T, or Bacteroides thetaiotaomicron JCM5827 every other day for 2 weeks.
(I) Serum ALT levels in the indicated mice following Con A injection.
(H and I) Data show mean ± SEM (n = 4–6/group). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
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9. Figure 7
Commensal Lactobacillus Species Activates Gut Innate Lymphoid Cells to Produce IL-22, which, in Turn, Elicits Liver Tolerance
(A) Gut-sterilized mice treated with broad-spectrum antibiotics for 3 weeks were either left untreated (recovery) or inoculated with an isolated L. johnsonii strain (Lj) every other day for 2 weeks. Shown is representative intracellular IL-22 and RORγt staining on TCRβ− CD3− CD11b− gated colonic lamina propria mononuclear cells (LPMCs).
(B) Mean percentage of IL-22+ cells in RORγt+ TCRβ−CD3− CD11b− ILC3s of colonic LPMCs (n = 6/group).
(C) The concentration of IL-22 in the serum derived from the indicated mice. Data show mean ± SEM (n = 6/group). ∗∗∗p < by Student’s t test.
(D) Intestinal permeability measured by serum fluorescein isothiocyanate (FITC)-dextran concentration following Con A injection in recovery (triangles), L. johnsonii (rectangles), and PBS-injected control (circles) mice. Data show mean ± SEM (n = 5/group).
(E) Gene expression of occludin (left) and ZO-1 (right) mRNA normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the colon epithelium derived from the indicated groups. Data show mean ± SEM (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < by ANOVA with Tukey’s multiple comparisons correction.
(F) Mice were intravenously injected with recombinant IL-22 (0.1 μg/g, , BioLegend) or PBS, followed by Con A challenge. Shown is representative CD11b and CD11c staining on liver mononuclear cells.
(G) Mean percentages (left) and absolute numbers (right) of cDCs in the liver. Data show mean ± SEM (n = 5/group). ∗∗∗p < by Student’s t test.
(H) Study design. Gut-sterilized mice were either left untreated (recovery) or transplanted with L. johnsonii every other day for 2 weeks. Both groups of mice were administered IL-22-neutralizing antibody (2.5 μg/g mouse, AF582, R&D Systems) 6 hr prior to Con A injection. All mice were sacrificed 12 hr after Con A administration.
(I) Serum ALT levels. Data show mean ± SEM (n = 8/group). Data are combined from two independent experiments.
See also Figures S6 and S7.
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