Lc exerts anti-inflammatory effect on LPS-activated macrophage cell line RAW 264.7.

<p>(A) Lc decreases the production of TNF-α in LPS-activated macrophages while Lp does not. TNF-α production by cells stimulated with 1 mg/l of LPS is set as 100% and data are expressed as means ± standard error of the mean of three independent experiments. *P<0.05: the means were compared against a hypothetical mean of 100% by one sample t-test (B) The effect of Lc on NF-κB binding activity in LPS-stimulated RAW 264.7 cells. Lc and Lp was co-cultured with LPS-activated cells for 24 h, and then the binding activity of NF-κB subunit p65 was analyzed by colorimetric assay. Data are expressed as mean ± standard deviation of three independent experiments. One-way ANOVA with Dunnett's multiple comparison test was used to evaluate the significance of differences between experimental groups and the LPS-treated cells group (**P<0.01, ***P<0.001). (C, D) Lc counteracts the LPS mediated M1 polarization. Expression of F4/80, CD206, IL-7R was determined by flow cytometry. One-way ANOVA with Dunnett's multiple comparison test was used to evaluate the significance of differences between experimental groups and the LPS-treated cells group (**P<0.01).</p

Oral treatment with Lc changes the intestinal microbiota composition.

<p>Normalized and z scored heat map and clustering dendrogram comparing relative abundance of the top 50 most abundant bacterial species in fecal microbiota of PBS (pool of 5 mice) and Lc-treated mice (pool of 5 mice) before the treatment (Day 0), before colitis induction (Day 28) and at the end of the experiment (Day 35). Horizontal columns represent the day of the experiment and or the treatment; vertical rows depict genus sorted from the most abundant species from left to right. The color scale for the heat maps is shown in upper left corner. The samples were clustered on the basis of their similarity by unsupervised clustering in the package CLUTO 2.1.1 (<a href="http://glaros.dtc.umn.edu/gkhome/cluto/cluto/download" target="_blank">http://glaros.dtc.umn.edu/gkhome/cluto/cluto/download</a>), as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027961#pone.0027961-Zhao1" target="_blank">[56]</a>.</p

Oral treatment with Lc increases the number of CD4⁺FoxP3⁺ T_regs in MLNs.

<p>No significant changes were found in spleen or Peyer's patches. The plots shows the expression of CD4 <i>versus</i> FoxP3 on gated Th cells (CD3⁺CD8⁻), and the values within the plots represent the mean ± standard deviation of the total numbers of CD4⁺FoxP3⁺ T cells from one representative experiment out of three independent experiments (3–5 mice per group). One-way ANOVA with Dunnett's multiple comparison test was used to evaluate the significance of differences in numbers of CD3⁺CD8⁻CD4⁺FoxP3⁺ cells between DSS/Lc-treated groups and the DSS/PBS-treated (control) group (*P<0.05).</p

Oral treatment with Lc strengthens the gut barrier function as compared to PBS control mice.

<p>(A) Measurement of intestinal permeability by FITC-dextran. Serum levels of 4.4-kDa FITC-dextran 5 hour after administration by gavage in DSS/PBS, DSS/Lc-treated group and healthy controls. Immunohistological detection of tight junction proteins ZO-1 (B) and occludin (C) in representative sections of colon and terminal ileum from DSS/PBS-, DSS/Lc-treated group and healthy controls. Fluorescent signal of ZO-1 or occludin (red) is merged with DAPI counterstained nuclei (blue). mRNA expression of ZO-1(D) and occludin (E) evaluated in DSS/PBS, DSS/Lc treated group and healthy controls in colon and terminal ileum. RT-PCR was performed using TaqMan® gene expression assay for ZO-1. β-actin was used as the internal control. One-way ANOVA with Dunnett's multiple comparison test was used to evaluate the significance of differences between experimental groups and the DSS/PBS-treated control group (*P<0.05, **P<0.01, ***P<0.001). Data represent means (bar) ± SD (whisker) of five mice of one representative experiment out of three independent experiments. Scale bars are 10 µm in ZO-1 and 20 µm in occludin figures.</p

Pretreatment with Lc changes cytokine production in different parts of the gut.

<p>After DSS treatment and 24 hours cultivation, the production of cytokines TNF-α, TGF-β, IL-6, IL-10, IFN-γ differs in various parts of the gut as measured by ELISA. *P<0.05, **P<0.01 between DSS/PBS and DSS/Lc-treated mice in the same part of the gut was compared by unpaired Student's t-test (n = 10 per group).</p

Lc improves the severity of DSS-induced colitis in BALB/c, but not in SCID mice.

<p>Values are expressed as means ± SD (5 BALB/c mice per group) of one representative experiment out of three independent experiments. Unpaired Student's t-test in BALB/c mice was used to evaluate the significance of differences between experimental groups and the PBS-treated control group (*P<0.05, ***P<0.001).</p