Lysate of Probiotic Lactobacillus casei DN-114 001 Ameliorates Colitis by Strengthening the Gut Barrier Function and Changing the Gut Microenvironment

BACKGROUND: Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4(+)FoxP3(+) regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4(+)FoxP3(+) regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway. CONCLUSION/SIGNIFICANCE: Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment

COVID-19, Vaccination, and Female Fertility in the Czech Republic

The fast-track process to approve vaccines against COVID-19 has raised questions about their safety, especially in relation to fertility. Over the last 2 years, studies have appeared monitoring female fertility, especially from assisted reproduction centers or in animal experiments. However, studies monitoring healthy populations are still limited. The aim of our study was to monitor the relevant parameters of female fertility (sex and other steroids, LH, FSH, SHBG, Antimüllerian hormone and antral follicle count) before and then 2–4 months after the third dose of vaccination against COVID-19 in a group of 25 healthy fertile woman. In addition, anti-SARS-CoV-2 and anti-SARS-CoV-2S antibodies were determined. We did not observe significant changes in the measured parameters before and after the third dose of vaccination. By comparing levels of the analytes with antibodies indicating a prior COVID-19 infection, we found that women who had experienced the disease had statistically lower levels of estrone, estradiol, SHBG and 5α-dihydroprogesterone, and conversely, higher levels of androgen active dehydroepiandrosterone and dihydrotestosterone. Our results confirm that vaccination does not affect female fertility, and that what fertile women should be worried about is not vaccination, but rather COVID-19 infection itself

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 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 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 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