Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes

The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the Tcell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment

Colonic Immune Stimulation by Targeted Oral Vaccine

<div><h3>Background</h3><p>Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that <em>Lactobacillus gasseri</em> expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against <em>Bacillus anthracis</em> Sterne challenge.</p> <h3>Methodology/Principal Finding</h3><p>In the present study, we investigated the effects of a dose dependent treatment of mice with <em>L. gasseri</em> expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of <em>L. gasseri</em> expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ T cell immune responses. Notably, high doses of <em>L. gasseri</em> expressing PA-DCpep (10¹² CFU) were not toxic to the mice. Treatment of mice with <em>L. gasseri</em> expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with <em>L. gasseri</em> expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG₁, IgG_2b, IgG_2c and IgG₃. <em>L. gasseri</em> expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors.</p> <h3>Conclusion/Significance</h3><p>These findings suggest that <em>L. gasseri</em> expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.</p> </div

<i>L. gasseri</i> expressing PA-DCpep is safe for mice at high doses.

<p>C57BL/6 mice were orally gavaged with 10⁹ CFU of <i>L. gasseri</i> expressing PA-DCpep once and the CFU per gram of feces was determined (A) in MRS plates with erythromycin (5 µg/mL). C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS, serum was collected after days 1, 3 and 7, and the enzyme activities of ALT/SGPT (B) and AST/SGOT (C) were analyzed by ELISA. CCl₄ was used as a positive control for toxicity (B & C). Tissues from C57BL/6 mice orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS were collected and sections stained with H&E at days 1, 3 and 7 of treatment. (D) Photomicrographs of H&E sections of liver A–D, kidney E–H, spleen I–L, colon M–P, and cecum Q–T. Insets of a representative glomerulus and surrounding proximal tubules are included in each of the kidney photomicrographs. Insets of high magnification of the lymphoid tissue are included in each of the spleen photomicrographs. For each of the organs, the PBS control is pictured in the left column A, E, I, M and Q, respectively. The three photomicrographs on the right are from increasing concentrations of bacteria from 10⁷, 10⁹ and 10¹² from left to right.</p

<i>L. gasseri</i> expressing PA-DCpep promotes Th17 responses.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, and stained with antibodies against CD4, CD8, RORγT, IL-17 and IL-22 (A&B) before analysis by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS. (C) C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; colonic sections were stained with RORγT (red) and CD4 (green) antibodies, and visualized using confocal microscopy.</p

<i>L. gasseri</i> expressing PA-DCpep enhances the induction of regulatory T cells.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, stained with antibodies against CD4, CD8, IFNγ (A&B), TGFβ and FoxP3 (C&D), and analyzed by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS.</p

Activation of colonic DCs by <i>L. gasseri</i> expressing PA-DCpep.

<p>Groups of C57BL/6 mice (n = 3) were fed with different doses (10⁷, 10⁹ and 10¹² CFU) of <i>L. gasseri</i> expressing PA-DCpep and sacrificed on days 1, 3, 7 and 14 post-inoculation. The activation of colonic DCs was evaluated by the surface expression of CD40, CD86 and B7H1, and analyzed by flow cytometry (A). Lamina propria lymphocytes (LPLs) were also stained with antibodies against CD11c, CD11b, IL-10 and IL-12, and analyzed by flow cytometry (B). Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS. (C) C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS and colonic sections were stained with CD11c (magenta), CD11b (red) and either TNFα, IL-12 or IL-10 (green) antibodies, and visualized using confocal microscopy.</p

Activation of PRR-genes by <i>L. gasseri</i> expressing PA-DCpep.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU), or PBS; RNA was isolated from colon tissue after days 1, 3 and 7 of treatment. Quantitative real-time PCR was performed to measure changes in gene expression. Hierarchical matrix cluster analysis (A) and comparative gene analysis presented as a heat map (B). Data are representative of two independent experiments with duplicates.</p