15 research outputs found

    CD4 T cells and iTreg cells in mice with T-cell transfer-induced colitis.

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    <p>Splenocytes of mice with T-cell transfer-induced colitis, harvested six weeks after T-cell transfer, were stained for CD4 and FoxP3 expression and analyzed by flow cytometry. (A) absolute numbers of splenocytes; (B, D) percentages, and (C, E) numbers of CD4+ T-cells and CD4+FoxP3+ T-cells in the spleen. Results for individual mice and means+SEM (n = 3–6 per group) are depicted. *p<0.05.</p

    Colitis development in T-cell transferred mice.

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    <p>Flow cytometry-sorted naĂŻve CD4<sup>+</sup> T-cells of B6 (WT) or ÎČ2i/MECL-1<sup>−/−</sup>ÎČ5i/LMP7<sup>−/−</sup>(IS−/−) mice were transferred into RAG1<sup>−/−</sup> or RAG1<sup>−/−</sup>ÎČ2i/MECL-1<sup>−/−</sup>ÎČ5i/LMP7<sup>−/−</sup> (IS−/− x RAG1−/−) mice and colitis development was determined 6 weeks by histological scoring of H&E stained tissue samples (see M&M). (A) overall colitis scores, (B) colitis scores in the proximal −, (C) mid −, and (D) distal colon sections. Scores for individual mice and means+SEM (n = 3–6 per group) are depicted. *p<0.05. Data are representative of two independent experiments.</p

    Cytokine expression in mice with T-cell transfer-induced colitis.

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    <p>Splenocytes of mice with T -cell transfer-induced colitis were stimulated with anti-CD3 mAb for 4 hrs. mRNA was extracted and cytokine expression was quantified by RT-PCR. Expression of (A) IL17a, (B) IFNy, (C) IL10, and (D) TNFα relative to 18S rRNA is shown for individual mice per experimental group. Bars represent means+SEM (n = 3–6). *p<0.05. Data are representative of two independent experiments.</p

    Innate immune memory through TLR2 and NOD2 contributes to the control of <i>Leptospira interrogans</i> infection

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    <div><p><i>Leptospira interrogans</i> are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many <i>Leptospira</i> serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with <i>Lactobacillus plantarum</i> confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of <i>Lactobacillus</i>, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced <i>ex vivo</i> more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of <i>Leptospira</i>, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train <i>ex vivo</i> human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.</p></div

    Protection correlates with early production of pro-inflammatory mediators.

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    <p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. Mice were sacrificed at 0, 12, 18, 24, 48, 72, and 144 h p.i. The time point 0 h p.i. of the Re-Inf mice corresponds to the day 10 of the SL mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153829#pone.0153829.s001" target="_blank">S1 Fig</a>). The BAL and lungs tissue homogenates were were assessed by ELISA for quantification of (A) IL-1α, andIL-1ÎČÎČ, (B) G-CSF, TNF α and IL-6, and (C) IL-17. Data (mean ± SEM) represent 2 to 3 independent experiments with n = 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01, +++p<0.001 SL vs LL dose; *p<0.05, **p<0.01, ***p<0.001 Re-Inf vs SL dose; #p<0.05, ##p<0.01, ###p<0.001 LL dose vs Re-Inf mice).</p

    Phagocytes from protected mice internalize and kill the conidia efficiently.

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    <p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153829#pone.0153829.s001" target="_blank">S1 Fig</a>). (A) Representative pictures showing phagocytosis of conidia by BAL neutrophils (left panel) and macrophages (right panel) in the three infection settings at 48 h post infection. Mice were infected with FITC (green) labelled conidia, and the non-phagocytosed conidia were stained using an anti-conidia antibody detected by a secondary Texas Red-conjugated antibody (red). DNA was labelled with Hoechst stain (blue). Using fluorescence microscopy, the phagocytosed conidia were detected only in green while the outside FITC and Texas red conidia appear in yellow. Germination was indicated by the arrowheads pointing to non-phagocytozed and germinating conidia and to piercing hyphae (in red). The percentage of phagocytosis was estimated as the ratio of the number of ingested conidia to the total number of conidia bound to 100 phagocytes (B) Percentages of neutrophils (PMNs) and alveolar macrophages (AMs) involved in the phagocytosis in the lethal and re-infected groups at D1 and D2 h p.i. Within each histogram, is represented the percentage of either neutrophils or alveolar macrophages participating to the phagocytosis (C) BALs were collected and analyzed by flow cytometry to evaluate the killing potential. Plots are gated on GR1<sup>high</sup> CD11b<sup>+</sup> neutrophils and show expression level of the ROS (left panel) and MPO (right panel) production. Data (mean ± SEM) represent 2 to 3 independent experiments with 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01, +++p<0.001 L dose <i>vs</i> SL dose; *p<0.05, **p<0.01, ***p<0.001 Re-Inf <i>vs</i> SL dose; #p<0.05, ###p<0.001 LL dose <i>vs</i> Re-Inf mice).</p

    Mice recovering from sublethal <i>A</i>. <i>fumigatus</i> conidia inoculum are protected in a T-, B-lymphocytes or NK independent manner.

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    <p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. The mice survival was followed on a daily basis during 15 days p.i. (A) <i>A</i>. <i>fumigatus-</i>emitted bioluminescence images from the thorax of representative BALB/c mice taken using the IVIS spectrum (Perkin) (B) Quantification (photons per second) of bioluminescence signal from mice chest areas using Living Image software (Perkin). (C) Modulation of the body weight in the three infection settings. (D) Colony forming unit from lungs homogenates. Mice were sacrificed from the “SL” group at day 1 and 10 and lung homogenates were collected and plated to determine the colony forming units (CFU). (E) Survival rate of BALB/c mice in the three infection settings. (F) Representative lung sections from mice at 48 h p.i. Left panels show Hemalun and right panel methenamine silver stained lung sections. Hemalun staining visualizes the inflammatory foci (purple), whereas silver staining visualizes fungal elements (black). (G) Survival rate of C57BL/6 mice in in the three conditions (H) Survival rate RAG<sup>-/-</sup>γc<sup>-/-</sup>mice in the three conditions. (****p = 0.0001 L dose <i>vs</i> SL dose; ####p<0.0001 L dose <i>vs</i> Re-Inf mice; n = 10 to 15 mice per group).</p

    A sublethal infection primes bone marrow and blood phagocytes to increased Dectin-1 and CXCR2 expression.

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    <p>To investigate the priming process following the subethal infection, mice were either sacrificed at day 0 (NaĂŻve) or at day 10 following infection with the sublethal dose (Primed). (A) CXCR2 expression on bone marrow and blood neutrophils (GR1<sup>high</sup> CD11b<sup>+</sup> population). Dectin-1 expression levels on bone marrow and blood neutrophils (GR1<sup>high</sup> CD11b<sup>+</sup> population) (B), and macrophages (F4/80<sup>+</sup>CD11b<sup>+</sup>GR-1<sup>-</sup> population) (C). (D) Dectin-1 expression levels on BAL neutrophils and macrophages. Representative histograms show level of expression (left panel) and quantification of respective values in graphs (right panel). (E) Dynamic of Dectin-1 level expression on both neutrophils (left panel) and macrophages (right panel) in the BALs in the three infection settings. “SL” or “L” mice were sacrificed at 0 h (naĂŻve), 12 18, 24, 48, 72 and 144 h p.i. and “Re-inf” mice were sacrificed at 0 h (10 days after the SL infection, day of the re-infection), 12, 18,24, 48, 72 and 144 h p.i. Data (mean ± SEM) represent 2 to 3 independent experiment with n = 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01; *p<0.05, **p<0.01, ***p<0.001Re-Inf <i>vs</i> SL dose; #p<0.05, ##p<0.005 ###p<0.001 LL dose <i>vs</i> Re-Inf mice).</p

    Similar antigen presentation capacity of BMDCs from WT and CatB<sup>-/-</sup> mice.

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    <p>WT and CatB<sup>-/-</sup> were infected subcutaneously with 3x10<sup>6</sup> stationary phase promastigotes of <i>L</i>. <i>major</i> in the footpads. On days 21 and 28, lymph nodes were harvested and whole cells or purified CD4 cells from these were co-cultured overnight with WT or CatB<sup>-/-</sup> BMDCs that were non-stimulated as control or activated by PMA, killed or live <i>L</i>. <i>major</i>. Supernatants were harvested for cytokine determination by specific ELISA and unadherent cells were harvested for intracellular cytokine staining by FACS. <b>(A-B)</b> IFNÎł levels secreted by lymph node (dLNs) cells and (<b>C-D)</b> percentage of IFNÎł+ cells gated on CD4+ cells in these dLNs from <i>L</i>. <i>major</i>-infected WT <b>(A, C)</b> or CatB<sup>-/-</sup> <b>(B, D)</b> mice after co-culture with WT (filled bars) or CatB<sup>-/-</sup> (empty bars) BMDCs. <b>(E-F)</b> IFNÎł levels secreted by CD4+ cells and <b>(G-H)</b> percentage of IFNÎł+ cells gated on CD4+ cells from purified CD4+ cells of <i>L</i>. <i>major</i>-infected WT <b>(E, G)</b> or CatB<sup>-/-</sup> <b>(F, H)</b> mice that were co-cultured with WT (filled bars) or CatB<sup>-/-</sup> (empty bars) BMDCs. Data depicted represent the mean and SEM of at least 3 independent experiments with n ≄ 4 donor mice/group.</p
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