MSU crystals induce trained anti-mycobacterial innate immunity.

<p>(<b>A</b>) Diagram showing the course of <i>in vitro</i> preincubation experiment. THP-1 cells were cultured with 5 or 50 μg/ml MSU for 3 days. Thereafter, the medium was changed to remove MSU stimulus and cells cultured for further 4 days. Finally, cells were exposed to BCG at the MOI of 10 for 3 hours (T0), washed and cultured for further 3 days (T3). (<b>B</b>) Intracellular mycobacterial growth was monitored in BCG infected THP-1 cells prestimulated or not with 5 or 50 μg/ml MSU. Results are shown as mean ± SD of CFU values performed in triplicate and are representative of three independent experiments. ° p < 0.05 and * p < 0.001 in comparison with same time non pre-stimulated control cells. (<b>C</b>) Intracellular mycobacterial growth was monitored in THP-1 cells, pre-stimulated or not with 50 μg/ml MSU, and exposed or not to 10 μM chloroquine after BCG infection for 3 days. Results are shown as mean ° SD of CFU values performed in triplicate. * p < 0.05 and ° p < 0.01 in comparison with same time non pre-stimulated control cells. (<b>D</b>) IL-1β production in the supernatant of BCG infected THP-1 cells pre-stimulated or not with either 5 or 50 μg/ml MSU. Results are shown as mean ± SD of values performed in triplicate and are representative of three independent experiments. * p < 0.05 in comparison with same time non pre-stimulated control cells.</p

MSU crystals enhance Ca²⁺ dependent ROS production.

<p>(<b>A</b>) dTHP-1 cells were incubated with 3 μM Fluo-3/AM at 37°C for 1 hour in the dark and were stimulated with 5 μg/ml MSU. After stimulation, fluorescence emission was continuously monitored for 30 minutes and expressed as to determine relative alteration in intensity. (<b>B</b>) dTHP-1 cells were incubated for 1 hour at the dark with 10 μM DCF, or with 3 μM Fluo-3/AM (inset of the figure), and were stimulated with 5 μg/ml MSU. Ca^<b>2+</b> dependence ROS generation was assessed by adding 20 μM BAPTA-AM or 3 mM EGTA 30 minutes and 15 minutes before MSU addition, respectively. Fluorescence emission was monitored at 20 minutes after stimulation. Results are expressed as mean ± SD of arbitrary fluorescence units performed in triplicate and are representative of three separate experiments. * p < 0.01 in comparison with non-stimulated control cells</p

Co-administration of BCG with MSU crystals enhances the clearance and efficacy of BCG vaccination.

<p>Five mice per group were vaccinated with i) 10^<b>6</b> CFU BCG, ii) 10^<b>6</b> CFU BCG+MSU [200 μg], or iii) PBS alone. (<b>A</b>) Mice were sacrificed after 15 days from immunization and BCG colonies enumerated by the draining axillary lymph nodes. Immunized and control mice were infected 10 weeks post immunization with <i>M</i>. <i>tuberculosis</i> Erdman (≈ 100 CFU/mouse) by the aerogenic route. Twenty-eight days later, mice were sacrificed and bacterial loads were determined by CFU counting in the lungs (<b>B</b>) and spleens (<b>C</b>). * p < 0.05 in comparison with BCG vaccinated mice.</p

MSU crystals reduce intracellular BCG viability in a phagolysosome maturation dependent and ROS mediated manner.

<p>BCG infected dTHP-1 cells were stimulated or not with 5 μg/ml MSU and CFU assays were performed at day 3 post-infection. (<b>A</b>) In order to ascertain whether phagolysosome maturation was responsible for intracellular mycobacterial killing, 10 μM chloroquine (Cq) or 20 mM NH₄Cl was added to BCG-infected cells together with MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells. (<b>B</b>) In order to ascertain the role of ROS in intracellular mycobacterial killing, 100 U/ml PEG-catalase was added to BCG infected cells together with MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells.</p

MSU crystals enhance antimycobacterial activity.

<p>(<b>A</b>) Differentiated THP-1 (dTHP1) cells were infected with BCG at the MOI of 1 and then stimulated or not with 0.5, 5, 50 μg/ml of MSU for 3 and 5 days. The results are expressed as means ± Standard Deviation (SD) of CFU values performed in triplicate and are representative of three independent experiments. * p ≤ 0.001 in comparison with non-stimulated control cells. (<b>B</b>) Stimulation of human macrophages with MSU enhances phagocytosis of BCG. Differentiated THP-1 cells were exposed to BCG at the MOI of 1 for 3 hour in the presence or not of 0.05, 0.5, 5 μg/ml MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.05 in comparison with non-stimulated control cells.</p

MSU crystals induce phagolysosome dependent ROS generation.

<p>dTHP-1 cells were infected or not with BCG at the MOI of 5 and then labelled with 10 μM DCF for 60 min. Thereafter, cells were washed twice, stimulated or not overnight with 5 μg/ml MSU in the presence or absence of 10 μM Chloroquine (Cq). Results are expressed as means ± SD of arbitrary fluorescence units of triplicate values and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells. ° p < 0.001 in comparison with MSU stimulated cells or with MSU-stimulated BCG-infected cells.</p

Seroprotection and seroconversion rates.

<p>(A) Proportion of responders who achieved IgG levels ≥ 0.35μg/mL and ≥1μg/mL or serotype-specific clinically validated thresholds (correlates of protection) at the different follow-up time points (a clinical correlate of protection for serotype 5 is unavailable). (B) Percentage of patients who reached IgG seroconversion defined as a ≥ 4-fold rise from baseline antibody levels at the different follow-up time points. Stars indicate a significant difference. Error bar represents the confidence intervals (CIs 95%).</p

Geometric mean concentration of vaccinated groups, PCV13 and PPSV23, at 48 weeks compared to level of unvaccinated HIV-negative control group.

<p>Stars indicate significant differences between each HIV vaccinated group (PCV13 and PPSV23) and the HIV-negative unvaccinated control group. Error bar represents the confidence intervals (CIs 95%).</p

GMCs of IgGs against <i>S</i>.<i>pneumoniae</i> serotypes at baseline (BL) vs 48 weeks.

<p>(A) comparison within the PCV13 group and (B) within the PPSV23 group. Stars indicate statistically significant differences. Error bar represents the confidence intervals (CIs 95%).</p

MSU crystals promote maturation of phagosomes containing BCG.

<p>(<b>A</b>) dTHP-1 cells were infected with NHS labelled BCG at the MOI of 1 and then stimulated overnight with 5 μg/ml of MSU, in the presence or absence 10 μM Chloroquine. Results are expressed in terms of mean ± SD of arbitrary fluorescence units of triplicate values and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with MSU stimulated cells. (<b>B</b>) dTHP-1 cells were infected with BCG, stimulated overnight with 5 μg/ml of MSU, in the presence or absence of 10 μM Chloroquine (Cq), and then labelled with 1 μM Lysosensor green DND 189. Results are expressed as mean ± SD of pH values from cultures performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with BCG infected MSU stimulated cells (<b>C</b>) Protease activity of BCG infected dTHP-1 cells stimulated overnight with 5 μg/ml of MSU was analysed by loading of 10 μg/ml DQ red BSA for 2h at 37°C. Results are expressed as mean ± SD of triplicate values and are representative of three independent experiments. * p ≤ 0.01 in comparison with non-stimulated control cells. (<b>D</b>) Confocal microscopy representative images out of 10 per condition showing the increase of Auramine-stained BCG (green) residing in LAMP-3 positive vacuoles (red) after stimulation with 5 μg/ml of MSU. One representative experiment out of three is shown. (<b>E</b>) Summary of the mean percentage ± standard deviation (SD) of BCG co-localizing in LAMP-3-positive vacuoles determined by acquiring at least 10 images per condition and by counting ≥ 50 dTHP-1 cells per sample, after stimulation or not with MSU. Three different experiments were assessed. * p < 0.05 in comparison with BCG-infected cells (data were analyzed using the unpaired Student’s <i>t</i>-test).</p