Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: ( A ) 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 ) 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.

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: Infection, Standard Deviation

Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: ( A ) 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 ) 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 2+ 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

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: ( A ) 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 ) 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 ( C ) 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. ( D ) 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. ( E ) 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 t -test).

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: Infection, Fluorescence, Activity Assay, Confocal Microscopy, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: 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.

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: Infection, Fluorescence

Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: BCG infected dTHP-1 cells were stimulated or not with 5 μg/ml MSU and CFU assays were performed at day 3 post-infection. ( A ) In order to ascertain whether phagolysosome maturation was responsible for intracellular mycobacterial killing, 10 μM chloroquine (Cq) or 20 mM NH 4 Cl 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 ) 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.

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: Infection

Journal: PLoS ONE

Article Title: Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis

doi: 10.1371/journal.pone.0127279

Figure Lengend Snippet: ( A ) Diagram showing the course of in vitro 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 ) 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. ( C ) 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. ( D ) 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.

Article Snippet: After removal of extra-cellular bacilli, cells were stimulated or not with MSU (Enzo Life Sciences Inc.) at the concentration of 0.5, 5, 50 μg/ml and colony forming unit (CFU) assays were performed at day 3 and 5 post-infection, as previously described [ ].

Techniques: In Vitro, Cell Culture, Infection

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Natural IgM binds to MSU and CPPD crystals. ( a ) Serum proteins bound to each indicated particle were analyzed by LC–MS. Fraction of each antibody isotype bound to each indicated particle is shown. Data from 2 individual serum samples is depicted. In one serum sample, IgA2 was not detected; therefore, only one data point is shown. ( b ) Fraction of immunoglobulin isotypes bound to MSU or t-CPPD crystals from 14 normal human serum samples quantified from Western blot analysis. ND = not detected. Raw data (blot images) is shown in Supplementary Fig. . ( c ) Individual normal human sera (NHS, including samples from B) or IgM/IgA-deficient serum samples were incubated with the indicated particles, and bound IgM and IgG were detected using anti-IgM PE or anti-IgG PE, respectively. Median fluorescence intensity (MFI) of the particles was normalized by subtracting the MFI of the negative control (FBS). Means of the NHS and IgM/IgA-deficient serum samples were compared by an unpaired t-test. ( d ) Binding of purified polyclonal (poly.) or monoclonal (mono.) IgM to three distinct preparations of MSU crystals in IgM/IgA-def. serum or HBSS + 10% BSA. ( e ) Pooled human serum collected either from healthy adults or from human cord blood was incubated with the indicated particles, and bound IgM was detected as in (c). Values for each particle preparation and the mean are shown. ( f ) MSU (lot 2), silica (SiO 2 ), or calcium carbonate (CaCO 3 ) crystals were incubated in FBS or 4 normal human pool serum samples. Bound human IgM was detected as in (c) and MFI for each serum sample is shown including the negative controls (FBS).

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Incubation, Fluorescence, Negative Control, Binding Assay, Purification, Calcium Carbonate

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Polyclonal IgM activates complement in the presence of MSU crystals. IgM/IgA-deficient serum obtained from three individuals was depleted of CRP and then reconstituted with polyclonal IgM, IgA, or monoclonal IgM at 0.4 mg/ml. Serum samples were incubated for 30 min at 37 °C with vehicle or MSU (lot 1, 20 mg/ml). The concentrations of anaphylatoxins C4a, C3a, and C5a are shown. Lines represent the means. p values for comparisons of the means were calculated using paired t-test. Arrows above the graphs indicate the sequence of production of C4, C3a, and C5a in the three main complement pathways.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Incubation, Sequencing

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: Non-redundant role of CRP in complement activation by MSU crystals. Four pooled serum samples obtained from healthy individuals and 11 IgM/IgA-deficient serum samples obtained from CVID patients were depleted of residual CRP and reconstituted with CRP (30 µg/ml) or vehicle. Sera were incubated with either MSU lot 1 ( a ) or t-CPPD ( b ) for 30 min at 37 °C. Concentrations of soluble complement system activation products C4a, C3a, C5a, and sC5b-9 in the supernatant are shown. Two serum samples from CVID patients that showed early classical pathway activation are displayed separately (IgM/IgA-def.*). Lines represent the means. For comparison of serum samples with or without CRP, p values were calculated by paired t-test; for comparison of IgM/IgA-sufficient and IgM/IgA-deficient serum samples, an unpaired t-test was used.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Activation Assay, Incubation, Comparison

Journal: Scientific Reports

Article Title: Natural antibodies and CRP drive anaphylatoxin production by urate crystals

doi: 10.1038/s41598-022-08311-z

Figure Lengend Snippet: CRP drives anaphylatoxin production in the presence of MSU crystals. IgM/IgA-deficient serum obtained from 3 individuals with CVID was depleted of CRP and then reconstituted with vehicle or CRP at 10 or 30 µg/ml. The serum samples were incubated for 30 min with nothing, MSU lot 2, or cholesterol crystals. The concentrations of anaphylatoxins C4a, C3a, and C5a in the serum after the incubation are shown. p values are shown for the comparison of the means of vehicle, 10 µg/ml CRP, and 30 µg/ml CRP as determined by one-way repeated-measures ANOVA.

Article Snippet: MSU crystals : Lot 1 (small; mean length: 9.2 ± 4.4 μm): 10 mM uric acid (Merck KGaA, #U0881) was dissolved by boiling in ultrapure water containing 10 mM NaOH.

Techniques: Incubation, Comparison