Fig 2: LDLR binds to both opsonized and unopsonized crystals. (A) MSU crystals (lot1-3) were opsonized in different solutions (unopsonized (HBSS), human serum, murine serum, or HBSS containing 5% BSA, 1 mg/ml fibrinogen, or 1 mg/ml LDL) at 37°C for 30 min. After washing with HBSS, the particles were incubated with 5 µg/ml recombinant His-tagged protein in HBSS + 5% BSA at 4°C for 60 min (hLDLR, mLDLR, hMARCO, hCD32b). Bound proteins were stained with anti-His Tag PE and analyzed using a flow cytometer. An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, *p < 0.05, **p < 0.01 ); MFI and SEM are shown. (B) Calcium carbonate and m-CPPD crystals were pre-incubated in HBSS or HBSS-containing 1 mg/ml hLDL and then incubated with hLDLR and stained as described in (A). An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, **p < 0.01). Mean MFI and SEM of n=3 samples is shown. (C) Indicated crystals were opsonized in human serum at 37°C for 30 min. Incubation with recombinant LDLR was done as in (A). Protein binding was analyzed using mouse anti-His Tag plus goat anti-mouse AlexaFluor488. Fluorescence of the samples was detected by fluorescent microscopy; scale bar = 40 µm. Representative of three independent experiments. (D) Indicated crystals were opsonized with human serum at 37°C for 30 min. After incubation, particles and supernatant were separated by centrifugation: the supernatant was collected, while the particles were extensively washed with HBSS to remove unbound proteins. Bound proteins were eluted. Both supernatants and eluates were subjected to Western blot analysis using ApoB, ApoAI, and ApoE antibodies. (E) Two distinct preparations of MSU crystals (lot1, lot2) were incubated with human serum or LDL-depleted human serum (both containing 10 µg/ml CRP) at 37°C for 30 min. Incubation with recombinant proteins (hLDLR and hCD32b) as well as detection and analysis of the bound proteins was performed as described in (A); MFI and SEM are shown. CRP binding was analyzed using CRP antibody and anti-rabbit-AlexaFluor488. Fluorescence of the crystals was detected by flow cytometry (MFI anti-CRP +SEM). A paired, two-sided t-test was used for statistical evaluation. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: LDLR and MARCO bind to MSU crystals. (A) Normal human serum (serum 1) and serum from an individual with an acute phase reaction (serum 2) were incubated with MSU crystals (lot1) or zymosan at 37°C for 45 min. Bound proteins were eluted, separated by SDS-PAGE, and subjected to LC-MS analysis. The relative intensity (log2-intensity) of transmembrane receptors and their respective ligands (ApoB is a ligand of LDLR and LBP is a ligand of CD14) bound to MSU crystals or zymosan is shown, compared to the intensity in the input serum; n.d., not detected. (B) Five distinct MSU crystal preparations (lot1-5) were incubated with recombinant Fc-proteins at room temperature (RT) for 60 min (vehicle = DMEM, Fc-mDectin-1, Fc-hMARCO, Fc-mMARCO, Fc-mClec12A, Fc-hClec12A). Crystals were washed with HBSS (always containing Ca2+); bound Fc-fusion proteins were stained with anti-human IgG AlexaFluor488 and the fluorescence of the particles was analyzed using a flow cytometer. An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, *p < 0.05); mean fluorescent intensity (MFI) and standard error of the mean (SEM) are shown. (C) MSU crystals (lot1; left) or S. cerevisiae particles (right) were incubated in HBSS (unopsonized) or human serum from three individual healthy donors with or without the addition of 40 µg/ml CRP at 37°C for 30 min. After washing with HBSS the particles were incubated with 5 µg/ml recombinant His-tagged protein in HBSS + 5% BSA at 4°C for 60 min (hLDLR, hMARCO, hCD32b). Bound proteins were stained with anti-His Tag PE and the fluorescence of the particles was analyzed using a flow cytometer. An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, *p < 0.05); MFI and SEM are shown.