Fig 1: 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.
Fig 2: MARCO binds to unopsonized crystals. (A) Confocal microscopy of MSU (lot2, lot3), cholesterol, t-CPPD, silica, and S. cerevisiae. Particles were incubated at RT for 60 min with either Fc-hMARCO or Fc-mDectin-1 recombinant protein. After washing with HBSS, bound proteins were visualized using anti-human IgG AlexaFluor488 (anti-Fc AF488, green). Representative of at least two independent experiments; DIC = digital interference contrast; scale bar = 40 µm. (B) Indicated crystals were incubated in HBSS (unopsonized) or human serum from three individual healthy donors 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. Median fluorescent intensity (FI) and SEM are shown. Negative control = S. cerevisiae.
Fig 3: MARCO enhances proinflammatory cytokine production in VZV-infected microglia. A HEK293T cells were co-transfected with Myc-tagged full-length MARCO and HA-tagged TLR2 plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-Myc antibodies to evaluate the interaction between MARCO and TLR2. WCL denotes whole cell lysates. B HEK293T cells were co-transfected with empty vector (EV) or HA-tagged TLR2 and increasing amounts of Myc-tagged MARCO plasmid. Relative NF-κB luciferase activity was measured. C HEK293T cells were transfected with VZV gE (100 ng) along with either EV, Myc-tagged MARCO, or HA-tagged TLR2 plasmids. NF-κB luciferase activity was measured. D HEK293T cells were transfected with Myc-tagged MARCO and/or HA-tagged TLR2 expression plasmids as indicated and treated with vehicle (Veh) or TNF-α. Cell lysates were harvested 24 h post-transfection and subjected to immunoblot analysis using antibodies against phospho-IκBα (pIκBα), total IκBα (IκBα), and phospho-p65 (pp65) to assess the activation of NF-κB signaling pathway. Numbers below the blot represent quantification by densitometric analysis. β-actin was used as a loading control. E HMC3 cells were transfected with EV, Myc-tagged MARCO, or HA-tagged TLR2 plasmids for 24 h, followed by YC01 infection (MOI 0.005) for 24 h. IL-6 and IL-8 levels in the cell culture supernatants were measured by ELISA. F HMC3 cells were transfected with control-(siCtl) or MARCO-specific siRNA (siMARCO) for 30 h, followed by infection with mock (m) or YC01 for 24 h. IL-6 and IL-8 levels in the supernatant were quantified by ELISA. Statistical analyses were performed using unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the EV-, or siCtl-transfected-m group
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