Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

doi: 10.1002/trc2.12319

Figure Lengend Snippet: The effects of monomeric C‐reactive proteins (mCRP) on the amyloid beta (Aβ)42 secretion in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E ( APOE ) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP or pentameric CRP (pCRP) for 16 hours. A, Primary neurons from different APOE mice were treated with control medium versus different concentrations of mCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by enzyme‐linkedimmunosorbent assay (ELISA). Values were the mean ± standard error (SE) from all experiments by using ANOVA with with Tukey's post hoc testing. Within each genotype, compared to the untreated neurons, differences of Aβ42 level with each concentration of mCRP are shown with statistical significance ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001. Within each concentration of mCRP, the comparisons of different APOE genotypes were conducted by using analysis of variance (ANOVA) with Tukey's post hoc testing. Statistical significance is shown with # P < .05, ## P < .01, ### P < .001, and #### P < .0001. B, Primary neurons from different APOE mice were treated with control medium versus different concentrations of pCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by ELISA. Values were the mean ± SE from all experiments by using ANOVA with with Tukey's post hoc testing. C, Real‐time polymerase chain reaction (PCR) assays were conducted to reveal the expressions of amyloid precursor protein (APP) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01. D, Real‐time PCR assays were conducted to reveal the expressions of β‐secretase (BACE1) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05. E, Western blot assays for the expressions of APP and BACE1 in different primary APOE neurons treated with mCRP were performed and quantified after normalization against β‐actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗∗∗ P < .001

Article Snippet: On day 14 of culture, E. coli recombinant mCRP or human blood purified pCRP (LeeBio, #140‐11A) was added in cell culture medium followed by cell incubation.

Techniques: Expressing, Isolation, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

doi: 10.1002/trc2.12319

Figure Lengend Snippet: The effects of monomeric C‐reactive proteins (mCRP) and pentameric CRP (pCRP) on the phosphorylated tau (p‐tau) expression in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E ( APOE ) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP for different time. A, Neurons from wild‐type (WT) mice or mice expressing different APOE genotypes were treated with control medium versus different concentrations of mCRP. The neurons were fixed, incubated with the p‐tau antibody, PHF1, to detect cellular tauopathy. The representative images were shown (scale bar: 50 μm). B, The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± standard error (SE) from all experiments and compared using analysis of variance (ANOVA) with Tukey's post hoc testing. Within each genotype, differences with different concentrations of mCRP for each genotype were shown with statistical significance ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. Within the same concentration of mCRP treated neurons, the differences among different APOE neurons were compared with ANOVA with Tukey's post hoc testing. Statistical significance is shown # P < .05. C, The neurons were treated with control medium or 5 μg/mL mCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance for each genotype were shown with ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. D, The neurons were treated with control medium versus different concentrations of pCRP. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. E, The neurons were treated with control medium versus 5 μg/mL pCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. F, Real‐time polymerase chain reaction (PCR) assay was conducted to reveal the expressions of CDK5 in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with ∗ P < .05. G, Real‐time PCR assay was conducted to reveal the expressions of GSK3β in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01. H, Western blot assays for the expressions of GSK3β, pGSK3β, and CDK5 in neurons were performed and quantified after normalization against β‐actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01

Article Snippet: On day 14 of culture, E. coli recombinant mCRP or human blood purified pCRP (LeeBio, #140‐11A) was added in cell culture medium followed by cell incubation.

Techniques: Expressing, Isolation, Cell Culture, Control, Incubation, Phospho-proteomics, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures

doi: 10.3389/fimmu.2020.596103

Figure Lengend Snippet: 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 Ca 2+ ); 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.

Article Snippet: Human serum containing 10 μg/ml purified human CRP (Merck KGaA, #AG723) was vigorously mixed with HBSS or LipoSep Immunoprecipitation Reagent (Bio-Connect Diagnostics, #LS-01) at a 1:1 ratio.

Techniques: Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Recombinant, Staining, Fluorescence, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures

doi: 10.3389/fimmu.2020.596103

Figure Lengend Snippet: 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).

Article Snippet: Human serum containing 10 μg/ml purified human CRP (Merck KGaA, #AG723) was vigorously mixed with HBSS or LipoSep Immunoprecipitation Reagent (Bio-Connect Diagnostics, #LS-01) at a 1:1 ratio.

Techniques: Incubation, Recombinant, Staining, Flow Cytometry, Calcium Carbonate, Protein Binding, Fluorescence, Microscopy, Centrifugation, Western Blot, Binding Assay