Supplementary Figure. S1 Supplementary Figure S1. Correlation of phagocytic ability measured with YG and YO beads. Fresh human monocytes (2 10 6 /ml) were labelled with APC conjugated anti CD14 mab alone (for YG beads uptake) or in combination with FITC conjugated anti CD16 mab (for YO beads uptake). Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of fluorescent beads in gated monocyte as indicated (n = 29). Linear regression was used to compare YG and YO beads uptake.
Supplementary Figure. S2 Supplementary Figure S2. Correlation between the basal phagocytic ability of monocyte subsets and the Aβ amyloid burden. Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of fluorescent beads in gated monocyte as indicated (n = 87). Linear regression between the basal phagocytic ability and PET Aβ score is shown for intermediate(top) and classic monocytes (bottom). There was no correlation between Aβ amyloid burden and basal phagocytic ability of non classical monocytes (data not shown).
Supplementary Figure. S3 Supplementary Figure S3. Aβ peptides promote phagocytosis of YG beads by human monocytes. Fresh human monocytes (2 10 6 /ml) were labelled with APC conjugated CD14 and the fluorescence intensity of CD14 + monocytes was analysed by real time flow cytometry. Monocytes were incubated with freshly prepared Aβ 40 or Aβ 42 (50 µg/ml in DMSO) for 15 minutes before the addition of beads. (a) A typical YG beads uptake curve shows the promoting effect of Aβ 40 and Aβ 42 on phagocytosis of beads. (b) Quantitative analysis of the promoting effect of Aβ 40 and Aβ 42 on phagocytosis of YG beads by monocytes as compared with untreated cells (n = 4).
Supplementary Figure. S4 Supplementary Figure S4. CPX promotes phagocytosis of YO beads by monocytes. (a) Fresh human monocytes (2 10 6 /ml) were labelled with APC conjugated anti CD14 and FITCconjugated anti CD16 antibodies and the fluorescence intensity of CD14 + CD16 monocytes was analysed by real time flow cytometry. Cells were pretreated with or without 100 µg/ml CPX for 10 min at 37 o C and left on ice or kept at 37 o C for another 5 min. The 6 min YO bead uptake was performed either at 37 o C or 5 o C as indicated. Results are representative data from three individuals. (b) Concentration dependent promoting effect of CPX on phagocytosis of YG beads. Human monocytes were pre treated with different concentrations of CPX for 10 min at 37 o C. The increment of phagocytosis stimulated by CPX is normalized as % of basal phagocytosis level (n = 3).
Supplementary Figure. S5 Phagocytosis ability (area under curve for YO beads uptake in the first 6 min) Supplementary Figure S5. The effect of Copaxone on phagocytosis of YO beads by human monocytes from HC, MCI and AD patitipants. Fresh human PBMCs were collected from participants with MCI or AD, as well as age and ethnicity matched healthy controls. Cells were labelled with APC conjugated CD14 mab and FITCconjugated CD16 mab and incubated with or without Copaxone 100 µg/ml for 10 minute prior to the addition of 1 µm YO beads. Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of YO beads. Monocyte subsets are gated as indicated.
Supplementary Figure. S6 Phagocytosis ability (area under curve for YO beads uptake in the first 6 min) Supplementary Figure S6. The effect of Copaxone on phagocytosis of YO beads by human monocytes from Aβ, Aβ + and Aβ ++ participants. Fresh human PBMCs were collected from participants who had PET Aβ imaging results. Cells were labelled with APC conjugated CD14 mab and FITC conjugated CD16 mab and incubated with or without Copaxone 100 µg/ml for 10 minute prior to the addition of 1.0 µm YO beads. Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of YO beads. Monocyte subsets are gated as indicated.
Supplementary Figure. S7 Phagocytosis ability (area under curve for YO beads uptake in the first 6 min) Supplementary Figure S7. The effect of ATP on phagocytosis of YO beads by human monocytes from HC, MCI and AD participants. Fresh human PBMCs were collected from participants with MCI or AD, as well as age and ethnicity matched healthy controls. Cells were labelled with APC conjugated CD14 mab and FITC conjugated CD16 mab and incubated with or without 1 mm ATP for 10 15 minute prior to the addition of 1.0 µm YO beads. Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of YO beads. Monocyte subsets are gated as indicated.
Supplementary Figure. S8 Phagocytosis ability (area under curve for YO beads uptake in the first 6 min) Supplementary Figure S8. The effect of ATP on phagocytosis of YO beads by human monocytes from Aβ, Aβ + and Aβ ++ participants. Fresh human PBMCs were collected from participants who have had PET Aβ imaging results. Cells were labelled with APC conjugated CD14 mab and FITC conjugated CD16 mab and incubated with or without 1 mm ATP for 10 15 min prior to the addition of 1 µm YO beads. Phagocytosis ability was quantitated as arbitrary units of area under bead uptake curve in the first 6 min after the addition of YO beads. Monocyte subsets are gated as indicated.
Supplementary Figure. S9 Supplementary Figure S9. P2X7 surface expression on monocyte subsets. Whole peripheral blood (100 µl) was incubated with FITC conjugated mouse anti CD16 mab, PE conjugated mouse anti human CD14 mab and Alexa647 conjugated mouse anti human P2X7 mab (clone L4) for 20 min at 4 o C in dark, followed by addition of 2 ml 1 FACS lysing buffer (BD Bioscience). After 10 min, equal volume of PBS was added and leukocytes were centrifuged at 1400rpm for 4 min. Cells were analysed by flow cytometry on gated subsets of monocytes according to surface CD14 and CD16 expression.
Supplementary Figure. S10 a. b. Ethidium uptake c. Supplementary Figure S10. The effect of CPX is independent of the P2X7 receptor. (a) ATP induced ethidium uptake measured by real time flow cytometry. Human PBMCs were pre treated with or without 20 or 100 µg/ml CPX for 10 min prior to the addition of ethidium and ATP. CD14 + monocytes were gated for analysis. (b) Human PBMCs were pre treated with 1.0 mm ATP for 15 min and/or 100 µg/ml CPX for 10 min prior to the addition of YG beads. CD14 + monocytes were gated for analysis. (c) PBMCs from P2X7 knockout mice were pretreated with or without 100 µg/ml CPX for 10 min prior to the addition of YG beads. CD11b + monocytes were gated for analysis.
Supplementary Figure. S11 a. % of Max Autofluorescence Isotype control with secondary Ab W0 2 staining (untreated) W0 2 staining (fresh Aβ 1 42 treated) W0 2 staining (old Aβ 1 42 treated) b. W0 2 binding on monocyte surface Supplementary Figure S11: (a). Aβ 1 42 binds to monocyte surface. Heparin anti coagulated human blood was incubated with 50 μg/ml of Aβ 1 42 for 5 min prior to washing and the addition of anti Aβ mab (clone W0 2, Ida et al, J. Biol. Chem. 271:22908, 1996) for 20 min at 4 o C. After two washes, cells were stained with FITC anti mouse Ig Ab. (b). Carboxylated YO beads were covalently coupled with fresh prepared Aβ 1 42 peptides by the Carbodiimide method according to the manufacturer s instruction, and resuspended to the same density as unopsonized beads. CD14 and CD16 labeled PBMCs were incubated with or without 100 μg/ml CPX for 10 min at 37 o C then cooled down in ice for another 10 min. The uptake assay was performed at either 4 o C or 37 o C with time zero module.
Supplementary Figure. S12 Supplementary Figure S12: Comparison of YO beads uptake and Aβ YO beads uptake by monocyte subset from the same individual. PBMCs were prelabeled with FITC CD16 and APC CD14 mab and incubated with or without 100 μg/ml CPX or 1 mm ATP for 10 15 min, followed by the addition of 5 µl 1 µm carboxylated YO beads or Aβ 1 42 opsonized YO beads. Typical uptake curves by one each of Aβ, Aβ + and Aβ ++ subjects are shown.
Supplementary Figure. S13 Supplementary Figure S11. Copaxone and ATP indices are independent of ApoE isoforms. Participants are grouped according to their ApoE ε4 allele status (ε4 : ε2 or ε3 carriers; ε4 + : ε4 heterozygotes or homocygotes). Basal phagocytic ability as well as the CPX stimulated phagocytosis index (CPX index) and ATP inhibited phagocytosis index (ATP index) are shown in different monocyte subsets.