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Repression of Phagocytosis by Human CD33 Is Not Conserved With Mouse CD33

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Repression of Phagocytosis by Human CD33 Is Not Conserved With Mouse CD33

Abhishek Bhattacherjee et al. Commun Biol.

Abstract

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aβ1-42 - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

Keywords: Microglia; Neuroimmunology.

Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Expression profile of mCD33 on immune cell subsets. ai A monoclonal rat IgG1 anti-mouse antibody was used to probe the expression of mCD33 on splenic leukocytes by flow cytometry. For each cell type, a representative histogram (gray: isotype control; black: αmCD33) is shown on the top and a bar graph of the median fluorescence intensity (MFI) for three individual WT (mCD33+/+) and CD33-deficient (mCD33−/−) mice. j Analysis of mCD33 expression on primary microglia isolated directly from an adult brain via a Percoll gradient from three independent WT and mCD33−/− mice. k Levels of mCD33 expression of microglia derived from the brain of neonatal WT (mCD33+/+; black) and CD33-deficient (mCD33−/−; gray) mice. l Levels of mCD33 expression on microglia expanded from the brain of neonatal WT mice cultured with TGF-β1 M-CSF, GM-CSF/ INFγ/LPS, or M-CSF/IL-4/IL-13. Statistical significance calculated based on an unpaired Student’s T-test.
Fig. 2
Fig. 2
Dap12-dependent cell surface expression of mCD33. a Sequence alignment of the transmembrane segment of three murine Siglecs bearing a transmembrane lysine residue, shown in bold, along with hCD33. b Flow cytometry analysis of mCD33 expression on WT and CRISPR/Cas9-generated Dap12−/− RAW264.7 cells (gray: isotype control; black: αmCD33). c WT and Dap12−/− RAW264.7 cells were transiently transfected with mDap12 (red) or empty vector (black) and stained with αmCD33 (black and red) or isotype (gray). d Expression of mCD33 on WT (black), mCD33−/− (gray), and Dap12−/− (red) peripheral blood neutrophils (CD11b+Ly-6G+Ly-6C). e Expression analysis of Dap12 and hCD33 on WT (black) and Dap12−/− U937 cells (red); isotype is shown in gray and was at the same levels for both cell types.
Fig. 3
Fig. 3
Phagocytosis in mCD33+/+ and mCD33−/− cultured macrophages and microglia. a, b Abrogated expression of mCD33 expression in a RAW264.7, b BV-2 cells by flow cytometry. cf Flow cytometry-based analysis of c dextran particles, d polystyrene beads, e myelin, f, and aggregated Aβ1-42 in mCD33+/+ (black; six independent clones) and mCD33−/− (red; nine independent clones) RAW264.7 cells. For each cargo, a representative flow cytometry data and summary plots for each clone, where each data point represents the average of at least three replicates for each clone. Note that for the polystyrene beads, cells incubated without beads are not shown as they overlay directly under the major peak on the left, which represent cells that have not taken up the beads. Statistical significance calculated based on an unpaired Student’s T-test. gi Microscopy-based analysis of polystyrene bead uptake in mCD33+/+ (n = 4 independent clones) and mCD33−/− (n = 8 independent clones) RAW264.7 clones. g Representative image of cells imaged following uptake (red = Calcein, blue = polystyrene bead). h Results for a single experiment where results for each clone are average for three different wells. i Summary of five independent experiments setting the average levels in the WT clones to 100%; N.S. represents no statistical significance based on a paired Student’s T-test. j, k Phagocytosis of polystyrene beads (j) and aggregated Aβ1-42 (k) in mCD33+/+ (black; six independent clones) and mCD33−/− (red; six independent clones) BV-2 cells in the absence and presence of 10 μM Cytochalasin-D. Statistical significance calculated based on an unpaired Student’s T-test.
Fig. 4
Fig. 4
A competitive phagocytic assay to examine cargo uptake in WT and mCD33−/− primary microglia. a Gating strategy for a flow cytometry-based competitive phagocytosis assay in microglia directly isolated from CD45.2+/+ WT or mCD33−/− mice tested in competition verses microglia from CD45.1+/+ WT mice. be Results of the competitive flow cytometry-based uptake of b dextran particles (11 independent experiments), c polystyrene beads (9 independent experiments), d myelin (9 independent experiments), and e aggregated Aβ1-42 (5 independent experiments). Shown are representative flow cytometry histograms for WT (CD45.1+) versus mCD33−/− (CD45.2+) and summary plots for each clone for each genotype plotted as a percentage compared with WT CD45.1+ cells. f, g Flow cytometry-based phagocytosis of polystyrene beads (f) and aggregated Aβ1-42 (g) from WT and mCD33−/− microglia expanded from the brain of neonatal mice polarized with GM-CSF/INFγ/LPS where each point represents a different mouse (n = 8). N.S. represents no statistical significance based on an unpaired Student’s T-test. All error bars represent +/− the standard deviation.
Fig. 5
Fig. 5
hCD33 negatively regulates cargo uptake in human monocytic cell lines. Targeted deletion of CD33 by CRISPR-Cas9 in a U937 and b THP-1 cells as demonstrated by flow cytometry staining with αhCD33 (clone WM53). Seven different clones of U937 (cf) or three different clones of THP-1 (gj) were tested in flow cytometry-based uptake of c, g dextran particles, d, h polystyrene beads, e, i myelin, and f, j aggregated Aβ1-42. For U937 cells (cf), a representative flow cytometry histogram is shown for each cargo and summary plot of each clone. k Representative images of phagocytosis of polystyrene beads by U937 cells visualized by imaging flow cytometry. l In three independent experiments, hCD33−/− U937 cells have a higher percentage of cells with internalized polystyrene beads. m Lentiviral expression of hCD33M in hCD33−/− cells demonstrating that hCD33 expression levels (red) are higher than those of the parent parental U937 cell line expressing endogenous hCD33 (black). Statistical significance based on an unpaired Student’s T-test. n Three independent stable cell lines transduced to express empty lentivirus (black) or hCD33M (red) were analysis for phagocytosis with polystyrene beads by flow cytometry. Statistical significance based on a paired Student’s T-test.
Fig. 6
Fig. 6
Transgenic expression of hCD33M in mouse microglial dampens phagocytosis. a Flow cytometry-based competitive phagocytosis gating strategy for WT (GFPhCD33) and hCD33M-Tg (GFP+hCD33M+) microglia. The competitive phagocytotic assay was carried out with hCD33M-Tg microglia on a mCD33+/+ background (be) or mCD33−/− background (fi) where the non-transgenic (hCD33M) microglia are WT or mCD33−/−, respectively. Flow cytometry-based uptake of b, f dextran particles (14 independent experiments for mCD33+/+ and 4 independent experiments for mCD33−/−), c, g polystyrene beads (11 independent experiments and 6 independent experiments for mCD33−/−), d, h myelin (5 independent experiments and 5 independent experiments for mCD33−/−), and e, i aggregated Aβ1-42 (5 independent experiments and 5 independent experiments for mCD33−/−). For cells on a mCD33+/+ background (be), a representative flow cytometry histogram is shown for each cargo and summary plots for each assay, where the average value for the hCD33 non-expressing cells was set to 100%. Statistical significance based on a paired Student’s T-test.

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