Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 1 CD33 knockout (CD33−/−) and lentiviral shRNA-mediated PTPN6 knockdown in THP1 macrophages. (a) Exemplary flow cytometry histogram graph showing expression levels of CD33 in wild type (WT) and CD33−/−macrophages. CD33 expression is absent in CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, shRNA, Knockdown, Flow Cytometry, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 2 Transcriptome analysis of CD33 knockout and PTPN6 knockdown in THP1 macrophages. (a) Principal component analysis (PCA) of THP1 macrophage RNA sequencing. PCA resulted in four clusters representing the four individual groups wild type (WT) shCTRL, WT shPTPN6, CD33−/−shCTRL CD33−/−shPTPN6. (b) Heatmap of the top 200 most varying genes with hierarchical clustering for rows and columns stressed that the most extreme differences were between WT and CD33−/−. Minor changes were observed for PTPN6 knockdown. (c) Dotblot of KEGG pathway enrichment analysis revealed an upregulation of the inflammation-related transcription profile in comparisons 1–3 (i.e., 1. WT shCTRL vs. CD33−/−shCTRL, 2. WT shPTPN6 vs. CD33−/−shPTPN6, 3. WT shCTRL vs. WT shPTPN6) but not in comparison 4 (CD33−/−shCTRL vs. CD33−/−shPTPN6)

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Knockdown, RNA Sequencing, Comparison

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 3 Activation of SYK and ERK1/2 signaling pathways as a consequence of CD33 knockout in THP1 macrophages and human-induced pluripotent stem cell-derived microglia (iPSdMiG). (a) Western blot showing phosphorylated ERK1/2 (pERK1/2, top) and total ERK1/2 (tERK1/2, bottom) in wild type (WT) and CD33−/−macrophages. One representative image of five independent experiments is shown. (b) Quantification of pERK1/2 to tERK1/2 ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pERK1/2 in THP1 macrophages. FcγRI antibody treatment tended to increase pERK1/2 levels slightly in WT THP1 macrophages. CD33 antibody treatment following FcγRI antibody treatment tended to decrease the FcγRI antibody-driven increase in pERK1/2 to tERK1/2 levels only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3–5). *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (c) Western blot showing phosphorylated SYK (pSYK, top) and total SYK (tSYK, bottom) in WT and CD33−/−macrophages. One representative image of three independent experiments is shown. (d) Quantification of pSYK to tSYK ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pSYK in THP1 macrophages. FcγRI antibody treatment tended to increase pSYK levels slightly and co-treatment with a CD33 antibody tended to counteract this increase, both only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3). *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test. (e) SYK phosphorylation relative to total SYK assessed by AlphaLISA. Untreated iPSdMiG revealed an increased pSYK/tSYK ratio followed by knockout of CD33 (dark gray) or expression of CD33ΔE2 (light gray) compared to WT iPSdMiG (black). Data are shown as mean + SEM (n = 6). **p ≤.01, *p ≤.05 determined by one-way ANOVA followed by Bonferroni post hoc test. (f) Treatment of iPSdMiG with activating antibodies for TREM2 resulted in an increased pSYK/tSYK ratio in WT (black), CD33−/−(dark gray) and CD33ΔE2 iPSdMiG (light gray) compared to isotype antibody-treated controls. Further, pSYK/tSYK was sharply increased in CD33−/−iPSdMiG following anti-TREM2 treatment. Data are shown as mean + SEM (n = 3). ***p ≤.001, *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Activation Assay, Protein-Protein interactions, Knock-Out, Derivative Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 4 Inflammatory cytokine gene transcription is elevated after CD33 knockout or PTPN6 knockdown. (a) Semiquantitative real-time polymerase chain reaction (PCR) of TNFA gene transcription in THP1 macrophages. Gene transcription levels of TNFA were unchanged in untreated wild type (black) and CD33−/−(dark gray) THP1 macrophages regardless of PTPN6 knockdown (shPTPN6). Following LPS treatment TNFA levels were increased. (b) IL1B mRNA levels in THP1 macrophages determined by semiquantitative real-time PCR. Knockout of CD33 increased gene transcription of IL1B in THP1 macrophages (dark gray), which was further elevated by treatment with LPS. IL1B levels tended to be increased after PTPN6 knockdown (shPTPN6) in wild-type THP1 macrophages (black). (c) IL8 gene transcription in THP1 macrophages analyzed via semiquantitative real-time PCR. IL8 mRNA levels were increased after knockout of CD33 (dark gray) and elevated in all cell lines after LPS treatment. PTPN6 knockdown (shPTPN6) attenuated the increase in IL8 transcription in CD33−/−THP1 macrophages. (d) Semiquantitative real-time PCR of IL10 mRNA levels in THP1 macrophages. Gene transcription levels of IL10 were increased in CD33−/−THP1 macrophages (dark gray). Knockdown of PTPN6 decreased IL10 gene transcription below the control vector levels (shCTRL) in both, untreated and LPS-treated THP1 macrophages. Data are shown as mean + SEM (n = 3–7). ***p ≤.001, **p ≤.01, *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (e–i) Gene transcription levels of the inflammatory cytokines TNFA, IL1B, IL8, IL10, and IL6 analyzed in wild type (WT) (black), CD33−/−(dark gray) and CD33ΔE2 human-induced pluripotent stem cell-derived microglia (iPSdMiG) (light gray) by semiquantitative real-time PCR. Knockout of CD33 as well as expression of CD33ΔE2 in iPSdMiG resulted in constitutively elevated mRNA levels of (e) TNFA, (f) IL1B, (g) IL8, (h) IL10, and (i) IL6. TNFA, IL10 and IL6 mRNA levels were decreased in CD33ΔE2 iPSdMiG compared to CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Knockdown, Real-time Polymerase Chain Reaction, Control, Plasmid Preparation, Derivative Assay, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 5 Increased gene transcription of INPP5D (SHIP1) caused by loss of CD33.(a + b) Gene transcription of SIRPA measured by semiquantitative real-time polymerase chain reaction (qRT-PCR). SIRPA mRNA levels were increased in CD33−/−(dark gray) and PTPN6 knockdown (shPTPN6) wild type (WT) THP1 macrophages (black). PTPN6 knockdown in CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 7 Knockout of CD33 increased radical production in human macrophages and microglia. (a) Production of radicals in THP1 macrophages was assessed by dihydroethidium (DHE) staining and confocal microscopy. Radical production was increased by Aβ1-42 in all four cell lines indicated by DHE staining intensity. Thereby, Aβ1-42-induced radical production in CD33−/−shCTRL macrophages (dark gray) was enhanced compared to the corresponding wild-type (black) shCTRL line. Treatment with scavengers SOD1 and Trolox attenuated the Aβ1-42- driven effect to untreated levels. Data are shown as mean + SEM (n = 4). ***p ≤.001, **p ≤.01, *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (b) Reactive oxygen species (ROS) production in human-induced pluripotent stem cell- derived microglia (iPSdMiG) analyzed by DHE staining. Wild type (WT) (black), CD33−/−(dark gray) and CD33ΔE2 (light gray) iPSdMiG exhibited similar constitutive ROS production. Treatment of the phagocytes with bacterial particles from S. aureus resulted in an elevated production of ROS in all iPSdMiG lines with a higher magnitude in CD33−/−iPSdMiG. Treatment with the scavenger NAC decreased ROS production to untreated levels. Data are shown as mean + SEM (n = 3–6). ***p ≤.001, **p ≤.01 determined by two-way ANOVA followed by Bonferroni post hoc test

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Staining, Confocal Microscopy, Derivative Assay