Additional file 1 of TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

Additional file 1 : Figure S1. Validation of R47H genotype. (A) CRISPR single guide RNA used for insertion of R47H mutation by Bioneer. (B) Chromatograms from sequencing of WT line BIONi010-C and R47H TREM2 line BIONi010-C-7. Red asterisk indicates the R47H mutation, black asterisks are silent mutations added by Bioneer to prevent re-cutting. Figure S2. SNP microarray of iPSCs. Chromosome karyograms from Illumina microarray SNP analysis, showing (A) BIONi010-C line, (B) BIONi010-C-7 R47H TREM2 line, (C) BIONi010-C-17 TREM2 KO line. Figure S3. Validation of R47H TREM2 and TREM2 KO pMac. (A) Macrophage surface markers CD11b, CD14, and CD45 measured by flow cytometry. Median fluorescence intensity (MFI) for each sample was normalized to the relevant isotype IgG, and then to the average for the three genotypes. Histogram shows means ± SEM, for n=3-4 harvests. 1-way ANOVA with Dunnett’s post-hoc test, comparisons to WT line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. (B) Total levels of TREM2 protein shown in a representative western blot (WB). (C-D) Surface TREM2 measured by immunofluorescence staining (IF): live pMac were stained with TREM2 antibody, followed by fluorescent secondary antibody, and subsequently fixed. Images are maximum projections from a z-stack of 5 slices, 1-5 μm, taken on an Opera Phenix microscope (Perkin Elmer). Quantified mean fluorescence (per μm2), for triplicate wells, was normalised to the average for the three genotypes, and then expressed as a ratio of whole-cell TREM2 staining from separate permeabilised wells on the same plate (D). Means ± SEM, for N=3 harvests, p = 0.047 in one-tailed paired t-test. (E-F) Kinetics of pMac calcium responses to 0.5 mM ATP (E), and 10 μg/mL TREM2 antibody (F). Means ± SEM, for N=3-5 harvests. Figure S4. Validation of antibodies for TREM2 immunocytochemistry. Fixed and permeabilized WT, R47H, and TREM2 KO pMac were stained for 1 hour at RT with three different TREM2 antibodies at the concentrations indicated, followed by staining with Alexa Fluor 488-conjugated secondary antibody (1:1000, Invitrogen). Cells were counterstained with DAPI nuclear dye and imaged on an EVOS FL Auto automated microscope (Thermo Fisher). Ab209814 showed cytoplasmic staining in all three genotypes, 13,483–1-AP showed nuclear staining in all three genotypes, whereas AF1828 stained cytoplasm and plasma membrane in WT and R47H TREM2 pMac but not TREM2 KO pMac. Scale bar is 100 μm. Figure S5. Validation of dead SH-SY5Y phagocytosis assay. (A) Freshly-fixed SH-SY5Ys stain uniformly for phosphatidylserine exposure (annexin V-FITC), but have limited cell permeability (propidium iodide). Live SH-SY5Ys do not stain for annexin V-FITC or propidium iodide, except for focal staining present on the few dead cells in culture. (B) No TREM2 expression in an SH-SY5Y not undergoing phagocytosis, marked with a white arrow. (C) No RAB9 expression in non-engulfed SH-SY5Ys, marked with a white arrow. (D) Dose-dependent uptake of dead SH-SY5Ys after 5 hours of phagocytosis with WT line BIONi010-C, means quantified from three independent experiments for % of spot positive (phagocytic) cells per well. Means ± SEM, for N=3 harvests. (E) Phagocytosis of 3 hours is inhibited with 10 μM cytochalasin D, 1 μM bafilomycin A1, 1 μM jasplakinolide, all with 1 hour pre-treatment, and 13 μg/mL recombinant annexin V added simultaneously to the dead SH-SY5Ys. Data was normalized to mean for each genotype per experiment. Means ± SEM, for N=3-6 harvests and with two WT cell lines (SFC840-03-03, the characterisation of this line is described in Fernandes et al [32], and BIONi010-C). 1-way ANOVA with Dunnett’s post-hoc test, comparisons to untreated cells. * p < 0.05, *** p < 0.001. Figure S6. Validation of synaptosome phagocytosis assay. (A) Two whole synaptosomes surrounded by cell debris in the cryopreserved prep, visualised by negative staining electron microscopy. White asterisks label the pre-synaptic termini, with many pre-synaptic vesicles, whereas purple asterisks label the post-synaptic termini. A dark post-synaptic density can be seen between connected pre- and post-synaptic termini. (B) Synaptosomes stain uniformly for phosphatidylserine exposure (annexin V-FITC), comparison is with unstained synaptosomes. An area magnified by 5X is shown inset. (C) Dose-dependent uptake of dead SH-SY5Ys after 3 hours of phagocytosis with WT line BIONi010-C, reaching saturation above 30 μg. (D) Phagocytosis in BIONi010-C pMac is inhibited by 10 μM cytochalasin D and 1 μM bafilomycin A1, and increased by prior opsonisation of synaptosomes for 30 minutes with 20% human serum. Data was normalized to mean for each genotype per experiment, and is represented as sum of spot areas (μm2) per cell. Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett’s post-hoc test, comparisons to untreated cells. * p < 0.05, ** p < 0.01. Figure S7. Validation for cytokine ELISAs and transwell chemotaxis assay. Cytokine ELISAs: (A) Secretion of TNF in response to 4 hours of 0.1-1 μg/mL LPS. (B) Secretion of IL-6 in the same supernatants as (A). Means ± SEM, for N=3 harvests. 2-way ANOVA with Dunnett’s post-hoc test. Comparisons with the coloured annotations are stimulations versus untreated cells (None) for each genotype. Comparisons with the black annotations are R47H or KO versus the WT line for each stimulation, all unannotated comparisons are not significant. Transwell chemotaxis assay: (C) Migration of WT pMac in transwell chemotaxis assay in the presence of four concentrations of ADP or C5a, for 6 hours. (D) Migration of WT pMac for 6 hours in the presence of 30 μM ADP is attenuated by 30 minutes pre-treatment with a P2RY12-selective inhibitor (PSB0739), but not a P2RY1 (MRS2179) or P2RY13 (MRS2211) inhibitor. (E) Migration of WT pMac for 6 hours in the presence of 3 nM C5a is attenuated by 30 minutes pre-treatment with a C5aR inhibitor (PMX-53), or a Syk inhibitor (OXSI-2, 3 μM). Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett’s post-hoc test, pairwise comparisons to control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure S8. RNA-seq differentially-expressed genes (DEGs). Volcano plots shown for DEGs relative to WT: (A) TREM2 KO and (B) R47H TREM2 pMac. Dashed lines show cut-offs at log2-fold-change=2 and p=0.001. Enrichment of Gene Ontology (GO) terms in significant (adjusted p value

附加文件 1：图 S1. R47H 基因型的验证。（A）由 Bioneer 插入 R47H 突变的 CRISPR 单向导 RNA。（B）WT 线 BIONi010-C 和 R47H TREM2 线 BIONi010-C-7 的测序色谱图。红色星号表示 R47H 突变，黑色星号是 Bioneer 添加的沉默突变，以防止二次切割。（图 S2. iPSCs 的 SNP 微阵列。来自 Illumina 微阵列 SNP 分析的染色体核型图，显示（A）BIONi010-C 线，（B）BIONi010-C-7 R47H TREM2 线，（C）BIONi010-C-17 TREM2 KO 线。（图 S3. R47H TREM2 和 TREM2 KO pMac 的验证。（A）通过流式细胞术测量的巨噬细胞表面标记 CD11b、CD14 和 CD45。每个样本的中位荧光强度（MFI）均以相应的同种型 IgG 为基准进行了标准化，然后与三种基因型的平均值进行了比较。直方图显示平均值 ± SEM，n=3-4 收获。单因素方差分析（ANOVA）及 Dunnett 的事后检验，与 WT 线比较。** p < 0.01，*** p < 0.001，**** p < 0.0001，所有未标记的比较均不显著。（B）代表性蛋白质印迹（WB）中显示的 TREM2 蛋白总水平。（C-D）通过免疫荧光染色（IF）测量的表面 TREM2：活化的巨噬细胞用 TREM2 抗体染色，随后用荧光二抗染色，并固定。图像是从 5 切片 z 堆叠的最大投影，1-5 μm，在 Opera Phenix 显微镜（Perkin Elmer）上获得。定量平均荧光（每 μm2），对于重复孔，以三种基因型的平均值为基准进行了标准化，并表达为与同一平板上单独透化孔的整细胞 TREM2 染色比率（D）。平均值 ± SEM，N=3 收获，单侧配对 t 检验 p = 0.047。（E-F）pMac 对 0.5 mM ATP（E）和 10 μg/mL TREM2 抗体（F）的钙反应动力学。平均值 ± SEM，N=3-5 收获。（图 S4. TREM2 免疫细胞化学中抗体的验证。固定和透化的 WT、R47H 和 TREM2 KO pMac 用三种不同浓度的三种 TREM2 抗体在室温下染色 1 小时，然后与 Alexa Fluor 488 标记的二抗（1:1000，Invitrogen）染色，细胞用 DAPI 核染料复染，并在 EVOS FL Auto 自动显微镜（Thermo Fisher）上成像。Ab209814 在所有三种基因型中均显示细胞质染色，13,483–1-AP 在所有三种基因型中均显示核染色，而 AF1828 在 WT 和 R47H TREM2 pMac 中染色细胞质和细胞膜，但在 TREM2 KO pMac 中不染色。标尺栏为 100 μm。（图 S5. 死亡 SH-SY5Y 吞噬作用的验证。（A）新鲜固定的 SH-SY5Ys 对磷脂酰丝氨酸暴露（annexin V-FITC）进行均匀染色，但细胞通透性有限（碘化丙啶）。活化的 SH-SY5Ys 对 annexin V-FITC 或碘化丙啶无染色，除了在培养中少数死亡细胞上存在的局灶性染色。（B）未经吞噬作用的 SH-SY5Ys 中无 TREM2 表达，用白色箭头标记。（C）非吞噬作用的 SH-SY5Ys 中无 RAB9 表达，用白色箭头标记。（D）WT 线 BIONi010-C 吞噬作用 5 小时后，死亡 SH-SY5Ys 的剂量依赖性摄取。从三个独立实验中量化得出的平均值，每孔阳性（吞噬作用）细胞的百分比。平均值 ± SEM，N=3 收获。（E）使用 10 μM 胞质素 D、1 μM 脂质体 A1、1 μM 茶树油，所有这些在预处理 1 小时后，同时向死亡 SH-SY5Ys 中添加 13 μg/mL 重组 annexin V，抑制 3 小时吞噬作用。数据按每个基因型的平均值进行了标准化。平均值 ± SEM，N=3-6 收获，与两个 WT 细胞系（SFC840-03-03，此线的特征在 Fernandes 等人 [32] 中描述，和BIONi010-C）进行比较。单因素 ANOVA 及 Dunnett 的事后检验，与未处理细胞比较。* p < 0.05，*** p < 0.001。（图 S6. 神经突触吞噬作用测定的验证。（A）在冷冻保存的制备中，两个完整的神经突触被细胞碎片包围，通过负染色电子显微镜可视化。白色星号标记突触前终末，有许多突触前囊泡，而紫色星号标记突触后终末。在连接的突触前和突触后终末之间可以看到暗色的突触后密度。（B）神经突触对磷脂酰丝氨酸暴露（annexin V-FITC）进行均匀染色，与未染色的神经突触进行比较。显示 5 倍放大的区域见插页。（C）使用 WT 线 BIONi010-C 吞噬作用 3 小时后，死亡 SH-SY5Ys 的剂量依赖性摄取，在 30 μg 以上达到饱和。（D）BIONi010-C pMac 的吞噬作用被 10 μM 胞质素 D 和 1 μM 脂质体 A1 抑制，并被预先用 20% 人血清对神经突触进行调理素化 30 分钟所增强。数据按每个基因型的平均值进行了标准化，并以每个细胞每孔斑点面积（μm2）的总和表示。平均值 ± SEM，N=3-4 收获。单因素 ANOVA 及 Dunnett 的事后检验，与未处理细胞比较。* p < 0.05，** p < 0.01。（图 S7. 细胞因子 ELISA 和 Transwell 趋化性测定的验证。细胞因子 ELISA：（A）对 0.1-1 μg/mL LPS 4 小时反应分泌的 TNF。（B）与（A）相同的上清液中 IL-6 的分泌。平均值 ± SEM，N=3 收获。双因素 ANOVA 及 Dunnett 的事后检验。与彩色注释的比较是针对每个基因型的刺激与未处理细胞（None）的比较。与黑色注释的比较是 R47H 或 KO 与每个刺激的 WT 线的比较，所有未标记的比较均不显著。Transwell 趋化性测定：（C）WT pMac 在 Transwell 趋化性测定中在 ADP 或 C5a 四种浓度存在下迁移 6 小时。（D）在存在 30 μM ADP 的情况下，WT pMac 的迁移在预处理 30 分钟后使用 P2RY12 选择性抑制剂（PSB0739）被减弱，但不是 P2RY1（MRS2179）或 P2RY13（MRS2211）抑制剂。（E）在存在 3 nM C5a 的情况下，WT pMac 的迁移在预处理 30 分钟后使用 C5aR 抑制剂（PMX-53）或 Syk 抑制剂（OXSI-2，3 μM）被减弱。平均值 ± SEM，N=3-4 收获。单因素 ANOVA 及 Dunnett 的事后检验，与对照进行成对比较。* p < 0.05，** p < 0.01，*** p < 0.001，**** p < 0.0001。（图 S8. RNA-seq 差异表达基因（DEG）。相对于 WT 的 DEG 的火山图：A）TREM2 KO 和（B）R47H TREM2 pMac。虚线表示 log2-变化倍数=2 和 p=0.001 的截止值。显著（调整后的 p 值