Microglia and monocyte-derived macrophages (MDM) are key players in dealing with Alzheimer’s disease. In amyloidosis mouse models, activation of microglia was found to be TREM2 dependent. Here, using Trem2−/−5xFAD mice, we assessed whether MDM act via a TREM2-dependent pathway. We adopted a treatment protocol targeting the programmed cell death ligand-1 (PD-L1) immune checkpoint, previously shown to modify Alzheimer’s disease via MDM involvement. Blockade of PD-L1 in Trem2−/−5xFAD mice resulted in cognitive improvement and reduced levels of water-soluble amyloid beta1–42 with no effect on amyloid plaque burden. Single-cell RNA sequencing revealed that MDM, derived from both Trem2−/− and Trem2+/+5xFAD mouse brains, express a unique set of genes encoding scavenger receptors (for example, Mrc1, Msr1). Blockade of monocyte trafficking using anti-CCR2 antibody completely abrogated the cognitive improvement induced by anti-PD-L1 treatment in Trem2−/−5xFAD mice and similarly, but to a lesser extent, in Trem2+/+5xFAD mice. These results highlight a TREM2-independent, disease-modifying activity of MDM in an amyloidosis mouse model.
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The single-cell RNA-seq data were deposited at the National Center for Biotechnology Information’s GEO with accession no. GSE176085. For the differential gene expression analysis presented in Extended Data Fig. 1b, we used the single-cell RNA-seq dataset published by Keren-Shaul et al.20 available at GEO with accession no. GSE176085. All underlying data used for generation of figures are collated in the associated source files. All other data are available from the corresponding authors upon reasonable request.
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Research in the laboratory of M.S. is supported by Advanced European Research Council grants (no. 741744), Israel Science Foundation (ISF)-research grant no. 991/16 and ISF-Legacy Heritage Bio-Medical Science Partnership-research grant no. 1354/15. M.S. thanks the Adelis and Thompson Foundations for their generous support of our AD research. I.A. is an incumbent of the Eden and Steven Romick Professorial Chair, supported by Merck KGaA, the Chan Zuckerberg Initiative, the HHMI International Scholar award, the European Research Council Consolidator Grant (ERC-COG, no. 724471- HemTree2.0), an SCA award of the Wolfson Foundation and Family Charitable Trust, the Thompson Family Foundation, an MRA Established Investigator Award (no. 509044), the ISF (no. 703/15), the Ernest and Bonnie Beutler Research Program for Excellence in Genomic Medicine, the Helen and Martin Kimmel award for innovative investigation, the NeuroMac DFG/Transregional Collaborative Research Center Grant, an International Progressive MS Alliance Grant/NMSS (no. PA-1604 08459) and an Adelis Foundation Grant. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The anti-CCR2 clone (MC21) was created by M. Mack and generously given to M.S.; we thank S. Schwarzbaum for editing the manuscript. Schematic presentations of the experimental design (Figs. 1a,e, 2a, 5a and 6a) were created with BioRender.com.
M.S. is an inventor of the intellectual property that forms the basis for development of PD-L1 immunotherapy for AD. K.B. is coinventor of the intellectual property that forms the basis for development of PD-L1 immunotherapy for AD. The remaining authors declare no competing interests.
Peer review information Nature Aging thanks Markus Kummer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 The transcriptomic signature of microglia in Trem2+/+5xFAD mice is not altered upon anti-PD-L1 treatment.
(a) Cumulative bar graphs showing the distribution of homeostatic microglia, stage-I DAM and DAM cells per animal in anti-PD-L1/IgG2b, mouse number, experiment batch and treatment indicated below each bar. (b) Scatter plot comparing DAM cells gene expression from this study and Keren-Shaul et al., 2017 study20, presented as average log2 UMI counts. (c,d) 2D projection plots as in Fig. 1b; highlighting DAM, colored for treatment (anti-PD-L1/IgG2b) (c), or showing the relative expression level of selected marker genes across all microglia (d), color scale indicate the normalized value of log2 fold change. (e) Scatter plots of log2 gene expression (average UMI counts) in homeostatic microglia (i, 1406 cells), stage-I-DAM (ii, 461 cells), and DAM (iii, 1064 cells) derived from the brains of anti-PD-L1 treated and IgG2b-control treated mice. Differential gene expression analysis was performed by Mann-Whitney U test with false-discovery rate (FDR) correction. Red dots represent values that pass the threshold of FDR corrected p < 0.05.
Extended Data Fig. 2 Monocyte-derived macrophages do not contribute to the pool of activated microglia in Trem2+/+5xFAD mice.
(a) FACS gating strategy for isolating all leukocytes (CD45+) and bone marrow (BM)-derived leukocytes (CD45+GFP+) from whole brains of GFP-BM transplanted Trem2+/+5xFAD mice. (b) Cumulative bar graph showing the percentage of each cell population out of all the cells collected using the CD45+ gate or the CD45+GFP+ gate. (c) Cumulative bar graphs of individual animals showing the percentage of each cell population out of all the cells collected using the CD45 + gate or CD45 + GFP + gate in IgG2b or anti-PD-L1 treated animals, corresponding to Fig. 1f,g; also shown are additional 2 WT samples.
Extended Data Fig. 3 Hippocampal levels of Aβ plaques and Triton X-100 Aβ1-42 are not affected by anti-PD-L1 treatment in Trem2-/-5xFAD mice.
(a) Graphic presentation of our RAWM procedure. (b) Graphic presentation of our NOR procedure. (c) RAWM performance of Trem2+/+WT (n=9) and Trem2−/−WT mice (n=12) presented (Y-axis; mean ± SEM) as the number of errors made by the mice (upper panel) and latency to platform (sec, lower panel), in 3-trial bins (x axis) analyzed by two-way ANOVA with repeated measures yielding only main effect of trial bins (upper panel: F(9,171)=45.03, p < 0.0001, 95% CI -1.031-0.8198; lower panel: F(9,171)=66.04, p < 0.0001, 95% CI -6.329-5.767). (d, upper panel) ELISA assessment of Aβ1-40 TBS-soluble hippocampal fractions from anti-PD-L1 (n=9; 7 females (F), 2 males (M)), and IgG2b (n=9; 6 F, 3 M) treated Trem2−/−5xFAD and untreated Trem2−/−WT (n=7; 5 F, 2 M); data derived from 3 cohorts of mice, pooled together after normalization per cohort. (d, lower panel) ELISA assessment of Aβ1-40 Triton X-100-soluble hippocampal fractions from anti-PD-L1 (n=11; 9 F, 2 M) and IgG2b (n=11, 8 F, 3 M) treated Trem2−/−5xFAD and untreated Trem2−/−WT (n=9; 7 F, 2 M); data derived from 4 cohorts of mice, pooled together after normalization per cohort. (e) ELISA assessment of Aβ1-40 TBS-soluble (upper panel) and Triton X-100-soluble (lower panel) hippocampal fractions from anti-PD-L1 (n=7 F) and IgG2b (n=8 F) treated Trem2+/+5xFAD and untreated Trem2+/+WT (n=5 F); data derived from 3 cohorts of mice, pooled together after normalization per cohort. Data were analyzed using one-way ANOVAs followed by a Fisher’s LSD test: (d) TBS-soluble: F(2,22)=25.0, p < 0.0001, R2=0.6944; Triton X-100-soluble: F(2,28)=9.568, p < 0.0001, R2=0.6862; (e) TBS-soluble: F(2,17)=3.479, p=0.0008, R2=0.5653; Triton X-100-soluble: F(2,17)=3.214, p=0.0003, R2=0.623. No significant difference (n.s.) was found following Fisher’s LSD tests. Box plots represent the minimum and maximum values (whiskers), first and third quartile (box boundaries), median (box internal line), and mean (cross); data in all other graphs are shown as mean ± SEM.
Extended Data Fig. 4 No differences in the transcriptomic profile between MDM-1 identified in Trem2−/−5xFAD and Trem2+/+5xFAD and anti-PD-L1 or IgG2b treated mice.
(a) FACS gating strategy for enriching monocyte-derived macrophages (MDM) from the brains of 7-9-month-old Trem2+/+5xFAD and Trem2−/−5xFAD mice, 14 days following injection of anti-PD-L1 or IgG2b. (b) A cumulative bar graphs of individual samples showing the percentage of each cell subpopulation out of all total non-microglial cells collected. (c) A volcano plot showing differentially expressed genes between MDM-1 and DAM, according to MARS-seq data corresponding to Fig. 4. (d) A scatter plot comparing the gene expression profile (log2) of MDM-1 cells collected from the brains of Trem2+/+5xFAD mice (83 cells) and Trem2−/−5xFAD mice (174 cells). (e) A scatter plot comparing the gene expression profile (log2) of MDM-1 cells collected from the brains of mice treated with anti-PD-L1 (83 cells) or IgG2b (102 cells). In figures (c-e) differential gene expression analysis was performed by Mann-Whitney U test with false-discovery rate (FDR) correction.
(a) Flow cytometry plots demonstrating the gating strategy for T cells (CD3+) from the blood of WT mice. (b) Representative histogram plots of anti-rat IgG2b FITC fluorescent intensity, as a measurement for PD-L1 occupancy, in the saturated tube (‘sat’, orange) and in the tested tube (‘test’, purple) (see ‘Methods’) on CD3+ T cells in blood samples from WT mice, 7 days following treatment with anti-PD-L1 antibody in dose of 0.1, 0.5 or 1.5 mg/mouse, or with 1.5 mg/mouse of IgG2, or untreated (n/group=6; 3 males, 3 females). (c,d) Quantification of the percentage of PD-L1 receptor occupancy (%RO) on T cells (mean ± SEM) from the blood (c) and spleen (d). The %RO is calculated as geometric mean of ‘test’ divided by geometric mean of ‘sat’. (e) Flow cytometry plots demonstrating the gating strategy for PD-1+ effector memory T cells (TEM, CD4+CD44+), measured in the blood of the same mice as detailed above. (f) Percentage of PD-1+ TEM cell out of total TEM (Mean ± SEM). One sample from the group of anti-PD-L1 1.5 mg/mouse was not included for technical reason. (c,d,f) were analyzed using one-way ANOVAs ([c] F(4,25)=7.23, p=0.0005, R2=0.536; [d] F(4,25)=7.16, p=0.0005, R2=0.534; [f] F(4,24)=10.71, p < 0.0001, R2=0.641) followed by Fisher’s LSD tests (n.s. – not significant, ** p < 0.01, *** p < 0.0001). data in all graphs are shown as mean ± SEM.
(a,b) Locomotor activity measured by total distance that each mouse moved (cm; mean ± SEM) (a), and anxiety measured by time (sec; mean ± SEM) spent in the center of the arena (b), both measured on the habituation phase (day 1) of the NOR task (Trem2−/−WT n=5, Trem2−/−5xFAD/IgG2b n=5, Trem2−/−5xFAD/anti-PD-L1 n=7, Trem2−/−5xFAD/anti-PD-L1 + anti-CCR2 n=8), analyzed using one-way ANOVAs ([a] F(3,21)=0.4152, p=0.744; [b] F(3,21)=0.323, p=0.8). (c) ELISA assessment of hippocampal Aβ1-42 Triton X-100-soluble from anti-PD-L1 (n=7; 5 females, 2 males) and anti-PD-L1+ anti-CCR2 (n=9; 6 females, 3 males) treated Trem2−/−5xFAD mice was analyzed using one-tailed Student’s t test: t(14)=0.126, p=0.45); data derived from 3 cohorts of mice, pooled together after normalization. (d) Flow cytometry plots demonstrating the gating strategy for live single MDM cells in Trem2−/−5xFAD brains. (e) Quantification (presented as mean ± SEM) of MDM (CD45+CD11b+CD44+Ly6G-CD38-) in Trem2−/−5xFAD treated with IgG, anti-PD-L1 or anti-PD-L1 together with anti-CCR2 and untreated Trem2−/−WT; n/group=5. One-way ANOVA yielded a significant main effect (F(3,16)=4.629, p=0.016, R2=0.4647) which was followed by Fisher’s LSD tests. # relates to the comparison between anti-PD-L1 and IgG2b; Φ relates to the comparison with WT; n.s - not significant.,#,Φ indicate p < 0.05.
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Dvir-Szternfeld, R., Castellani, G., Arad, M. et al. Alzheimer’s disease modification mediated by bone marrow-derived macrophages via a TREM2-independent pathway in mouse model of amyloidosis. Nat Aging 2, 60–73 (2022). https://doi.org/10.1038/s43587-021-00149-w
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