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Cited by 143Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsPhagocytosis and Immune Regulation

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Microglial PD-1 stimulation by astrocytic PD-L1 suppresses neuroinflammation and Alzheimer’s disease pathology

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Kummer, Markus P, Ising, Christina, Kummer, Christiane, Sarlus, Heela, Griep, Angelika, Vieira‐Saecker, Ana, Schwartz, Stephanie, Halle, Annett, Brückner, Matthias, Händler, Kristian, Schultze, Joachim L, Beyer, Marc, Latz, Eicke, Heneka, Michael T·2021·The EMBO Journal
DOI 10.15252/embj.2021108662PMC PMC8672180
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Abstract

Chronic neuroinflammation is a pathogenic component of Alzheimer’s disease (AD) that may limit the ability of the brain to clear amyloid deposits and cellular debris. Tight control of the immune system is therefore key to sustain the ability of the brain to repair itself during homeostasis and disease. The immune‐cell checkpoint receptor/ligand pair PD‐1/PD‐L1, known for their inhibitory immune function, is expressed also in the brain. Here, we report upregulated expression of PD‐L1 and PD‐1 in astrocytes and microglia, respectively, surrounding amyloid plaques in AD patients and in the APP/PS1 AD mouse model. We observed juxtamembrane shedding of PD‐L1 from astrocytes, which may mediate ectodomain signaling to PD‐1‐expressing microglia. Deletion of microglial PD‐1 evoked an inflammatory response and compromised amyloid‐β peptide (Aβ) uptake. APP/PS1 mice deficient for PD‐1 exhibited increased deposition of Aβ, reduced microglial Aβ uptake, and decreased expression of the Aβ receptor CD36 on microglia. Therefore, ineffective immune regulation by the PD‐1/PD‐L1 axis contributes to Aβ plaque deposition during chronic neuroinflammation in AD. Neuroinflammation, a hallmark of Alzheimer’s disease (AD), is involved in beta‐amyloid peptide (Aβ) plaque deposition and clearance. Here, the PD‐1/PD‐L1 axis is found as an important pathway for regulating the immune system in the brain, sustaining microglial Aβ uptake and reducing chronic neuroinflammation. The immune‐checkpoint receptor/ligand pair PD‐1/PD‐L1 sustains phagocytic function of microglia to prevent Aβ plaque deposition in APP/PS mouse models and patient tissues.

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Figure 1 Expression of PD‐L1 in AD and in APP/PS1 transgenic mice AELISA quantification of PD‐L1, pTau 181, and Aβx‐42 in the CSF of control and AD patients (n = 10 ± SEM, Student's t‐test, *P < 0.05 and ***P < 0.001, PD‐L1: t = 2.47 df = 18, Aβx‐42: t = 4.83 df = 18, pTau 181: t = 5,768 df = 18).B, C(B) Colocalization of PD‐L1 and GFAP in AD and (C) in control patients (MX‐O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar = 30 µm).DImmunohistochemical detection of PD‐L1 and GFAP in 9‐month‐old female APP/PS1 mice (bar = 50 µm).EImmunohistochemical detection of PD‐L1 and GFAP in 4‐month‐old female APP/PS1 mice (bar = 20 µm).FInduction of PD‐L1 on astrocytes using 1 μM aggregated Aβ1‐42 or 10 μg/ml TNF‐α/100 U/μl IFN‐γ analyzed by flow cytometry.
Figure 1 Expression of PD‐L1 in AD and in APP/PS1 transgenic mice AELISA quantification of PD‐L1, pTau 181, and Aβx‐42 in the CSF of control and AD patients (n = 10 ± SEM, Student's t‐test, *P < 0.05 and ***P < 0.001, PD‐L1: t = 2.47 df = 18, Aβx‐42: t = 4.83 df = 18, pTau 181: t = 5,768 df = 18).B, C(B) Colocalization of PD‐L1 and GFAP in AD and (C) in control patients (MX‐O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar = 30 µm).DImmunohistochemical detection of PD‐L1 and GFAP in 9‐month‐old female APP/PS1 mice (bar = 50 µm).EImmunohistochemical detection of PD‐L1 and GFAP in 4‐month‐old female APP/PS1 mice (bar = 20 µm).FInduction of PD‐L1 on astrocytes using 1 μM aggregated Aβ1‐42 or 10 μg/ml TNF‐α/100 U/μl IFN‐γ analyzed by flow cytometry.CC BY-NC-ND · PubMed Central
Figure 2 Secretion of soluble PD‐L1 from astrocytes C6 cells expressing PD‐L1‐myc treated with the γ‐secretase inhibitor DAPT for 18 h. PD‐L1 was detected using a myc antibody.Immunoblot of HEK293 cells expressing PD‐L1‐myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).Evaluation of three independent experiments of B (n = 3, mean + SEM, Student's t‐test, *P = 0.012, t = 3.4803, df = 4).18 h 1 μM PMA treatment of C6 cells expressing PD‐L1‐myc. Secretion of PD‐L1 in the conditioned medium (CM) was detected using antibody AF1019.Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNF‐α/100 U/μl IFN‐γ. Lysates and the CM were immunoblotted using antibody AF1019.Scheme of PD‐L1 secretion: PMA‐induced S2 cleavage results in the formation of sPD‐L1 and a C‐terminal fragment (PD‐L1‐CTF), that is cleaved by γ‐secretase.
Figure 2 Secretion of soluble PD‐L1 from astrocytes C6 cells expressing PD‐L1‐myc treated with the γ‐secretase inhibitor DAPT for 18 h. PD‐L1 was detected using a myc antibody.Immunoblot of HEK293 cells expressing PD‐L1‐myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).Evaluation of three independent experiments of B (n = 3, mean + SEM, Student's t‐test, *P = 0.012, t = 3.4803, df = 4).18 h 1 μM PMA treatment of C6 cells expressing PD‐L1‐myc. Secretion of PD‐L1 in the conditioned medium (CM) was detected using antibody AF1019.Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNF‐α/100 U/μl IFN‐γ. Lysates and the CM were immunoblotted using antibody AF1019.Scheme of PD‐L1 secretion: PMA‐induced S2 cleavage results in the formation of sPD‐L1 and a C‐terminal fragment (PD‐L1‐CTF), that is cleaved by γ‐secretase.CC BY-NC-ND · PubMed Central
Figure 3 PD‐1 expression in APP/PS1 and AD AColocalization of PD‐1 and CD11b (MX‐04 = methoxy‐XO4, stains amyloid deposits) in AD by immunohistochemistry (bar = 50 µm).BColocalization of PD‐1 and Iba1 or PD‐1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar = 20 µm).CPD‐1 mRNA quantification in laser‐dissected microglia from CX3CR1‐EGFP+/− mice (wt) or plaque‐associated (plaque) and plaque‐distant (distant) microglia from APP/PS1 CX3CR1‐EGFP+/− mice (n = 6, Mann–Whitney test, **P = 0.0043, W = 35).DInduction of PD‐1 on CD11b+ microglia in vitro using 1 μM Aβ or 100 ng/ml LPS, analyzed by flow cytometry.EELISA analysis of TNF‐α secretion by wild‐type (wt) and PD‐1−/− microglia induced by 0.5 µM Aβ1‐42 for 6 h in vitro (n = 3 biological replicates, one‐way ANOVA (df = 1, F = 8.29, P = 0.021), Tukey's HSD, *P < 0.05).F–HMicroglia from methoxy‐XO4‐injected, 8‐month‐old female, wild‐type (wt), and APP/PS1 mice were analyzed (F) for PD‐1 (n = 5 biological replicates per group, one‐way ANOVA (df = 2, F = 13.99, P = 0.0013), Tukey's HSD, **P < 0.01), (G) for CD11b (n = 5 biological replicates per group, one‐way ANOVA (df = 2, F = 30.06, P = 5.47 × 10−5), Tukey's HSD, ***P < 0.001), and (H) for CD45 by flow cytometry (n = 5 biological replicates per group, one‐way ANOVA (df = 1, F = 24.96, P = 0.00013), Tukey's HSD, ***P < 0.001) and normalized to the expression of wild‐type mice (represented by dashed lines). Data information: (C, E–H) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.
Figure 3 PD‐1 expression in APP/PS1 and AD AColocalization of PD‐1 and CD11b (MX‐04 = methoxy‐XO4, stains amyloid deposits) in AD by immunohistochemistry (bar = 50 µm).BColocalization of PD‐1 and Iba1 or PD‐1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar = 20 µm).CPD‐1 mRNA quantification in laser‐dissected microglia from CX3CR1‐EGFP+/− mice (wt) or plaque‐associated (plaque) and plaque‐distant (distant) microglia from APP/PS1 CX3CR1‐EGFP+/− mice (n = 6, Mann–Whitney test, **P = 0.0043, W = 35).DInduction of PD‐1 on CD11b+ microglia in vitro using 1 μM Aβ or 100 ng/ml LPS, analyzed by flow cytometry.EELISA analysis of TNF‐α secretion by wild‐type (wt) and PD‐1−/− microglia induced by 0.5 µM Aβ1‐42 for 6 h in vitro (n = 3 biological replicates, one‐way ANOVA (df = 1, F = 8.29, P = 0.021), Tukey's HSD, *P < 0.05).F–HMicroglia from methoxy‐XO4‐injected, 8‐month‐old female, wild‐type (wt), and APP/PS1 mice were analyzed (F) for PD‐1 (n = 5 biological replicates per group, one‐way ANOVA (df = 2, F = 13.99, P = 0.0013), Tukey's HSD, **P < 0.01), (G) for CD11b (n = 5 biological replicates per group, one‐way ANOVA (df = 2, F = 30.06, P = 5.47 × 10−5), Tukey's HSD, ***P < 0.001), and (H) for CD45 by flow cytometry (n = 5 biological replicates per group, one‐way ANOVA (df = 1, F = 24.96, P = 0.00013), Tukey's HSD, ***P < 0.001) and normalized to the expression of wild‐type mice (represented by dashed lines). Data information: (C, E–H) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.CC BY-NC-ND · PubMed Central
Figure 4 Deletion of PD‐1 in the APP/PS1 mouse model aggravates plaque pathology and behavioral deficits A, BELISA of the (A) RIPA‐soluble fraction of female APP/PS1 and APP/PS1 PD‐1−/− mice for Aβx‐40 (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 0.6, P = 0.45), Tukey's HSD, **P < 0.01, ***P < 0.001) and (B) of the SDS‐soluble fraction (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 32.8, P = 2 × 10−5), Tukey's HSD, **P < 0.01, ***P < 0.001).CThioflavin T histochemistry from sagittal brain sections in the hippocampus (Hc), and cortex (Cx) of female APP/PS1 and APP/PS1 PD‐1−/− mice. Nuclei were stained using Hoechst 33342 (bar = 500 μm).D, EMice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 0.905, P = 0.90), Tukey's HSD, ***P < 0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 4.08, P = 0.054), Tukey's HSD, **P < 0.01, ***P < 0.001).FWestern blot analysis of brain lysates from female APP/PS1 and APP/PS1 PD‐1−/− mice for the expression of APP and APP C‐terminal fragments using antibodies 6E10 (transgene expression) and CT15 (transgene and endogenous expression). Tubulin was used as a loading control.GTime needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean ± SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 7 for APP/PS1, and n = 8 for APP/PS1 PD‐1−/−, one‐way ANOVA (df = 3, F = 17.72, P = 1 × 10−9), Tukey's HSD, *P < 0.05, ***P < 0.001).HAnalysis of the area under the curve (mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 12 for APP/PS1, and n = 8 for APP/PS1 PD‐1−/−, ANOVA (df = 3, F = 6.98, P = 0.001), *P < 0.05 and **P < 0.01).
Figure 4 Deletion of PD‐1 in the APP/PS1 mouse model aggravates plaque pathology and behavioral deficits A, BELISA of the (A) RIPA‐soluble fraction of female APP/PS1 and APP/PS1 PD‐1−/− mice for Aβx‐40 (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 0.6, P = 0.45), Tukey's HSD, **P < 0.01, ***P < 0.001) and (B) of the SDS‐soluble fraction (biological replicates with n = 6 for APP/PS1 and n = 5 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 32.8, P = 2 × 10−5), Tukey's HSD, **P < 0.01, ***P < 0.001).CThioflavin T histochemistry from sagittal brain sections in the hippocampus (Hc), and cortex (Cx) of female APP/PS1 and APP/PS1 PD‐1−/− mice. Nuclei were stained using Hoechst 33342 (bar = 500 μm).D, EMice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 0.905, P = 0.90), Tukey's HSD, ***P < 0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n = 6 for APP/PS1 and n = 9 for APP/PS1 PD‐1−/−, mean ± SEM, one‐way ANOVA (df = 1, F = 4.08, P = 0.054), Tukey's HSD, **P < 0.01, ***P < 0.001).FWestern blot analysis of brain lysates from female APP/PS1 and APP/PS1 PD‐1−/− mice for the expression of APP and APP C‐terminal fragments using antibodies 6E10 (transgene expression) and CT15 (transgene and endogenous expression). Tubulin was used as a loading control.GTime needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean ± SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 7 for APP/PS1, and n = 8 for APP/PS1 PD‐1−/−, one‐way ANOVA (df = 3, F = 17.72, P = 1 × 10−9), Tukey's HSD, *P < 0.05, ***P < 0.001).HAnalysis of the area under the curve (mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 12 for APP/PS1, and n = 8 for APP/PS1 PD‐1−/−, ANOVA (df = 3, F = 6.98, P = 0.001), *P < 0.05 and **P < 0.01).CC BY-NC-ND · PubMed Central
Figure EV1 APP expression is unaltered in APP/PS1 PD‐1−/− mice Quantification of the APP expression normalized to tubulin shown in Fig 4F (biological replicates with n = 4 mice per group, one‐way ANOVA (df = 1, F = 2.776, P = 0.122), Tukey’s post hoc test).Quantification of the 6E10 Western blot and the calculation of the APP C‐terminal fragment (CTF)‐to‐APP ratio shown in Fig 4F (biological replicates with n = 4 mice per group, Student's t‐test (t = 0.9435, df = 6), P = 0.3818).Quantification of the CT15 Western blot and the calculation of the APP α‐C‐terminal fragment (α‐CTF)‐to‐APP and the APP β‐C‐terminal fragment (β‐CTF)‐to‐APP ratio shown in Fig 4F (biological replicates with n = 4 mice per group, ANOVA (df = 1, F = 1.184, P = 0.298), Tukey HSD). Data information: (A–C) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.
Figure EV1 APP expression is unaltered in APP/PS1 PD‐1−/− mice Quantification of the APP expression normalized to tubulin shown in Fig 4F (biological replicates with n = 4 mice per group, one‐way ANOVA (df = 1, F = 2.776, P = 0.122), Tukey’s post hoc test).Quantification of the 6E10 Western blot and the calculation of the APP C‐terminal fragment (CTF)‐to‐APP ratio shown in Fig 4F (biological replicates with n = 4 mice per group, Student's t‐test (t = 0.9435, df = 6), P = 0.3818).Quantification of the CT15 Western blot and the calculation of the APP α‐C‐terminal fragment (α‐CTF)‐to‐APP and the APP β‐C‐terminal fragment (β‐CTF)‐to‐APP ratio shown in Fig 4F (biological replicates with n = 4 mice per group, ANOVA (df = 1, F = 1.184, P = 0.298), Tukey HSD). Data information: (A–C) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.CC BY-NC-ND · PubMed Central
Figure EV2 Behavioral analysis of wild‐type, PD‐1−/−, APP/PS1, and APP/PS1 PD‐1−/− mice AMice were tested one day after the last trial day for 30 s in the absence of the platform in quadrant 4. The time in the quadrants was measured and averaged for quadrants 1–3.B–EEvaluation of the open field testing for (B) time spend in the center (center time) (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test), (C) corner time (ANOVA (F = 0.47, df = 6, P = 0.93), Bonferroni test), (D) corridor time (ANOVA (F = 1.487, df = 6, P = 0.193), Bonferroni test, *P < 0.05), and (E) distance (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test, *P < 0.05, **P < 0.01, and ***P < 0.001). Data information: For all panels: mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 7 for APP/PS1, and n = 5 for APP/PS1 PD‐1−/−.
Figure EV2 Behavioral analysis of wild‐type, PD‐1−/−, APP/PS1, and APP/PS1 PD‐1−/− mice AMice were tested one day after the last trial day for 30 s in the absence of the platform in quadrant 4. The time in the quadrants was measured and averaged for quadrants 1–3.B–EEvaluation of the open field testing for (B) time spend in the center (center time) (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test), (C) corner time (ANOVA (F = 0.47, df = 6, P = 0.93), Bonferroni test), (D) corridor time (ANOVA (F = 1.487, df = 6, P = 0.193), Bonferroni test, *P < 0.05), and (E) distance (ANOVA (F = 0.83, df = 6, P = 0.54), Bonferroni test, *P < 0.05, **P < 0.01, and ***P < 0.001). Data information: For all panels: mean + SEM of biological replicates with n = 12 for wt, n = 5 for PD‐1−/−, n = 7 for APP/PS1, and n = 5 for APP/PS1 PD‐1−/−.CC BY-NC-ND · PubMed Central
Figure 5 PD‐1 deletion reduces phagocytosis and CD36 expression in microglia A In vitro phagocytosis of FAM‐Aβ1‐42 by wild‐type or PD‐1−/− microglia for up to 6 h (mean ± SEM of a technical quadruplicate, Student's t‐test, **P < 0.01 and ***P < 0.001, representative result of an n = 2 biological replicate).B In vitro phagocytosis of FAM‐Aβ1‐42 by wild‐type microglia cultured in astrocyte‐conditioned medium (ACM) from wild‐type or PD‐L1−/− astroglial cultures (mean ± SEM of a technical quadruplicate, Student's t‐test, *P < 0.05 and ***P < 0.001, representative result of an n = 2 biological replicate).CFlow cytometry analysis of microglia from wild‐type and PD‐1−/− mice for the expression of CD36 and CD11b.DQuantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD‐1−/− mice at 8.5 months of age after intraperitoneal injection of methoxy‐XO4 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice, Student's t‐test (t = 2.1808, df = 6.9829), *P < 0.05).E–GRelative expression of the cell surface marker (E) CD36 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice, ANOVA (df = 1, F = 0.002, P = 0.94), Tukey's HSD, *P < 0.05, ***P < 0.001), (F) CD11b (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice), and (G) CD45 on methoxy‐XO4+ versus methoxy‐XO4− microglia in APP/PS1 and APP/PS1 PD‐1−/− mice normalized to methoxy‐X04‐negative cells from APP/PS1 mice (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice). Data information: (D–G) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.
Figure 5 PD‐1 deletion reduces phagocytosis and CD36 expression in microglia A In vitro phagocytosis of FAM‐Aβ1‐42 by wild‐type or PD‐1−/− microglia for up to 6 h (mean ± SEM of a technical quadruplicate, Student's t‐test, **P < 0.01 and ***P < 0.001, representative result of an n = 2 biological replicate).B In vitro phagocytosis of FAM‐Aβ1‐42 by wild‐type microglia cultured in astrocyte‐conditioned medium (ACM) from wild‐type or PD‐L1−/− astroglial cultures (mean ± SEM of a technical quadruplicate, Student's t‐test, *P < 0.05 and ***P < 0.001, representative result of an n = 2 biological replicate).CFlow cytometry analysis of microglia from wild‐type and PD‐1−/− mice for the expression of CD36 and CD11b.DQuantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD‐1−/− mice at 8.5 months of age after intraperitoneal injection of methoxy‐XO4 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice, Student's t‐test (t = 2.1808, df = 6.9829), *P < 0.05).E–GRelative expression of the cell surface marker (E) CD36 (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice, ANOVA (df = 1, F = 0.002, P = 0.94), Tukey's HSD, *P < 0.05, ***P < 0.001), (F) CD11b (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice), and (G) CD45 on methoxy‐XO4+ versus methoxy‐XO4− microglia in APP/PS1 and APP/PS1 PD‐1−/− mice normalized to methoxy‐X04‐negative cells from APP/PS1 mice (biological replicates with n = 5 for APP/PS1 and n = 4 for APP/PS1 PD‐1−/− mice). Data information: (D–G) Central bands represent median, boxes show interquartile range (IQR), and whiskers define the ± 1.5xIQR.CC BY-NC-ND · PubMed Central
Figure EV3 Aβ phagocytosis in PD‐1−/− microglial cells by flow cytometry Microglia from wild‐type or PD‐1−/− mice were incubated with FAM‐Aβ1‐42, and the increase in fluorescence was measured in the CD11b+ cells.Microglia were preincubated for 1 h with regular medium (RM), astrocyte‐conditioned medium (ACM), or serum‐free medium (SFM) followed by the addition of 0.5 μM FAM‐Aβ1‐42 for 4 h (median (central band) with interquartile range (IQR; boxes) and ± 1.5xIQR (whiskers) of one experiment in technical quadruplicates, one‐way ANOVA (df = 2, F = 1.493, P = 0.275), Tukey’s HSD).
Figure EV3 Aβ phagocytosis in PD‐1−/− microglial cells by flow cytometry Microglia from wild‐type or PD‐1−/− mice were incubated with FAM‐Aβ1‐42, and the increase in fluorescence was measured in the CD11b+ cells.Microglia were preincubated for 1 h with regular medium (RM), astrocyte‐conditioned medium (ACM), or serum‐free medium (SFM) followed by the addition of 0.5 μM FAM‐Aβ1‐42 for 4 h (median (central band) with interquartile range (IQR; boxes) and ± 1.5xIQR (whiskers) of one experiment in technical quadruplicates, one‐way ANOVA (df = 2, F = 1.493, P = 0.275), Tukey’s HSD).CC BY-NC-ND · PubMed Central
Figure 6 Transcriptome analysis of PD‐1−/− microglia Top 5 enriched KEGG pathways of differentially expressed genes (abs(fold change) > 2, P < 0.01, Benjamini–Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.Different expression of inflammasome‐related genes (abs(fold change) > 1.5, P < 0.05, Benjamini– Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.Different expression of complement system‐related genes (abs(fold change) > 1.5, P < 0.05, Benjamini–Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.
Figure 6 Transcriptome analysis of PD‐1−/− microglia Top 5 enriched KEGG pathways of differentially expressed genes (abs(fold change) > 2, P < 0.01, Benjamini–Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.Different expression of inflammasome‐related genes (abs(fold change) > 1.5, P < 0.05, Benjamini– Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.Different expression of complement system‐related genes (abs(fold change) > 1.5, P < 0.05, Benjamini–Hochberg‐adjusted) in PD‐1−/− vs. wild‐type microglia.CC BY-NC-ND · PubMed Central
Figure EV4 Gene expression analysis of wild type and PD‐1−/− microglia Gene expression in microglia from wild‐type or PD‐1−/− mice presented as a Volcano plot. Dark data points represent more than 1.5 times up‐ or downregulated genes that have a P‐value of 0.05 or less (one‐way ANOVA, dashed lines represent fold change ± 1.5 and P = 0.05).Same as A but only the top 50 regulated genes are depicted (one‐way ANOVA, dashed lines represent fold change ± 1.5 and P = 0.05).
Figure EV4 Gene expression analysis of wild type and PD‐1−/− microglia Gene expression in microglia from wild‐type or PD‐1−/− mice presented as a Volcano plot. Dark data points represent more than 1.5 times up‐ or downregulated genes that have a P‐value of 0.05 or less (one‐way ANOVA, dashed lines represent fold change ± 1.5 and P = 0.05).Same as A but only the top 50 regulated genes are depicted (one‐way ANOVA, dashed lines represent fold change ± 1.5 and P = 0.05).CC BY-NC-ND · PubMed Central
Figure EV5 Gene ontology (GO) enrichment analysis Visualization of gene ontology enrichment analysis (GOEA) of DE genes (PD‐1−/− vs. wild‐type microglia: blue = downregulated genes, red = upregulated genes) using BiNGO and the EnrichmentMap plugin.ClueGO network analysis of “Immune System Process” GO terms of DE genes (PD‐1−/− vs. wild‐type microglia: red = upregulated genes).
Figure EV5 Gene ontology (GO) enrichment analysis Visualization of gene ontology enrichment analysis (GOEA) of DE genes (PD‐1−/− vs. wild‐type microglia: blue = downregulated genes, red = upregulated genes) using BiNGO and the EnrichmentMap plugin.ClueGO network analysis of “Immune System Process” GO terms of DE genes (PD‐1−/− vs. wild‐type microglia: red = upregulated genes).CC BY-NC-ND · PubMed Central

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Authors
Kummer, M. P., Ising, C., Kummer, C., Sarlus, H., Griep, A., Vieira‐Saecker, A., Schwartz, S., Halle, A., Brückner, M., Händler, K., Schultze, J. L., Beyer, M., Latz, E., & Heneka, M. T.
Affiliations
  • University of Bonn (DE)
  • German Center for Neurodegenerative Diseases (DE)
  • Center of Advanced European Studies and Research (DE)
Corresponding author
  • Kummer, Markus P
  • Heneka, Michael T
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2021-11-26
Volume
40
Issue
24
Pages
e108662-e108662
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.15252/embj.2021108662
PMC
PMC8672180
OpenAlex
W3215791278
Cited by
127
References
55
Citations by year
2026: 22 · 2025: 28 · 2024: 42 · 2023: 24 · 2022: 10 · 2021: 1
FWCI
9.38
Type
journal article
Language
en
Open access
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Locations
  • The EMBO Journal (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Open Repository and Bibliography (University of Luxembourg) (submittedVersion, other-oa)
  • DZNE Pub (submittedVersion, other-oa)
Funders
  • European Union Seventh Framework Program (INMIND)
  • Deutsche Forschungsgemeinschaft (KFO177, IS 299/3‐1, EXC2151‐390873048)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
Keywords
Alzheimer's disease, neuroinflammation, PD-1/PD-L1 axis, microglia, astrocytes, amyloid plaques, amyloid-beta uptake, CD36, APP/PS1 mice, immune checkpoint, phagocytosis, amyloid-beta clearance, ectodomain shedding
MeSH terms
Aged, Aged, 80 and over, Alzheimer Disease, Animals, Astrocytes, Disease Models, Animal, Female, HeLa Cells, Humans, Male, Mice, Transgenic, Middle Aged, Up-Regulation, Case-Control Studies, Amyloid beta-Protein Precursor, Gene Deletion, Microglia, CD36 Antigens, Mice, HEK293 Cells, B7-H1 Antigen, Programmed Cell Death 1 Receptor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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