Use of peripheral blood macrophages in preparation of reagent and / or drug for diagnosis, prognosis and treatment of alzheimer's disease

Abstract

Disclosed in the present invention is the use of peripheral blood macrophages in the preparation of a reagent and / or a drug for the diagnosis, prognosis and treatment of Alzheimer's disease (AD). The present invention will open new doors for a better understanding of the disease mechanisms of Alzheimer's disease and may guide development in new directions for early diagnosis, prognosis and treatment, so as to enable early prevention and treatment of Alzheimer's disease.

Description

Application of peripheral blood macrophages in the preparation of reagents and / or drugs for diagnosis, prognosis and treatment of Alzheimer's disease Technical Field

[0001] The present invention relates to the use of peripheral blood macrophages in preparing reagents and / or medicines for diagnosis, prognosis and treatment of Alzheimer's disease. Background Art

[0002] Late-onset Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) in the brain. Immune system defects are believed to contribute to this accumulation, as human monocytes have been shown to be unable to effectively clear Aβ. Aβ aggregates are highly neurotoxic and are generally considered the primary culprit in Alzheimer's disease. A key factor contributing to Aβ aggregation is the gradual decline in the body's natural phagocytic capacity with aging, which prevents the effective clearance of Aβ and other denatured proteins.

[0003] Monocytes, macrophages and microglia are professional phagocytes in the body, responsible for clearing pathogens, denatured proteins, cell debris and apoptotic cells. They all originate from bone marrow stem cells and belong to the blood cell system. Human monocytes are divided into atypical (CD14 dim CD16 + ), intermediate (CD14 + CD16 + ) and the largest number of typical (CD14 + CD16 - Monocytes generally only exist in peripheral blood for about a month before migrating to tissues and becoming macrophages, with the main phenotype being CD14 + CD68 + CD163 + Microglia are a type of cell in the central nervous system, and their main phenotype is CD14 + CD16 + CD45 - TREM2 + Currently, textbooks generally believe that there are no macrophages in peripheral blood, and the academic community generally believes that CD45 + It is a common marker of peripheral blood leukocytes, and peripheral blood leukocytes of healthy people do not express TREM2. Summary of the Invention

[0004] The present study shows that the monocytes of patients with late-onset Alzheimer's disease may show disorder of basic cells and stimulated innate phagocytosis. In the blood circulation, a part of the intermediate type (CD14 + CD16+ ) monocytes carry large amounts of Aβ and express cell migration receptors (CX3CR1 and CCR2). This monocyte subset has also been found in human cerebrospinal fluid and brain as the main infiltrating cell type. Subsequent studies have shown that this type of cell is likely derived from intermediate monocytes that migrate to the central nervous system and then evolve into macrophages (CD68 + TREM2 + ) and return to the peripheral blood circulation after carrying large amounts of Aβ. Working with 165 volunteers from the Australian Imaging, Biomarkers and Lifestyle Study (AIBL), the applicants, through quantitative surface Aβ measurements, discovered that a specific type of monocyte in Alzheimer's patients carries large amounts of Aβ on their surface. Further research revealed that these cells possess the immune characteristics of macrophages. The number of these peripheral blood macrophages in Alzheimer's patients is approximately 26% lower than in healthy controls, and the magnitude of this decrease correlates with disease progression. Our findings will open new avenues for a better understanding of the mechanisms of Alzheimer's disease and may lead to the development of novel therapeutic approaches to prevent the disease.

[0005] The peripheral blood macrophages described in the present invention are peripheral blood macrophages that carry a large amount of β-amyloid protein on their surface, and are defined as CD14 + CD16 + Aβ ++ The CD14 + CD16 + Aβ ++ The cells also have the immune characteristics of central nervous system macrophages CD68 + , TREM2 and peripheral blood leukocyte characteristics: CD45 + The CD14 + CD16 + Aβ ++ The immune characteristics of cells include: CD45 + / CD14 + / CD16 + / CD68 + / CD163 + / TREM2 + The CD14 + CD16 + Aβ ++ The immune characteristics of cells include: Lin-1 + HLA-DR + , CCR2 ++ , CX3CR1 + , CD85a ++ , CD85d ++ , CD91+ , CR1 + , CD11b + , CD11c + , CD33 + , CD163 + , P2X7 ++ and MerTK dim The peripheral blood macrophages are used as scavengers of beta-amyloid protein in the preparation of reagents and / or drugs for the diagnosis, prognosis and treatment of Alzheimer's disease.

[0006] Therefore, the peripheral blood macrophages described in the present invention can be used to prepare reagents and / or drugs for the diagnosis, prognosis and treatment of Alzheimer's disease, and such peripheral blood macrophages are used as scavengers of β-amyloid protein in the preparation of reagents and / or drugs for the diagnosis, prognosis and treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1: Anti-Aβ monoclonal antibodies (Clone W0-2 and 4G8) recognize cell-surface Aβ: Flow cytometry was used to examine the ability of different antibodies to recognize cell-surface Aβ antigens in blood samples of cognitively normal elderly individuals from the AIBL cohort (n=6). Aβ fluorescence intensities (expressed as a percentage of Max) in monocytes and monocyte subsets were compared between the different antibodies. Representative flow cytometric plots are shown.

[0008] Figure 2: Aβ staining of surface Aβ ++ The vast majority of cells are CD14 + CD16 + Intermediate monocytes: A. Flow cytometric analysis of monocytes after Aβ, CD14, and CD16 staining. B. Z-stack analysis of monocytes after staining using fluorescence confocal microscopy. Aβ staining within the cells is shown.

[0009] Figure 3: Aβ ++ Cells exhibit higher natural phagocytic capacity: Mononuclear cells (PBMCs) were stained for CD14 and Aβ and then added with 1 μm YO fluorescent latex beads. The cells were analyzed in real time on a FACSCalibur flow cytometer for 7 minutes. Different cell populations in the same tube showed different phagocytic capacities during analysis.

[0010] Figure 4: Aβ ++ The cells also have the immune characteristics of central nervous system macrophages: CD68 + , TREM2 and peripheral blood leukocyte characteristics: CD45 + Typical flow cytometer two-dimensional dot plot showing four-color fluorescence analysis of TREM2 in human peripheral blood CD14+ CD16 + Expression levels of monocytes and CD14 in peripheral blood + CD16 + Aβ ++ The cells possess characteristics of central nervous system microglia; human peripheral blood cells were simultaneously stained with Qdot525-anti-human Aβ monoclonal antibody (clone W0-2), R-PE-anti-human CD16 antibody, PerCP-anti-human CD14 antibody, and APC-anti-human TREM2 antibody; flow cytometry experiments were performed using a BD FACSCalibur™ flow cytometer system.

[0011] Figure 5: Aβ ++ Cellular immune signature Lin-1 + , HLA-DR + , CX3CR1 + , CCR2 + , CD85A + , CD85D + , CD91 + , CD11b + , CD11c + , CD68 + ,CD35 + , P2X7 + , CD163 + , CD33 + , ABCA7 + , MerTK + / - ;

[0012] Figure 6: Aβ ++ immune characteristics of cell subsets;

[0013] Figure 7: Aβ ++ Cellular proteomics confirmed the presence of Aβ monomers and dimers: Peripheral blood leukocytes were stained for CCR2 and CD14 and then purified using a flow cytometer to separate different cell populations. a. The sorted cells were lysed and analyzed for Aβ using Western blot. b. SELDI-TOF mass spectrometry was used to analyze Aβ in different cell populations.

[0014] Figure 8: Human CSF contains a large number of peripheral blood immune cells: Leukocytes isolated from 10 mL of mixed CSF were stained with various fluorescently labeled antibodies and analyzed by flow cytometry. ah. Typical flow cytometric profiles; i. Pie chart showing the average proportions of different cell populations (n=11); j. CD45 expression in different populations. +The percentage of monocytes in the Aβ PET scan-negative (n = 6) and positive (n = 5) groups. Two-way ANOVA and Student's t-test were used for comparison between groups.

[0015] Figure 9: Synthetic Aβ peptides are mostly bound to intermediate monocytes: Whole blood was incubated with 10 µM Aβ42 (light color) or control (dark color) at 37 o Incubate at 4°C for 15 minutes. Cells were washed three times and then stained with W0-2 mAb and labeled with CD4+. Cells were grouped by CD14 and CD16 staining and forward and side scatter. One-way ANOVA was used to analyze the statistical significance of Aβ binding in different leukocytes and monocyte subsets. Tukey post hoc was used to compare Aβ binding between control and experimental groups. Representative flow cytometer histograms of lymphocytes (c), neutrophils (d), monocytes (e), and monocyte subsets (fh) show the binding capacity to Aβ peptides, as shown. Data are expressed as mean ± SD (n = 6).

[0016] Figure 10: Aβ ++ Association between monocytes and Aβ PET scan and cognitive function (episodic memory and complex cognition): Monocyte surface Aβ density and Aβ ++ Monocyte percentages are shown in a and b, respectively; the number of participants was 150 (CN = 74, MCI = 39, and AD = 37); bars are the mean ± SD of the mean, and P values ​​were determined by one-way analysis of variance followed by Tukey's HSD for multiple comparisons; r and P values ​​were determined by Pearson product-moment correlation analysis; c. Association between changes in the percentages of lymphocyte and monocyte subsets and the clinical stage of AD; bars are the mean ± SD of the mean, and P values ​​were determined by one-way analysis of variance followed by Tukey's HSD for multiple comparisons; *: P < 0.05; **: P < 0.01;

[0017] Figure 11: Correlation between cell surface Aβ and various cognitive tests;

[0018] Figure 12: Peripheral blood Aβ ++ ROC curve of cells predicting brain Aβ deposition;

[0019] Figure 13: In vitro culture experiments demonstrate that monocytes in peripheral blood can transform into macrophage-like cells: Human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured in vitro for 0 to 8 days. 0.1 µM Aβ protein or solvent was added to the culture medium as a negative control. The cultured cells were stained with APC-anti-human CD163 antibody, or Alexa Fluor 647-anti-human P2X7R antibody, as well as FITC-anti-human CD68 antibody, R-PE-anti-human CD16 antibody, and PerCP-anti-human CD14 antibody. The dark bars in the figure represent CD14 after 2 days of Aβ protein treatment. + CD16 + Expression levels of macrophage markers or central nervous system microglia markers on cells; light-colored histograms represent CD14 cells not treated with Aβ protein + CD16 + The expression levels of corresponding markers on cells; Flow cytometry experiments were performed using a BD FACSCalibur™ flow cytometer system;

[0020] Figure 14: Detection of the distribution of CFSE-labeled mouse peripheral blood mononuclear cells injected directly into the cerebral ventricle: CFSE-stained mouse peripheral blood mononuclear cells were injected into the cerebral ventricle of recipient mice; two days later, the (a) brain, (b) deep cervical lymph nodes, and (c) peripheral blood of the recipient mice were collected and detected for CFSE. + cell. Implementation Method

[0021] Whether monocyte infiltration into the central nervous system is a universal mechanism for Aβ clearance warrants further investigation. Even without a pathway for monocytes to enter the brain from the peripheral blood, altered monocyte phagocytosis of Aβ may provide a mirror image of changes in endogenous brain microglia during the lifespan. In this study, we first used three different anti-Aβ monoclonal antibodies (clones W0-2, 4G8, and 6E10) and an antibody against the N-terminus of amyloid precursor protein (APP) (clone 22C11) to detect the presence of Aβ on the cell surface. We found that W0-2 and 4G8 could recognize Aβ on the surface of a subset of intermediate-sized monocytes, with W0-2 showing the best effect (Figure 1). We subsequently quantified monocyte surface Aβ using the W0-2 monoclonal antibody assay and flow cytometry in 165 volunteers from the Australian Imaging, Biomarker and Lifestyle Study (AIBL) (84 cognitively normal, 41 patients with mild cognitive impairment (MCI), and 40 patients with Alzheimer's disease (AD), all Caucasian individuals. Brain Aβ positron emission tomography data were available for 154 of these individuals (see Table 1). The results showed that Aβ was present on the surface of peripheral blood monocytes (Figures 2A&B), with a small number of intermediate-sized monocytes carrying large amounts of amyloid-β. CD14 and CD16 staining confirmed these intermediate-sized monocytes (Figures 2A&B). Atypical and typical monocytes carried small amounts of Aβ (Figure 2A). Lymphocytes and neutrophils also carried trace amounts of Aβ.

[0022] Table 1: Demographic composition of the clinical diagnosis study cohort

[0023] a.

[0024] Stratification by amyloid-PET (Centiloid); CL: Centiloid.

[0025] 2 Aβ ++ Monocytes exhibit strong phagocytic function

[0026] We first quantitatively tested the ability of monocytes carrying different amounts of Aβ to phagocytose fluorescent YO latex microspheres by real-time multicolor flow cytometry. ++ Monocytes have the strongest phagocytic ability to microbeads, followed by Aβ + Monocytes, and Aβ + / - Monocytes have the weakest phagocytic ability (Figure 3).

[0027] 3 Aβ ++ Cell characteristics

[0028] We then used a variety of monoclonal antibodies to detect Aβ++ We first compared the immune characteristics of CD14 cells in peripheral blood and cerebrospinal fluid (CSF). + CD16 + cells, and found a large number of CD14 + CD16 + cells and most of these cells carried a large amount of Aβ (Figure 4A). + CD16 + Aβ ++ The cells have similarities, both are CD68 + TREM2 + (Figure 4A&B). CD68 is a characteristic marker of macrophages and is negative in normal peripheral blood leukocytes. TREM2 is a characteristic marker of microglia. We found that very low levels of TREM2 were found in MCI / AD patients. + All cells are CD14 + CD16 + Aβ ++ TREM2 is a known AD-related gene. Previously, researchers found that TREM2 is only expressed in microglia and macrophages in the central nervous system, and no TREM2 was found in the peripheral blood of healthy people. + Only a small amount of TREM2 is found in the peripheral blood of patients with AD or other neurodegenerative diseases. + We analyzed more than 20 samples of cerebrospinal fluid from healthy elderly people and MCI / AD patients and found that most of the free cells in cerebrospinal fluid and the CD14 + CD16 + Aβ ++ The cells have an almost identical immunophenotype, showing: CD45 + / CD14 + / CD16 + / CD68 + / CD163 + / TREM2 + This result shows that CD14 + CD16 + Aβ ++ The cells are most likely macrophages from the central nervous system.

[0029] Further analysis of CD14 in blood + CD16 + Aβ ++ Cell studies have found that these cells express Lin-1 + (a pan-leukocyte marker, so not a dendritic cell marker) and HLA-DR+ (MHC-II) (Figure 5a, b). They express chemotactic receptors, CX3CR1 and CCR2 (Figure 5c, d), and a small number express CD68 (Figure 5e). They can express a variety of β receptor proteins, such as CD85A and D receptors (Figure 5f, g) and low-density lipoprotein receptor CD91 (Figure 5h). They can express a variety of complement receptors, such as CD11b (Figure 5i), CD11c (Figure 5j) and CD35 (CR1, Figure 5k). They can also express a variety of scavenger receptors such as tyrosine protein kinase Mer receptor (MerTK Figure 5l), P2X7 (Figure 5m), CD163 (Figure 5n, also one of the macrophage markers) and ABCA7 (Figure 5p), as well as the mucopolysaccharide receptor CD33 (Figure 5o). Therefore, peripheral blood CD14 + CD16 + Aβ ++ Cellular immune signatures include Lin-1 + HLA-DR + , CCR2 ++ , CX3CR1 + , CD85a ++ , CD85d ++ , CD91 + , CR1 + , CD11b + , CD11c + , CD33 + , CD163 + , P2X7 ++ and MerTK dimLin-1 is a pan-leukocyte surface antigen, expressed on the surface of all peripheral blood leukocytes; HLA-DR is a receptor of the major histocompatibility complex on the surface of leukocytes, expressed on the surface of monocytes, macrophages and dendritic cells; CCR2 (or CD192) is a type of cytokine (monocyte chemoattractant protein 1, CCL2 or MCP-1) receptor, expressed on monocytes and macrophages, guiding cell migration; CX3CR1 is another type of cell chemokine CX3CL1 receptor, expressed on blood-derived cells; CD85a and CD85d are expressed on granulocytes, monocytes, macrophages and dendritic cells, controlling the body's inflammatory response; CD91 is an apolipoprotein receptor, expressed on monocytes, macrophages and epithelial cells, involved in the clearance of Aβ; CR1 (CD35) is a complement receptor, expressed on peripheral blood cells, macrophages and dendritic cells, and is one of the known risk factors for AD; CD11b and CD11c are expressed on blood-derived cells and are associated with cell activation; CD33 (Siglec-3) is a type of lectin that can bind to sialic acid and can activate microglia; CD163 is a scavenger receptor expressed on macrophages and is involved in the clearance of Aβ; P2X7 is strongly positive on monocytes, macrophages and microglia, is a receptor that triggers inflammation, and is also a scavenger receptor; MerTK is also a type of phagocytosis-related receptor, mainly expressed on epithelial cells and macrophages. These immune characteristics further confirm that CD14 + CD16 + Aβ ++ The cells are most likely a type of macrophage rather than a monocyte found in normal peripheral blood.

[0030] We further analyzed the expression of Aβ in monocyte subsets. ++ The immune characteristics of cells are + CD16 + ), atypical (CD14 dim CD16 + ) and typical (CD14 + CD16 - ) In monocyte subsets, Aβ ++ The immune characteristics of the cells are basically the same, all of which are CCR2 ++ , CX3CR1 + , CD85a ++ , CD85d ++ , CD91 + , CR1 + , CD11b + , CD11c + , CD163 +, P2X7 ++ and MerTK dim (Figure 6).

[0031] 4 Protein characteristics of Aβ in different cells

[0032] To further understand the characteristics of cell surface Aβ, we used flow cytometry to separate purified CCR2 ++ CD14 + Monocytes, CCR2 + / - CD14 + Monocytes, CCR2 - CD14 - Lymphocytes and mixed leukocytes were collected. After cell membrane lysis and protein electrophoresis, Aβ in the cell lysate was detected by Western blot. Aβ monomers (~4 kD) and dimers (~8 kD) were detected, along with a suspected C-terminal fragment of APP (CTF83, ~11 kD). These results were confirmed by SELDI-TOF mass spectrometry (Figure 7).

[0033] 5 Cellular Characteristics in CSF

[0034] The extent to which peripheral immune cells contribute to the core pathological changes in Alzheimer's disease is not fully understood, but accumulating data suggest that we may have underestimated their importance. In this study, we conducted a pilot study to demonstrate the presence of infiltrating peripheral immune cells in human cerebrospinal fluid (CSF) (n = 8). Ten mL of CSF samples were concentrated and analyzed for cell types and cell surface amyloid-β peptide using monoclonal antibody assays and flow cytometry. A small number of infiltrating peripheral immune cells (CD45 + ), that is, 317±84.3 lymphocytes and 74.3±19.6 monocytes per ml of CSF (Fig. 8a, b). No neutrophils (CD45 + CD15 +The cell count was 1.67±1.53). Monocytes were classified into subsets based on CD14 and CD16. Surprisingly, intermediate monocytes were the main monocyte subset present in the cerebrospinal fluid, accounting for as much as 70.2±11.8%, while atypical and typical monocytes accounted for only 7.78±6.14% and 1.41±0.37%, respectively (Figure 8c). In peripheral venous blood, we found that intermediate monocytes and typical monocytes accounted for 6.30±3.87% and 49.60±15.26% of the total monocytes, respectively (n=165). In other words, the proportions of intermediate monocytes and typical monocytes in peripheral blood and cerebrospinal fluid were opposite. We further studied the expression levels of monocyte chemoattractant receptors CX3CR1 and CCR2 in cerebrospinal fluid. The study found that cerebrospinal fluid monocytes expressed CX3CR1 + CX3CR1 expressed 92.2% to 3.61% of cells, while 54.8% to 5.53% also expressed CCR2 (Figure 8d). This suggests that CX3CR1 may play a role in facilitating the entry of peripheral monocytes into the CSF. Furthermore, Lin-1, HLA-DR (a MHC class II cell surface marker), CD11b, and CD11c are used to characterize monocytes. The study found that 77.20 ± 7.94% of CSF monocytes were double-positive for HLA-DR and Lin-1 (Figure 8e), while 88.60 ± 6.53% were double-positive for CD11b and CD11c (Figure 8f). Finally, we examined endogenous amyloid-β peptide in CSF monocytes. While collecting such a large number of cells is technically difficult, it appears that monocytes carry significant amounts of amyloid-β peptide (Figure 8g), providing ample opportunity for them to differentiate into or become macrophages.

[0035] 6. Amyloid Peptide Cell Surface Binding Test

[0036] We used healthy human blood leukocytes to test the ability of cells to adsorb and phagocytose beta-amyloid peptides in vitro (n=6). First, we compared the adsorption of beta-amyloid peptides by lymphocytes, monocytes, and neutrophils (Figure 9a, c, d, e). It was found that compared with the dimethyl sulfoxide control group, monocytes adsorbed a large amount of exogenous beta-amyloid peptides (Tukey test showed P<0.0001). Compared with lymphocytes and neutrophils, monocytes are the main cells in human leukocytes responsible for the adsorption and phagocytosis of beta-amyloid peptides (analysis of variance P<0.0001). Secondly, we divided monocytes into three subsets, namely typical monocytes (CD14 + CD16 - ), intermediate monocytes (CD14 + CD16 + ), and atypical monocytes (CD14 dimCD16 + ) and compared their adsorption and phagocytosis of amyloid-β peptide (Figure 9b). The results showed that both intermediate and atypical monocytes could absorb exogenous amyloid-β peptide (comparing the sham group with the control group, Tukey test showed P < 0.0001 and P = 0.0033, respectively). Among the three subsets, intermediate monocytes had the strongest adsorption and phagocytosis ability (ANOVA P < 0.0001) (Figure 9b, f, g, h).

[0037] 7 In Alzheimer's patients, Aβ ++ Decreased cell count and associated with cognitive function

[0038] To understand the significance of this phenomenon, we conducted a cross-sectional study to determine whether monocyte surface Aβ is associated with AD. Peripheral blood was collected from age-matched control CN (n=74) with normal cognition, patients with mild cognitive impairment (MCI) (n=39), and participants with AD dementia (n=37) through AIBL and cell surface immunophenotyping was performed. We found that total monocyte surface Aβ was decreased in patients with MCI and AD dementia. We first examined total monocytes. Amyloid β (Aβ) fluorescence intensity assay, Aβ + The percentage of cells (fluorescence intensity at 10 2 to 10 3.6 between ), Aβ ++ The percentage of cells (fluorescence intensity at 10 3.6 to 10 4 The results showed that the Aβ fluorescence intensity of patients with Alzheimer's disease decreased by 26.21% (Figure 10a, average values ​​of normal cognition 294.62±16.96, mild cognitive impairment 224.31±21.73, and Alzheimer's patients 217.41±18.87, variance analysis P=0.0049, Tukey test, Alzheimer's patients vs. normal cognition P=0.0096, mild cognitive impairment vs. normal cognition P=0.0223). At the same time, the Aβ fluorescence intensity of patients with Alzheimer's disease decreased by 26.21% (Figure 10a, average values ​​of normal cognition 294.62±16.96, mild cognitive impairment 224.31±21.73, and Alzheimer's patients 217.41±18.87, variance analysis P=0.0049, Tukey test, Alzheimer's patients vs. normal cognition P=0.0096, mild cognitive impairment vs. normal cognition P=0.0223). ++ The percentage of cells decreased by 43.15% (Fig. 10b, mean values ​​were 1.28±0.12% in subjects with normal cognitive level, 0.83±0.14% in subjects with mild cognitive impairment, and 0.73±0.14% in subjects with Alzheimer's disease; ANOVA P=0.0068, Tukey test, P=0.0089 for subjects with Alzheimer's disease versus subjects with normal cognitive level, and P=0.0425 for subjects with mild cognitive impairment versus subjects with normal cognitive level).

[0039] Compared with CN, the Aβ content on monocytes was reduced by 22% in MCI and 28% in AD dementia (Fig. 10a); ++ Monocyte percentages showed a downward trend compared to CN, with a 36% decrease in MCI patients and a 49% decrease in AD dementia patients (Figure 10b). These significant decreases were also correlated with brain Aβ levels, as measured by quantitative calculation of the Centiloid Score (CL) from brain Aβ positron emission tomography scans, and were closely associated with cognitive scores such as episodic memory and the Preclinical Alzheimer's Disease Cognitive Composite (PACC) score (Figures 10a,b).

[0040] Aβ in AD ++ Consistent with the decrease in the percentage of monocytes, we also found that the percentage of intermediate monocytes in MCI and AD dementia was also reduced by 21% compared with CN (P = 0.005, Figure 10c), which is likely due to the Aβ ++ A possible reason for the decrease in monocytes is the decrease in monocytes. Correspondingly, the percentage of classical monocytes in monocytes increased by 15% in the MCI and AD groups compared with CN (P = 0.003; Figure 10c). In addition to monocytes, we found an 18% decrease in the percentage of natural killer cells within the entire leukocyte population in MCI and AD dementia patients compared with CN (P = 0.008; Figure 10c).

[0041] In summary, we found that the parameters of monocyte subsets were less correlated with disease status than those of total monocytes, which may be due to the deviation of the number of small cells and atypical cells. Here, we selected two parameters of total monocytes, namely Aβ fluorescence intensity and Aβ ++ The percentage of cells was analyzed by using a variety of neuropsychological assessment tests, namely MMSE score, CDR score and Preclinical Alzheimer's Disease Composite Cognitive Test (PACC) (Figure 11). ++ The percentage of cells showed similar results, and was positively correlated with contextual memory, executive function, language function, attention, and PACC results, and negatively correlated with CDR score results (Figure 11). These results indicate that Aβ ++ Cells are closely related to the course of AD and can be used to evaluate and predict the course of AD.

[0042] 8 Amyloid-β peptide on monocytes may have diagnostic value as a biomarker

[0043] By optimizing multiple parameters of Aβ expression on the surface of peripheral blood leukocytes, we found that the fluorescence intensity of Aβ on the surface of monocytes, Aβ ++ Monocyte percentage and Aβ +The percentage of natural killer cells can be combined into a novel diagnostic model to replace brain Aβ PET scans (positron emission tomography scans). The curves showed excellent overall performance, with an area under the curve (AUC) of 87% (95% CI: 0.78 to 0.92), a sensitivity of 83% (95% CI: 0.71 to 0.92), a specificity of 77% (95% CI: 0.61 to 0.88), an accuracy of 81%, a positive predictive value of 83%, and a negative predictive value of 77%. The Youden index cutoff value was ≥ 0.56 (Figure 11 and Table 2).

[0044] Table 2: ROC curve AUC and critical value

[0045] b. The best performing W02-related biomarkers (including: monocyte surface Aβ fluorescence intensity, Aβ ++ Monocyte percentage and Aβ + A synthetic diagnostic marker was constructed by adding the percentage of natural killer cells (NK cells) to a mixed logistic regression model (the base model consisted of demographic information, including patient age, sex, years of education, and APOE genotype). Cutoff value 1: The cutoff value determined by the Youden Index (the maximum value of the combined sensitivity and specificity); cutoff value 2: The cutoff value for 95% sensitivity. PPV and NPV: Positive and negative predictive values.

[0046] 9 Cells cross the blood-brain barrier

[0047] In the case of Alzheimer's disease, whether peripheral leukocytes can infiltrate the brain is controversial. However, the importance of the peripheral innate immune system in the pathogenesis of Alzheimer's disease has been updated based on the latest mouse chimera experiments. ++Monocytes possess the chemotactic receptors CX3CR1 and CCR2, as well as the amyloid receptors CD85A / D and the low-density lipoprotein receptor CD91. These may be important for cross-blood-brain barrier transport and the targeted removal of neurotoxic Aβ deposits in the brains of Alzheimer's patients. Our findings suggest that peripheral monocytes, particularly intermediate monocytes, may infiltrate the central nervous system of Alzheimer's patients, where these peripheral immune cells may act to absorb and phagocytose Aβ. This hypothesis is particularly plausible given that our CSF donors had an average age of 77. Previous studies have shown that monocytes that infiltrate the brain rapidly transform into macrophages, and our in vitro cell conversion experiments confirm this. Our experiments showed that peripheral blood monocytes expressed CD68 after only 2-3 days of in vitro culture, with significantly increased expression of CD163 and P2X7 (Figure 13). P2X7 is a receptor that mediates inflammation and is also one of the scavenger receptors that control innate phagocytosis. During the process of monocyte-macrophage conversion, P2X7 expression is significantly increased, so it can be used to assess the conversion rate of monocytes to macrophages.

[0048] Our previous results showed that CD14 + CD16 + Aβ ++ The cells are likely derived from macrophages in the brain. Evidence from studies in APP / PS1 mice (15-80 weeks old) suggests that a subset of monocytes loaded with amyloid beta (Aβ) return to the peripheral circulation (Table 3, Figure 14). To investigate whether brain leukocytes can migrate to the peripheral circulation, we collected blood and isolated mononuclear cells (lymphocytes and monocytes) by Ficoll density gradient centrifugation. These cells were stained with CFSE fluorescent dye and injected directly into the brains of 13 mice intracerebroventricularly. Two days later, peripheral blood and deep cervical lymph nodes were collected and examined. CFSE-positive mononuclear cells (78%) were detected in the peripheral blood or deep cervical lymph nodes of seven of nine young mice (15-30 weeks old). In contrast, traces of CFSE-positive mononuclear cells were found in both the peripheral blood and deep cervical lymph nodes of four aged mice (68-80 weeks old) (100%). The positive rates for blood and lymph node samples were similar, at 62%. Our results demonstrate that blood-derived cells in the brain can migrate to the peripheral blood and lymph nodes.

[0049] Table 3

[0050] Note: ND: Not Detected.

[0051] The human brain is considered "immune privileged," meaning that peripheral immune cells are prohibited from entering the brain due to the blood-brain barrier, thus shielding them from immune surveillance. This assumption is not entirely correct, as microglia, the primary immune cells in the brain, contribute consistently to central immune defense. However, the extent to which peripheral immune cells contribute to central immune defense in neurodegenerative diseases such as AD remains unknown. This study first investigated the ability of monocytes to absorb and phagocytose Aβ and identified intermediate monocytes as the primary phagocytic cell population. Second, the study revealed that intermediate monocytes / macrophages are the predominant subset present in the cerebrospinal fluid (CSF) that infiltrates the brain parenchyma and carries significant amounts of Aβ. Finally, by examining Aβ levels in peripheral blood mononuclear cells from 165 volunteers, we found that monocytes in AD patients have reduced Aβ absorption and phagocytosis. These findings provide a deeper understanding of the pathogenesis of AD and may lead to novel therapeutic approaches and targets for its prevention.

[0052] Aβ in AD patients ++ The decrease in cells was attributed to Aβ in total monocytes ++ The percentage of cells decreased. ++ Whether this decrease in cell percentage is due to a weakened ability to absorb and phagocytose Aβ, a reduced proportion of intermediate monocytes relative to total monocytes, or both, warrants further investigation. Regardless of which hypothesis is correct, it suggests that AD patients have an impaired ability to clear Aβ, potentially reflecting changes in endogenous microglia and macrophages in the brain during aging. Interestingly, the proportion of intermediate monocytes relative to total monocytes is decreased in AD patients. A plausible explanation for this is that intermediate monocytes are highly mobile and enter the central nervous system in response to chemotactic factors triggered by Alzheimer's pathology.

[0053] Our further experimental data showed that CD14 + CD16 + Aβ ++ "At least some of these cells are likely macrophages that have returned from the central nervous system to the peripheral blood. Our findings will have a significant impact on the clearance mechanism of Aβ and provide new insights into the prediction and diagnosis of AD and its targeted treatment."

[0054] In summary, our further experimental data showed that CD14 + CD16 + Aβ ++ cells and the traditional sense of CD14 + CD16 +Intermediate monocytes are significantly different; they are likely macrophages derived from the brain and involved in clearing Aβ from the brain. Brain macrophages are known to originate from peripheral blood monocytes that migrate to the brain. This novel finding provides strong evidence for the first time that peripheral blood monocytes and macrophages play a crucial role in clearing Aβ from the brain. Our findings have broad implications for better understanding the development and progression of AD, as well as its diagnosis and treatment.

Claims

1. Use of peripheral blood macrophages in the preparation of reagents and / or drugs for diagnosis, prognosis and treatment of Alzheimer's disease.

2. The use according to claim 1, characterized in that The peripheral blood macrophages are peripheral blood macrophages that carry a large amount of β-amyloid protein on their surface and are defined as CD14 + CD16 + Aβ ++ cell.

3. The use according to claim 2, characterized in that CD14 + CD16 + Aβ ++ The cells also have the immune characteristics of central nervous system macrophages CD68 + , TREM2 and peripheral blood leukocyte characteristics: CD45 + .

4. The use according to claim 2, characterized in that CD14 + CD16 + Aβ ++ The immune characteristics of cells include: CD45 + / CD14 + / CD16 + / CD68 + / CD163 + / TREM2 + .

5. The use according to claim 2, characterized in that CD14 + CD16 + Aβ ++ The immune signature of the cells includes: Lin-1 + HLA-DR + , CCR2 ++ , CX3CR1 + , CD85a ++ , CD85d ++ , CD91 + , CR1 + , CD11b + , CD11c + , CD33 + ,CD163 + , P2X7 ++ and MerTK dim .

6. The use according to any one of claims 1 to 5, characterized in that The peripheral blood macrophages are used as scavengers of beta-amyloid protein in the preparation of reagents and / or drugs for diagnosing, prognosing and treating Alzheimer's disease.

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