CN117460956A - Application of peripheral blood macrophages in preparation of reagents and/or medicines for diagnosis, prognosis and treatment of Alzheimer's disease - Google Patents

Abstract

The invention discloses application of peripheral blood macrophages in preparing reagents and/or medicines for diagnosis, prognosis and treatment of Alzheimer's Disease (AD). Will open new gates for better understanding of the mechanism of Alzheimer's disease and possibly lead to the development of new early diagnosis, prognosis and therapeutic directions for early prevention and treatment of Alzheimer's disease.

Description

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

Technical Field

The invention relates to application of peripheral blood macrophages in preparing reagents and/or medicines for diagnosing, prognosing and treating Alzheimer's disease.

Background

Late-onset Alzheimer's Disease (AD) is characterized by the deposition and accumulation of beta amyloid (Abeta) in the brain, while defects in the immune system are thought to be responsible for this accumulation, as human monocytes have been shown to be ineffective in scavenging Abeta. While aβ aggregates have strong neurotoxicity and are generally considered to be the leading cause of senile dementia. One important factor that leads to the aggregation of aβ is that the natural phagocytic capacity of the human body gradually decreases with aging, and aβ and other denatured proteins cannot be effectively removed.

Monocytes, macrophages and glial cells are specialized phagocytes in the body and are responsible for the clearance of pathogenic, denatured proteins, cell debris and apoptotic cells. They are derived from bone marrow stem cells and belong to the blood cell system. Human monocytes are classified as atypical (CD 14) based on their surface CD14 and CD16 expression ^dim CD16 ⁺ ) Intermediate type (CD 14) ⁺ CD16 ⁺ ) The most abundant classical (CD 14 ⁺ CD 16-) subset. Monocytes generally only exist in peripheral blood for about one month and migrate into tissues to become macrophages, the major phenotype of which is CD14 ⁺ CD68 ⁺ CD163 ⁺ . Glial cells are a cell in the central nervous system and have a major phenotype of CD14 ⁺ CD16 ⁺ CD45 ^- TREM2 ⁺ . At present, textbooks consider that peripheral blood is free of macrophages, and the academic world also commonly considers CD45 ⁺ Is a common marker of peripheral blood leukocytes, and peripheral blood leukocytes of healthy people do not express TREM2.

Disclosure of Invention

The research of the invention shows that the disorder of basal cells and stimulated innate phagocytosis can be presented in monocytes of patients suffering from late senile dementia. In the blood circulation, a part of the intermediate form (CD 14 ⁺ CD16 ⁺ ) Monocytes carry a large amount of aβ and express cell migration receptors (CX 3CR1 and CCR 2). This monocyte subpopulation is also found as the predominant infiltrating cell type in human cerebrospinal fluid and brain. Subsequent further studies showed that these cells are most likely macrophages (CD 68) which become after migration from intermediate monocytes to the CNS ⁺ TREM2 ⁺ ) And carry a large amount of AbetaAnd then back to peripheral blood circulation. With the aid of a total of 165 volunteers from the Australian imaging, biomarkers and lifestyle research organization (AIBL), the applicant found that there was a special class of monocytes carrying a large amount of Aβ on their surface in Alzheimer's patients, and further studies found that these cells met the immune characteristics of macrophages. Such peripheral blood macrophages are reduced by about 26% in patients with Alzheimer's disease compared to normal persons, and the magnitude of the decline is related to the course of the disease. Our findings will open new gates for better understanding of the mechanism of alzheimer's disease and possibly lead to the development of new therapeutic directions in order to prevent alzheimer's disease.

The peripheral blood macrophage in the present invention is peripheral blood macrophage carrying large amount of beta-amyloid protein, and is defined as CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ And (3) cells. The CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The cells have the macrophage immune characteristic CD68 of the central nervous system at the same time ⁺ TREM2 and peripheral blood leukocyte characteristics CD45 ⁺ . The CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The immunological features of the cells include: CD45 ⁺ /CD14 ⁺ /CD16 ⁺ /CD68 ⁺ /CD163 ⁺ /TREM2 ⁺ . The CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The immunological features of the cells include: lin-1 ⁺ HLA-DR ⁺ ,CCR2 ⁺⁺ ,CX3CR1 ⁺ ,CD85a ⁺⁺ ,CD85d ⁺⁺ ,CD91 ⁺ ,CR1 ⁺ ,CD11b ⁺ ,CD11c ⁺ ,CD33 ⁺ ，CD163 ⁺ ,P2X7 ⁺⁺ And MerTK ^dim . The peripheral blood macrophage is used as a scavenger of beta amyloid protein for the preparation of reagents and/or medicines for diagnosing, prognosing and treating Alzheimer disease.

Thus, the peripheral blood macrophages of the present invention can be used in the preparation of reagents and/or medicaments for the diagnosis, prognosis and treatment of Alzheimer's disease, and such peripheral blood macrophages are used as scavengers of amyloid beta in the preparation of reagents and/or medicaments for the diagnosis, prognosis and treatment of Alzheimer's disease.

Drawings

Fig. 1: anti-Abeta monoclonal antibodies (Clone W0-2 and 4G 8) recognize Abeta on the cell surface: blood sample cell surface aβ antigens of the aged (n=6) with normal cognition of the AIBL cohort were examined using a flow cytometer and the aβ fluorescence intensities (expressed as a percentage of Max) between the different antibodies in monocytes and monocyte subpopulations were compared, shown as a representative flow chart;

fig. 2: aβ stained surface Aβ ⁺⁺ Is predominantly CD14 ⁺ CD16 ⁺ Intermediate monocytes: A. flow cytometry detects the monocytes stained with aβ, CD14 and CD16, b. after the monocytes are stained, with a fluorescence confocal microscope, Z-Stack shows the staining of aβ inside the cells;

fig. 3: aβ ⁺⁺ Cells exhibit higher native phagocytic capacity: mononuclear Cells (PBMC) were stained with CD14 and aβ, added with 1um YO fluorescent latex beads, and continuously detected in real time on a FACSCalibur flow cytometer for 7 minutes, with different cell populations in the same tube showing different phagocytic capacities when analyzed;

fig. 4: aβ ⁺⁺ The cells have the macrophage immune characteristic of the central nervous system, CD68 ⁺ TREM2 and peripheral blood leukocyte characteristics CD45 ⁺ : typical flow cytometer two-dimensional dot patterns show that four-color fluorescence analysis TREM2 is used for CD14 in human peripheral blood ⁺ CD16 ⁺ Expression level of monocytes and display of CD14 in peripheral blood ⁺ CD16 ⁺ Aβ ⁺⁺ Microglial characteristics of the central nervous system possessed by the cells; human peripheral blood cells were stained simultaneously with Qdot 525-anti-human aβ monoclonal antibody (clone W0-2), R-PE-anti-human CD16 antibody, perCP-anti-human CD14 antibody and APC-anti-human TREM 2; flow cytometry experiments were performed using BD FACSCalibur ^TM Flow cytometer systems are complete;

fig. 5: aβ ⁺⁺ Immune characteristics of cells Lin-1 ⁺ ,HLA-DR ⁺ ,CX3CR1 ⁺ ,CCR2 ⁺ ,CD85A ⁺ ,CD85D ⁺ ,CD91 ⁺ ,CD11b ⁺ ,CD11c ⁺ ,CD68 ⁺ ,CD35 ⁺ ,P2X7 ⁺ ,CD163 ⁺ ,CD33 ⁺ ,ABCA7 ⁺ ,MerTK ^+/- ；

Fig. 6: aβ ⁺⁺ Immune characteristics of the cell subpopulation;

fig. 7: aβ ⁺⁺ Cytoproteomics demonstrated aβ monomers and dimers: purifying different cell groups of peripheral blood white blood cells by a flow cell sorter after the peripheral blood white blood cells are dyed by CCR2 and CD 14; a. after dissolving the separated cells, detecting Abeta by Western blotting, and b.SELDI-TOF mass spectrometry for analyzing Abeta of different cell populations;

fig. 8: there are a large number of peripheral blood immune cells in human CSF: white blood cells centrifuged from 10mL of mixed CSF were stained with different fluorescent-labeled antibodies and detected with a flow cytometer. a-h. typical flow cytometry profile; i. pie charts show the average ratio of different cell populations (n=11); j. CD45 of different clusters ⁺ Percentage of monocytes in aβ PET scan negative (n=6) and positive (n=5) groups. Inter-group comparisons were performed using Two-Way ANOVA and student's t-test;

fig. 9: the synthesized aβ polypeptide binds mostly to intermediate monocytes: whole blood was incubated with 10 μ M A β42 (light) or control (dark) for 15 minutes at 37 ℃. Cells were washed 3 times, followed by W0-2 mAb staining and CD labeling; cells were stained by CD14 and CD16 and clustered by forward and side scatter; a statistically significant analysis of Abeta binding in different white blood cells and different monocyte subpopulations was performed using One-way ANOVA (One-way ANOVA); tukey post-hoc was used to compare Aβ binding between control and experimental groups; typical flow cytometer histograms in lymphocytes (c), neutrophils (d), monocytes (e) and monocyte subpopulations (f-h) show binding capacity to aβ peptide, as shown; data are expressed as mean ± SD (n=6);

fig. 10: aβ ⁺⁺ Association of monocytes with aβ PET Scan and cognitive functions (episodic memory and complex cognition): monocyte surface Aβ Density and Aβ ⁺⁺ The percentages of monocytes are shown in a. And b. Respectively; the number of participants was 150 (cn=74, mci=39, and ad=37); the bar graph is the mean value.+ -. Standard deviation of the mean value, and the P value is determined by one-way analysis of varianceDetermining, and then performing multiple comparisons by using Tukey HSD; the correlation r and P values are determined by Pearson moment correlation analysis; c. correlation between percent change in lymphocyte and monocyte subpopulations and clinical stage of AD; the histogram is the mean ± standard deviation of the mean, P values were determined by one-way anova, and then multiple comparisons were performed using Tukey HSD; * : p (P)<0.05；**：P<0.01；

Fig. 11: correlation of cell surface aβ and various cognitive tests;

fig. 12: peripheral blood Aβ ⁺⁺ The cells predict ROC curves for brain aβ deposition;

fig. 13: in vitro culture experiments prove that monocytes in peripheral blood can be transformed into macrophage-like cells: human peripheral blood mononuclear cells were subjected to density gradient centrifugation and cell culture in vitro for a period of 0 to 8 days, with the addition of 0.1 mu M A beta protein or solvent to the medium as a negative control. Cultured cells were stained simultaneously with either an APC-anti-human CD163 antibody, or an 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 bar graph shows CD14 after 2 days of Abeta protein treatment ⁺ CD16 ⁺ Macrophage marker or central nervous system microglial marker expression levels on cells; the light bar graph shows CD14 without Abeta protein treatment ⁺ CD16 ⁺ The level of expression of the corresponding marker on the cell; flow cytometry experiments were performed using BD FACSCalibur ^TM Flow cytometer systems are complete;

fig. 14: detection of distribution of CFSE-labeled mouse peripheral blood mononuclear cells directly injected into brain chamber: CFSE stained mouse peripheral blood mononuclear cells are injected into the ventricles of the recipient mice; two days later, (a) brain, (b) deep cervical lymph node, and (c) peripheral blood of recipient mice were taken and CFSE was detected ⁺ And (3) cells.

Detailed Description

1 beta amyloid peptide on the surface of peripheral blood mononuclear cells

This warrants further investigation whether monocyte infiltration into the central nervous system is a common mechanism of beta amyloid peptide (aβ) clearance or not. Even without a channel for monocytes to enter the brain from peripheral blood, the alteration of β amyloid peptide (aβ) phagocytosis by monocytes may provide a mirror image to account for changes in brain endogenous microglia during a human life cycle. In this study we first examined the presence or absence of Abeta on the cell surface using three different anti-Abeta monoclonal antibodies (clones W0-2,4G8 and 6E 10) and an anti-Amyloid Precursor Protein (APP) N-terminal monoclonal antibody (clone 22C 11), and found that W0-2 and 4G8 recognized part of Abeta carried on the surface of intermediate monocytes, with W0-2 being the most effective (FIG. 1). We then quantified aβ on the monocyte surface with the aid of australian imaging, biomarkers and lifestyle study organization (AIBL) 165 volunteers (84 normal cognitive levels CN,41 mild cognitive impairment patients MCI and 40 senile dementia patients AD, all caucasians, 154 of which had data from brain aβ positron scans, see table 1) using the W0-2 monoclonal antibody method and a flow cytometer. The results indicate that aβ is present on the surface of peripheral blood mononuclear cells (fig. 2a & b), and that a small fraction of intermediate mononuclear cells carry a large amount of β amyloid peptide (aβ). These were intermediate monocytes as evidenced by CD14 and CD16 staining (fig. 2a & b). Atypical and typical monocytes carry small amounts of aβ (fig. 2A). Lymphocytes and neutrophils can carry trace amounts of aβ.

Table 1: population composition of clinical diagnostic study cohorts

^a. Layering from amyloid-PET (Centiloid); CL: centilid.

2 Aβ ⁺⁺ Monocytes showed very strong phagocytic function

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. Studies have shown that Aβ ⁺⁺ Monocytes have the strongest phagocytic capacity for microbeads, and secondly Abeta ⁺ Monocytes, A beta ^+/- The phagocytic capacity of monocytes was the weakest (fig. 3).

3 Aβ ⁺⁺ Cell characterization

Subsequently we detected aβ using a variety of monoclonal antibodies ⁺⁺ Immune characteristics of the cells. We first compared CD14 in peripheral blood and cerebrospinal fluid (CSF) ⁺ CD16 ⁺ Cells found to have a high amount of CD14 in CSF ⁺ CD16 ⁺ Cells and such cells mostly carry large amounts of aβ (fig. 4A). Notably, CD14 in blood and CSF ⁺ CD16 ⁺ Aβ ⁺⁺ The cells share similarities, all being CD68 ⁺ TREM2 ⁺ (FIG. 4A)&B) A. The invention relates to a method for producing a fibre-reinforced plastic composite While CD68 is a characteristic marker for macrophages, negative in normal peripheral blood leukocytes. TREM2 is a characteristic marker of neuromicroglia. We found that very small amounts of TREM2 in MCI/AD patients ⁺ The cells were all CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ And (3) cells. TREM2 is a known AD-related gene, and researchers have found that TREM2 is expressed only in microglia and macrophages in the central nerve, and that TREM2 is not found in the peripheral blood of healthy people ⁺ Cells, only in the peripheral blood of AD patients or other neurodegenerative diseases, will find a small amount of TREM2 ⁺ And (3) cells. We examined over 20 parts of cerebrospinal fluid from healthy elderly and MCI/AD patients and found a large proportion of free cells in the cerebrospinal fluid and found CD14 in peripheral blood ⁺ CD16 ⁺ Aβ ⁺⁺ Cells have nearly identical immunophenotypes and exhibit: CD45 ⁺ /CD14 ⁺ /CD16 ⁺ /CD68 ⁺ /CD163 ⁺ /TREM2 ⁺ . This result indicates that CD14 in blood ⁺ CD16 ⁺ Aβ ⁺⁺ The cells are most likely macrophages from the central nervous system.

Further to CD14 in blood ⁺ CD16 ⁺ Aβ ⁺⁺ Cell studies have found that such cells are indicative of Lin-1 expression ⁺ (ubiquitin markers and thus not dendritic cells) and HLA-DR ⁺ (MHC-II) (FIGS. 5a, b). They express chemotactic receptorsCX3CR1 and CCR2 (FIGS. 5c, d) express CD68 in a small proportion (FIG. 5 e). They can express a variety of beta receptor proteins, such as CD85A and D receptors (fig. 5f, g) and the low density lipoprotein receptor CD91 (fig. 5 h). Multiple complement receptors can be expressed, such as CD11b (fig. 5 i), CD11c (fig. 5 j) and CD35 (CR 1, fig. 5 k). While also expressing a variety of scavenger receptors such as the tyrosine protein kinase Mer receptor (MerTK figure 5 l), P2X7 (figure 5 m), CD163 (figure 5n, also one of the markers for macrophages) and ABCA7 (figure 5P), and the mucopolysaccharide receptor CD33 (figure 5 o). Thus peripheral blood CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ Cellular immune features include Lin-1 ⁺ HLA-DR ⁺ ,CCR2 ⁺⁺ ,CX3CR1 ⁺ ,CD85a ⁺⁺ ,CD85d ⁺⁺ ,CD91 ⁺ ,CR1 ⁺ ,CD11b ⁺ ,CD11c ⁺ ,CD33 ⁺ ，CD163 ⁺ ,P2X7 ⁺⁺ And MerTK ^dim . Lin-1 is a leukocyte surface antigen expressed on the surface of all peripheral blood leukocytes; HLA-DR is a receptor for the major histocompatibility complex on the surface of leukocytes, expressed on the surface of monocytes, macrophages and dendritic cells; CCR2 (or CD 192) is a class of cytokine (monocyte chemotactic protein 1, ccl2 or MCP-1) receptors expressed on monocytes and macrophages that directs cell migration; CX3CR1 is a receptor for another class of cell chemokines CX3CL1 expressed in blood-borne cells; CD85a and CD85d are expressed in granulocytes, monocytes, macrophages and dendritic cells, controlling inflammatory responses in vivo; CD91 is an apolipoprotein receptor expressed on monocytes, macrophages and epithelial cells, involved in the clearance of aβ; CR1 (CD 35), a complement receptor, is expressed in peripheral blood cells, macrophages and dendritic cells, is one of the known risk factors for AD; CD11b and CD11c are expressed in blood-borne cells and are associated with cell activation; CD33 (Siglec-3) is a lectin that binds sialic acid and activates glial cells; CD163 is a scavenger receptor expressed on macrophages and involved in the clearance of aβ; P2X7 is strongly positive on monocytes, macrophages and glial cells, and is a receptor for triggering inflammation, and is also a scavenger receptor; merTK is also a class of phagocytosis-related receptors, mainly inEpithelial cells and macrophages. These immune features further confirm CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The cells are likely to be a class of macrophages, rather than monocytes in normal peripheral blood.

We further analyzed aβ in monocyte subpopulations ⁺⁺ Immune characteristics of cells, in the intermediate form (CD 14 ⁺ CD16 ⁺ ) Atypical (CD 14) ^dim CD16 ⁺ ) And typical (CD 14) ⁺ CD16 ^- ) Of monocyte subpopulations, abeta ⁺⁺ The immune characteristics of the cells are basically consistent and are CCR2 ⁺⁺ ,CX3CR1 ⁺ ,CD85a ⁺⁺ ,CD85d ⁺⁺ ,CD91 ⁺ ,CR1 ⁺ ,CD11b ⁺ ,CD11c ⁺ ,CD163 ⁺ ,P2X7 ⁺⁺ And MerTK ^dim (FIG. 6).

4 protein characterization of Abeta in different cells

To further understand the characteristics of cell surface aβ, we collected purified CCR2 with a flow cytometer ⁺⁺ CD14 ⁺ Monocytes, CCR2 ^+/- CD14 ⁺ Monocytes, CCR2 ^- CD 14-lymphocytes and mixed leukocytes. After cell membrane lysis and protein electrophoresis, the A.beta.in the cell lysate was detected by Western blotting, and monomers (. About.4 KD) and dimers (. About.8 KD) of A.beta.and C-terminal fragments (CTF 83,. About.11 KD) of suspected APP were found. These results were confirmed by SELDI-TOF mass spectrometry (FIG. 7).

5 cell Properties in cerebrospinal fluid

The extent to which peripheral immune cells are involved in the core pathological changes of Alzheimer's disease is not fully understood, but the accumulated data suggests that we may underestimate their importance. In this study, we performed a pilot study to show peripheral immune cells that penetrated in human cerebrospinal fluid (n=8). 10mL cerebrospinal fluid samples were concentrated and cell types and cell surface amyloid beta were detected by monoclonal antibody methods and flow cytometry. We found small amounts of infiltrated peripheral immune cells in all three human cerebrospinal fluid samples (CD 45 ⁺ ) I.e. 317+ -84.3 lymphocytes and 74.3+ -19.6 lymphocytes per ml cerebrospinal fluidIs shown (FIGS. 8a, b). Neutrophils were not found in cerebrospinal fluid (CD 45 ⁺ CD15 ⁺ Cell count was 1.67±1.53). Monocytes were classified into subsets by CD14 and CD16, surprisingly intermediate monocytes were the major subset of monocytes present in cerebrospinal fluid up to 70.2±11.8%, whereas atypical and typical monocytes were only 7.78±6.14% and 1.41±0.37%, respectively (fig. 8 c). In peripheral venous blood we found that the proportion of intermediate monocytes and typical monocytes to total monocytes was 6.30±3.87% and 49.60 ±15.26% (n=165), respectively. In other words, the proportion of intermediate monocytes and typical monocytes in peripheral blood and cerebrospinal fluid is reversed. We further studied the expression levels of monocyte chemotactic receptors CX3CR1 and CCR2 in cerebrospinal fluid. Research shows that the mononuclear cell of cerebrospinal fluid expresses CX3CR1 ⁺ (92.23.61%) whereas 54.85.53% of the cells also expressed CCR2 (fig. 8 d). This suggests that CX3CR1 may play a role in helping peripheral monocytes enter cerebrospinal fluid. In addition, lin-1, HLA-DR (MHC-II cell surface marker), CD11b and CD11c were used to characterize monocytes, and it was found that 77.20.94% of cerebrospinal fluid monocytes exhibited HLA-DR and Lin-1 double positivity (FIG. 8 e) and 88.60.+ -. 6.53% of cerebrospinal fluid monocytes exhibited CD11b and CD11c double positivity (FIG. 8 f). Finally we studied the endogenous β amyloid peptide of cerebrospinal fluid monocytes. Technically, it is very difficult to collect these many cells, but it appears that monocytes carry large amounts of beta amyloid peptide (fig. 8 g), with a sufficient chance to be or have become macrophages.

6 amyloid peptide cell surface binding assay

We tested the in vitro ability of amyloid β peptide cells to absorb phagocytic capacity (n=6) with healthy human blood leukocytes. First, the adsorption of amyloid beta by lymphocytes, monocytes and neutrophils was compared (FIGS. 9a, c, d, e). It was found that monocytes adsorbed a large amount of exogenous beta amyloid peptide compared to the dimethylsulfoxide control group (Du Kaishi assay Tukey test showed P<0.0001). Monocytes are the white blood cells of the human body responsible for adsorbing and phagocytizing beta amyloid peptide compared to lymphocytes and neutrophilsPrincipal cells (analysis of variance P)<0.0001). Second, we split monocytes into three subsets, i.e. typical monocytes (CD 14 ⁺ CD16 ^- ) Intermediate monocytes (CD 14) ⁺ CD16 ⁺ ) And atypical monocytes (CD 14) ^dim CD16 ⁺ ) And comparing their adsorptive phagocytosis of amyloid beta peptides (fig. 9 b). As a result, it was found that both intermediate monocytes and atypical monocytes were able to absorb exogenous beta amyloid peptide (comparison of pseudomodular and regulatory group, du Kaishi assay Tukey test showed P, respectively<0.0001 and p=0.0033), wherein the intermediate monocytes are the strongest in adsorption phagocytosis in three subsets (analysis of variance P<0.0001 (FIGS. 9b, f, g, h).

7 Aβ in Alzheimer's disease patients ⁺⁺ Decreased cell number and associated cognitive function

To understand the importance of this phenomenon, we performed a cross-sectional study to determine whether the monocyte surface aβ is associated with AD. Peripheral blood from cognitively normal coholic control CN (n=74), MCI (n=39) and AD dementia (n=37) participants in mild cognitive impairment were collected by AIBL and analyzed for cell surface immunophenotyping. We found that total monocyte surface Abeta was reduced in MCI and AD demented patients. We first examined total monocytes. Fluorescence intensity test of amyloid beta peptide (Abeta), abeta ⁺ Cell percentage (fluorescence intensity at 10) ² To 10 ^3.6 Between) Aβ ⁺⁺ Cell percentage (fluorescence intensity at 10) ^3.6 To 10 ⁴ Between) by clinical classification. As a result, it was found that the aβ fluorescence intensity was reduced by 26.21% in the patients with alzheimer's disease (fig. 10a average cognitive normal 294.62 ±16.96, mild cognitive impairment 224.31 ±21.73, and alzheimer's patient 217.41 ±18.87, analysis of variance p=0.0049, du Kaishi assay Tukey test, alzheimer's patient versus cognitive normal p=0.0096, mild cognitive impairment versus cognitive normal p= 0.0223). Meanwhile, abeta of Alzheimer disease patient ⁺⁺ Cell percentage was reduced by 43.15% (fig. 10b, average cognitive level normal 1.28±0.12%, mild cognitive impairment 0.83±0.14%, and alzheimer's patient 0.73±0.14%, analysis of variance PTest Tukey, alzheimer's patient versus cognitive normal p=0.0089, mild cognitive impairment versus cognitive normal p=0.0425, =0.0068, du Kaishi.

The aβ content on monocytes was reduced by 22% in MCI compared to CN, 28% in AD dementia (fig. 10 a); while Aβ ⁺⁺ The percentage of monocytes decreased by 36% compared to CN for MCI patients and 49% for AD dementia patients (fig. 10 b). These significant decreases were also shown to correlate with brain aβ levels by quantitative calculation of brain aβ positron emission tomography (CL) scores and to correlate closely with cognitive scores such as contextual memory and Preclinical Alzheimer's Cognitive Complex (PACC) scores (fig. 10a, b).

And Abeta in AD ⁺⁺ Consistent with the reduction 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 to CN (p=0.005, fig. 10 c), which is likely to be patient aβ ⁺⁺ Possible causes of monocyte depletion. In contrast, the percentage of classical monocytes in MCI and AD groups increased by 15% compared to CN (p=0.003; fig. 10 c). In addition to monocytes, we found a 18% decrease in the percentage of natural killer cells in the whole leukocyte population of MCI and AD dementia patients compared to CN (p=0.008; fig. 10 c).

In summary, we have found that the parameters of the monocyte subset are less relevant to the disease condition than the total monocytes, possibly due to the bias of the small cell number intermediate to atypical cells. Here we selected two parameters of total monocytes, namely Abeta fluorescence intensity and Abeta ⁺⁺ Cell percentage content additional auxiliary analysis was performed by using various neuropsychological measurement tests, namely MMSE score, CDR score and preclinical alzheimer's disease complex cognition (PACC) (fig. 11). The research shows that the fluorescence intensity of Abeta and Abeta ⁺⁺ The percentage of cells expressed closely, all correlated positively with memory, executive function, language function, attention, and PACC results, and correlated negatively with CDR scoring results (fig. 11). These results indicate Abeta ⁺⁺ The course of the disease of cells and AD is closely related and can be used toThe course of AD is assessed and predicted.

Amyloid beta on 8 monocytes may have diagnostic value as a biomarker

By optimizing a number of parameters of Abeta expression on the peripheral blood leukocyte surface, we found the Abeta fluorescence intensity on the monocyte surface, abeta ⁺⁺ Percentage of monocytes and aβ ⁺ The natural killer cell percentages can be combined into a new diagnostic model to replace brain aβ PET Scan (positron Scan). The curve is excellent in combination, with an area under the curve (AUC) of 87% (95%CI:0.78to 0.92), a sensitivity of 83% (95%CI:0.71to 0.92) and a specificity of 77%

(95%CI:0.61to 0.88), accuracy was 81%, positive predictive value was 83%, negative predictive value was 77%. The critical value of about log index was ≡0.56 (FIG. 11 and Table 2).

Table 2: ROC curve AUC and critical value

^b. The W02 related biomarker (including: abeta fluorescence intensity on monocyte surface, abeta) ⁺⁺ Percentage of monocytes and aβ ⁺ Natural killer cell percentage) was added to the mixed logistic regression model (the basic model consisted of population composition base information including age, sex, education age and APOE genotype of the patient). Critical value 1: a critical value judged from the about Index (maximum value after the integrated sensitivity and specificity); threshold 2: threshold of 95% sensitivity. PPV and NPV: positive and negative predictive values.

9 cells pass through the blood brain barrier

In the case of Alzheimer's disease, it is controversial whether peripheral leukocytes can penetrate the brain. However, according to the latest mouse chimera test study, the importance of the peripheral innate immune system in the pathogenesis of alzheimer's disease has been updated. Current research shows that aβ ⁺⁺ Monocytes possess the chemotactic receptor CX3CR1 andCCR2, which also possesses the amyloid fibrosis receptor CD85A/D and the low density lipoprotein receptor CD91, may be important for cross-blood brain barrier transport and targeted metastasis of neurotoxic aβ deposits in the brains of alzheimer's disease patients. From the results of the study, it was hypothesized that peripheral monocytes, particularly intermediate monocytes, may penetrate into the central nervous system of Alzheimer's patients, and that these peripheral immune cells exert adsorptive phagocytosis of Abeta. This assumption may be correct especially when our cerebrospinal fluid donors have an average age of 77 years. Many previous studies have shown that monocytes penetrating the brain will rapidly transform into macrophages, as demonstrated by our in vitro cell transformation experiments. Our experiments showed that monocytes in peripheral blood expressed CD68 after 2-3 days of in vitro culture, while also significantly increased expression of CD163 and P2X7 (fig. 13). P2X7 is a class of receptors that mediate inflammation and is also one of the scavenger receptors that dominate natural phagocytosis. During the conversion of monocytes to macrophages, P2X7 expression was significantly enhanced. And thus can be used to assess monocyte to macrophage conversion.

Our previous results show CD14 in peripheral blood ⁺ CD16 ⁺ Aβ ⁺⁺ The cells are most likely macrophages from the brain. We have evidence from experimental studies in APP/PS1 mice (15-80 weeks) that some of the beta amyloid peptide (Abeta) -loaded monocytes return to the peripheral circulation (Table 3, FIG. 14). To investigate whether leukocytes in the brain can migrate to the peripheral blood circulation, we collected mouse blood and isolated mononuclear cells (lymphocytes and monocytes) by Ficoll density gradient centrifugation and stained with CSFE fluorescent dye and injected directly from the ventricles into 13 mouse brains. Two days later, peripheral blood and deep cervical lymph nodes were taken for detection, and among 9 young mice of 15-30 weeks, 7 found CFSE positive mononuclear cells (78%) in peripheral blood or deep cervical lymph nodes; whereas traces (100%) of CFSE positive mononuclear cells were found in peripheral blood or deep cervical lymph nodes of 4 aged mice (68-80 weeks). The detection positive rates of the blood sample and the lymph node sample are approximately 62%. Our resultsIt was demonstrated that blood-derived cells in the brain can migrate to peripheral blood and lymph nodes.

TABLE 3 Table 3

No

Surgical time

Mouse numbering

Sex (sex)

Year and month of birth

Age (week)

Deep cervical lymph node

Peripheral blood

1

19/02/2023

480

♀

11/11/2022

15

42(666K)

1(1.03M)

2

19/02/2023

482

♀

11/11/2022

15

13(40K)

3(916K)

3

05/02/2023

465

♂

10/10/2022

17

ND

ND

4

14/03/2023

475

♂

11/11/2022

18

1(303K)

ND

5

14/03/2023

479

♂

11/11/2022

18

1(817K)

ND

6

12/02/2023

472

♀

10/10/2022

18

ND

ND

7

12/02/2023

473

♀

10/10/2022

18

5(869K)

ND

8

26/03/2023

481

♀

11/11/2022

20

5(131K)

10(1.5M)

9

05/02/2023

328

♂

11/07/2022

30

ND

1(1M)

10

07/03/2023

92

♂

21/11/2021

68

31(169K)

21(663K)

11

07/03/2023

80

♂

19/10/2021

72

ND

4(560K)

12

04/06/2023

132

♀

12/01/2022

73

ND

2(573K)

13

04/06/2023

96

♂

26/11/2021

80

2(334K)

4(845K)

Note that: ND, undetected.

The human brain is considered to have "immune privileges", i.e. peripheral immune cells are prohibited from entering the brain due to the blood brain barrier, and thus, peripheral immune cells are disconnected from immune monitoring. It is known that this is not entirely correct, since microglial cells act as the primary immune cells in the brain in concert with central immune defenses. However, we do not know how much peripheral immune cells are involved in central immune defenses in neurodegenerative diseases such as AD. This study first investigated the adsorption and phagocytosis capacity of monocytes for aβ and determined that intermediate monocytes play a major role in phagocytosis of aβ. Second, studies have found that intermediate monocytes/macrophages are a major subset of those found in cerebrospinal fluid, which infiltrate the brain parenchyma and carry large amounts of aβ. Finally, through studies on peripheral blood mononuclear cells aβ from 165 volunteers, we found that the ability of monocytes to adsorb and phagocytose aβ was reduced in AD patients. These findings give us a better understanding of the pathogenesis behind AD and may lead to new therapeutic approaches and targets for preventing AD.

Aβ of AD patient ⁺⁺ The decrease in cells is due to Aβ in total monocytes ⁺⁺ The percentage of cells is reduced. Regardless of Abeta ⁺⁺ It is worth discussing whether the decrease in the percentage of cells is due to a decrease in the ability of Abeta to adsorb phagocytosis or a decrease in the proportion of intermediate monocytes to total monocytes or both. Whichever prosthesis is correct, it is indicative of an impaired ability of AD patients to clear aβ, which may reflect changes in endogenous microglia and macrophages in the brain during aging. Interestingly, the proportion of intermediate monocytes to total monocytes was reduced in AD patients. An appropriate explanation for this is that intermediate monocytes are highly fluid and they enter the central nervous system due to chemokines caused by pathological changes in Alzheimer's disease.

Our further experimental data demonstrate CD14 in peripheral blood ⁺ CD16 ⁺ Aβ ⁺⁺ At least a portion of the cells are most likely macrophages that return 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 ideas for disease course prediction and diagnosis and targeted therapy.

Taken together, our further experimental data indicate CD14 in peripheral blood ⁺ CD16 ⁺ Aβ ⁺⁺ Cell and traditional meaningCD14 of (C) ⁺ CD16 ⁺ The intermediate monocytes are very different, and these cells are most likely macrophages from the brain and participate in the process of eliminating aβ in the brain, which is known to evolve after peripheral blood mononuclear cells migrate into the brain. This new finding provides strong evidence for the first time that peripheral blood mononuclear cells and macrophages play a very important role in the process of clearing brain aβ. Our findings have a broad impact on better understanding of the development and progression of AD, as well as diagnosis and treatment.

Claims (6)

1. The application of peripheral blood macrophages in preparing reagents and/or medicines for diagnosis, prognosis and treatment of Alzheimer's disease.

2. The use of claim 1, wherein the peripheral blood macrophages are peripheral blood macrophages carrying a substantial amount of β -amyloid protein on their surface, defined as CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ And (3) cells.

3. The use of claim 2, wherein said CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The cells have the macrophage immune characteristic CD68 of the central nervous system at the same time ⁺ TREM2 and peripheral blood leukocyte characteristics CD45 ⁺ 。

4. The use of claim 2, wherein said CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The immunological features of the cells include: CD45 ⁺ /CD14 ⁺ /CD16 ⁺ /CD68 ⁺ /CD163 ⁺ /TREM2 ⁺ 。

5. The use of claim 2, wherein said CD14 ⁺ CD16 ⁺ Aβ ⁺⁺ The immunological features of the cells include: 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 1to 5, wherein the peripheral blood macrophages are used as scavengers of amyloid β for the diagnosis, prognosis and preparation of reagents and/or medicaments for the treatment of alzheimer's disease.