WO2021072650A1

WO2021072650A1 - Application of intermediate monocyte in preparation of drug for diagnosis and prediction of ad

Info

Publication number: WO2021072650A1
Application number: PCT/CN2019/111302
Authority: WO
Inventors: 戴正乾; 顾柏俊; 黄欣
Original assignee: 湖南乾康科技有限公司
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2021-04-22
Also published as: CN111727260A

Abstract

An application of an intermediate monocyte in the preparation of a drug for diagnosis and prediction of AD.

Description

Application of intermediate monocytes in preparation of drugs for diagnosis and prediction of AD

Technical field

The invention relates to the application of intermediate mononuclear cells in preparing drugs for diagnosis and prediction of AD.

Background technique

Delayed-onset Alzheimer’s disease is characterized by the deposition and accumulation of β-amyloid peptide (Aβ) in the brain, and the defect of the immune system is believed to be the cause of this accumulation, because human monocytes have been shown to be unable to effectively eliminate Amyloid β peptide Aβ. The specific mechanism of this is not yet clear, but genome-wide association analysis studies strongly indicate that this mechanism can change cell phagocytosis/endocytosis in the microglia channel. Intermediate monocytes refer to a subtype of monocytes that are CD14-positive and CD16-positive (CD14+CD16+) in the human body. The "intermediate monocytes" in the human body are the same.

Summary of the invention

The research of the present invention shows that the monocytes of patients with delayed-type Alzheimer's disease can present basic cell disorder and stimulated innate phagocytosis. In the blood circulation, a subtype of monocytes (CD14+CD16+) carries a large amount of β-amyloid polypeptide (Aβ) and expresses cell migration receptors (CX3CR1 and CCR2). This subtype of monocytes is also found in the human cerebrospinal fluid and brain as the main osmotic cell type. With the assistance of a total of 165 volunteers from the Australian Organization for Imaging, Biomarkers and Lifestyle Research (AIBL), through the quantitative determination of β-amyloid peptide (Aβ) on the surface of monocytes, the applicant found that it was in the Alzheimer’s The phagocytosis of β-amyloid peptide by monocytes in the patient's body decreased by 26%. It will open new doors for a better understanding of Alzheimer's disease mechanisms and may lead the development of new therapeutic directions to prevent Alzheimer's disease.

The accumulation of Aβ amyloid peptide in the brain is a major pathological feature of Alzheimer's disease. The Aβ aggregates have strong neurotoxicity and are generally considered to be the culprit leading to Alzheimer's disease. An important reason for the aggregation of Aβ amyloid peptide is that the human body's natural phagocytic ability gradually decreases with aging, and it cannot effectively remove Aβ amyloid peptide and other denatured proteins. We found that there is a subgroup of monocytes in peripheral blood-intermediate monocytes, which are the new force for the human body to eliminate Aβ amyloid peptide. The ability of such cells to adhere to Aβ amyloid peptide in patients with Alzheimer's disease is nearly 1/4 lower than that of healthy controls of the same age. Through comparison with the normal interval value, and multiple follow-ups (2-3 times, each time interval is 18 Months), we can diagnose Alzheimer’s early and predict the course of the disease.

Therefore, intermediate monocytes can be used to prepare drugs for diagnosis and prediction of AD, and intermediate monocytes are used as scavengers of beta amyloid peptide to prepare drugs for diagnosis and prediction of AD.

Description of the drawings

Figure 1: β-amyloid peptide on the surface of peripheral blood mononuclear cells;

Figure 2: In Alzheimer's patients, monocytes have reduced phagocytosis of β-amyloid peptide;

Figure 3: In the case of Alzheimer's disease, the ability of intermediate monocytes to phagocytose β-amyloid peptide is reduced;

Figure 4: In the case of Alzheimer's disease, atypical monocytes have reduced phagocytic ability of β-amyloid peptide;

Figure 5: In the case of Alzheimer's disease, typical monocytes have reduced phagocytic ability of β-amyloid peptide;

Figure 6: Cell phagocytic ability test for β-amyloid peptide;

Figure 7: Monocytes penetrated into the cerebrospinal fluid;

Figure 8: Phagocytosis of YO latex microspheres by ^{Aβ++ monocytes;}

Figure 9: Aβ ⁺⁺ monocyte phenotype;

Figure 10: The relevance of neuropsychological tests;

Figure 11: ROC curve (receiver operating characteristic curve);

Figure 12: Recombination of brain beta amyloid peptide by peripheral immune cells.

Detailed ways

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

Regardless of whether the infiltration of monocytes into the central nervous system is a common mechanism of β-amyloid peptide (Aβ) clearance, this ensures further research. Even if there is no channel for monocytes to enter the brain from the peripheral blood, the changes in the phagocytosis of amyloid β peptide (Aβ) by monocytes may provide a mirror image to illustrate the endogenous microglia of the brain during a person’s life cycle. Cell changes. In this study, we used the monoclonal antibody method and flow cytometry to establish a total of 165 volunteers (84 with normal cognition CN and 41 with mild CN) at the Australian Organization for Imaging, Biomarker and Lifestyle Research (ALBL). With the help of MCI in patients with high-degree cognitive impairment and 40 Alzheimer's patients with AD, all white people), quantified the amyloid β peptide (Aβ) of monocytes (Fig. 1a and e). The results showed that peripheral monocytes captured endogenous β-amyloid peptide (Aβ) (Fig. 1d), and a small part of monocytes carried a large amount of β-amyloid peptide (Aβ). CD14 and CD16 staining proved that these were intermediate monocytes. Cells (Fig. 1d and h, surrounding). Lymphocytes and neutrophils can carry a small amount of beta amyloid peptide (Aβ) (Fig. 1b and c). Atypical and typical monocytes carry a small amount of beta amyloid peptide (Aβ) (Fig. 1f and g).

2In Alzheimer's patients, monocytes have reduced phagocytosis of β-amyloid peptide

We first detected total monocytes. Amyloid β peptide (Aβ) fluorescence intensity test, the ^{percentage of Aβ +} cells (fluorescence intensity between 10 ² and 10 ^3.6 ), the ^{percentage of Aβ ++} cells (fluorescence intensity between 10 ^3.6 and 10 ⁴ ), through three methods Compare. Method 1-Simple clinical classification, Method 2-Amyloid β peptide (Aβ) positron tomography cerebellar standardized uptake rate (SUVR) BecKet score and Method 3, Amyloid β peptide (Aβ) brain deposition (uptake rate ratio SUVR increase /year). The results showed that the Aβ fluorescence intensity of patients with Alzheimer's disease was reduced by 26.21% (Fig. 2a Method 1 average cognitive normal 294.62±16.96, mild cognitive impairment 224.31±21.73, and Alzheimer's 217.41 ±18.87, analysis of variance P = 0.0049, Tukey test, Alzheimer's patients with normal cognition P = 0.0096, patients with mild cognitive impairment compared with normal cognition P = 0.0223; Method 2r =- 0.1974, P = 0.0261; Method 3r = -0.2127, P = 0.0430). At the same time, the ^{percentage of Aβ ++} cells in patients with Alzheimer's disease decreased by 43.15% (Fig. 2g Method 1 averaged cognitive level of persons with normal 1.28±0.12%, mild cognitive impairment 0.83±0.14%, and Alzheimer patients 0.73±0.14%, analysis of variance P = 0.0068, Tukey test by Tukey test, Alzheimer's patients have normal cognition (P=0.0089, mild cognitive impairment compared with normal cognitive P=0.0425; Fig. 2h method 2r=-0.1793, P=0.437; Fig. 2i method 3r=-0.2392, P=0.224). The ^{percentage of Aβ +} cells did not change (Fig. 2d-f).

After that, we used CD14 and CD16 to classify the mononuclear cell set. Through the observation of the middle mononuclear cell population, Alzheimer’s disease can reduce the fluorescence intensity of Aβ (Fig. 3a, method 1 analysis of variance P=0.353 [people with mild cognitive impairment vs. people with normal cognition]; Fig. 3c Method 3 shows r=-0.2682, P=0.0336), and the ^{percentage of Aβ++} cells (Figure 3i Method 3 shows r=-0.2694, P=0.0328). (Fig. 3d-f). In the middle monocyte population, the ^{percentage of Aβ +} cells did not change (Fig. 3d-f). In atypical monocytes, Alzheimer’s disease reduces the fluorescence intensity of Aβ (Fig. 4c Method 3 shows r=-0.2623, P=0.378), and the ^{percentage of Aβ++} cells (Fig. 4i Method 3 shows r= -0.2569, P=0.421). Alzheimer’s disease can increase the ^{percentage of Aβ +} cells in atypical monocytes (Fig. 4d Method 1 shows the analysis of variance P = 0.0260, Tukey test Alzheimer's test Alzheimer's patients compared to cognitive normal population P = 0.0146). In typical monocytes, Alzheimer reduces the fluorescence intensity of Aβ (Figure 5b method 2 shows r = -0.2084P = 0.0427; Figure 5c method 3 shows r = -0.315, P = 0.0119), and the percentage of ^{Aβ ++ cells} Content (Fig. 5h Method 2 shows r=-0.2156, P=0.0358). In the middle monocyte population, the ^{percentage of Aβ +} cells did not change (Fig. 5d-f).

In summary, we found that compared with total monocytes, the correlation between the parameters of the subset of monocytes and the disease condition is weaker, which may be caused by the deviation between the number of small cells and atypical cells. Here, we have selected two parameters of total monocytes, namely Aβ fluorescence intensity and Aβ ⁺⁺ cell percentage, by using a variety of neuropsychological evaluation tests, namely MMSE score, CDR score and preclinical Alzheimer’s Acute Complex Cognition (PACC) for additional auxiliary analysis. (Suppl.Fig.1d r = -0.2482, P = 0.0035). The study found that Aβ fluorescence intensity was positively correlated with the PACC results (Supplementary, Fig. 1m r = 0.3200, P = 0.0005), but it was negatively correlated with the CDR score. Correlation (Fig. 10n r = 0.3412, P = 0.0002). The ^{percentage of Aβ++} cells is positively correlated with the MMSE score (Fig. 10br = 0.1858, P = 0.0297), and also positively correlated with the PACC result (= Fig. 10n r = 0.3412, P = 0.0002), and negatively correlated with the CDR score. Correlation (Fig. 10d r = -0.2482, P = 0.0035).

3 Amyloid peptide cell surface binding test

We used immunofluorination chemistry and flow cytometry (CN n = 6) to do two in vitro β-amyloid peptide cell phagocytosis experiments. First, compare the phagocytosis of β-amyloid peptide by lymphocytes, monocytes and neutrophils (Fig. 6a). It was found that monocytes absorbed a large amount of exogenous β-amyloid peptide (comparing the dummy module treated with DMSO and the control group, Tukey test showed P<0.0001), and the mononuclear cell Cells are the main phagocytic cells responsible for β-amyloid peptide in human leukocytes (compared with lymphocytes and neutrophils, analysis of variance P<0.0001). Secondly, we divide monocytes into three subsets, namely typical monocytes (CD14+CD16-), intermediate monocytes (CD14+CD16+), and atypical monocytes (CD14-CD16+), and treat them Compare the phagocytosis of β-amyloid peptide (Fig. 6b). The results showed that both intermediate monocytes and atypical monocytes can absorb exogenous β-amyloid peptide (comparing the fake module and the control group, the Tukey test showed P<0.0001 and P=0.033 respectively), and the intermediate mononuclear cells Nuclear cells have the strongest phagocytic ability in the three subsets (ANOVA P<0.0001).

4 Cell characteristics in cerebrospinal fluid

The extent to which peripheral immune cells are involved in the core pathological changes of Alzheimer’s disease is not yet fully understood, but the accumulated data indicate that we may underestimate their importance. In this study, we did a pilot study to show the infiltration of peripheral immune cells in human cerebrospinal fluid (n=8). Concentrate 10ml of cerebrospinal fluid sample, use monoclonal antibody method and flow cytometry to detect cell type and β-amyloid peptide on cell surface. In the three samples of human cerebrospinal fluid, we found a small amount of infiltrating peripheral immune cells (CD45+), that is, 317.00±84.30 lymphocytes and 74.30±19.60 monocytes per milliliter of cerebrospinal fluid (Fig. 7a and b). No neutrophils were found in the cerebrospinal fluid (CD45+CD15+ cell count is 1.67±1.53). Monocytes are classified into subsets by CD14 and CD16. Surprisingly, intermediate monocytes are the main monocyte subsets present in the cerebrospinal fluid, up to 70.2±11.8%, instead of atypical and typical monocytes. The cells were only 7.78±6.14% and 1.41±0.37% respectively (Fig. 7c). In peripheral venous blood, we found that intermediate monocytes and typical monocytes accounted for 6.30±3.87% and 49.60±15.26% of total monocytes (n=165). In other words, the ratios of intermediate monocytes and typical monocytes in peripheral blood and cerebrospinal fluid are opposite. We further studied the expression levels of monocyte chemotactic receptors CX3CR1 and CCR2 in cerebrospinal fluid. The study found that cerebrospinal fluid monocytes express CX3CR1+ (92.23.61%) and 54.85.53% of cells also express CCR2 (Figure 7d). This indicates that CX3CE1 may play a role in helping peripheral monocytes enter the cerebrospinal fluid. In addition, Lin-1, HLA-DR (MHC-II cell surface marker), CD11B and CD11C are used to describe the characteristics of monocytes. The study found that 77.207.94% of cerebrospinal fluid monocytes behaved as HLA-DR and Lin-1 Double positive (Fig. 7e) and 88.60±6.53% of CSF mononuclear cells were double positive for CD11B and CD11C (Fig. 7f). Finally, we studied the endogenous β-amyloid peptide of cerebrospinal fluid monocytes. Technically, it is very difficult to collect these many cells, but it seems that monocytes carry a large amount of β-amyloid peptide (Fig. 7g).

5 Phagocytosis related to Aβ ^+/- , Aβ ⁺ and Aβ ^{++ monocytes}

We first tested the ability of Aβ ⁺⁺ monocytes to swallow YO latex microspheres by real-time flow cytometry. Studies have shown that Aβ ⁺⁺ monocytes have the strongest phagocytic ability of microbeads (Fig. 8).

Aβ ⁺⁺ monocyte characteristics

Then we used monoclonal antibodies to detect Aβ ⁺⁺ monocytes. The study found that Aβ ⁺⁺ monocytes are composed of Lin-1 ⁺ (so they are not dendritic cells) and HLA-DR ⁺ (MHC-II) (Fig. 9a and b). They express chemotactic receptors, CX3CR1 and CCR2 (Fig. 9c and d), and a small portion express CD68 (Fig. 9e). They can express a variety of beta receptor proteins, such as CD85A and D receptors (Fig. 9f and g) and low-density lipoprotein receptor CD91 (Fig. 9h). A variety of complement receptors can be expressed, such as CD11B (Fig. 9i), CD11C (Fig. 9j) and CD35 (CR1 Fig. 9k). It can also express a variety of scavenger receptors such as the tyrosine protein kinase Mer receptor (MerTK Fig. 13l), P2X7 (Fig. 9m) and CD163 (Fig. 9n).

6 Amyloid β peptides on monocytes may have diagnostic value as biomarkers

We presume to use the plasma amyloid β peptide (Aβ) contained in Alzheimer's disease as a biomarker. However, this contradicts the published results of the correlation between Alzheimer’s disease and plasma amyloid β (Aβ), and the measurement of plasma amyloid β (Aβ) is more variable than that of cerebrospinal fluid plasma amyloid β (Aβ). The impact is greater and greater. Technically speaking, it is simpler and more repeatable to measure beta amyloid peptide (Aβ) in leukocytes by flow cytometry (FACS). We are trying to study whether β-amyloid peptide (Aβ) has the diagnostic value of early warning of brain β-amyloid peptide (Aβ) load. The load status of amyloid β peptide (Aβ) in the brain is expressed in binary form, with 0 representing AβPET score<1.4 and 1 representing AβPET score>1.4. The first expected variable is the participant’s demographic data such as age, gender, apolipoprotein E genotype, and years of education, and results in an area under the curve (AUC) of 0.706 and a 95% confidence interval of 0.574-0.837 (Fig.11) ). Age and apolipoprotein E are confirmed to be risk factors for Alzheimer's disease, and years of education are preventive factors for Alzheimer's disease. This variable evaluation is better than random probability 0.5 (Fig. 11). The predicted variable for the second test is the combined consideration of demographic data, MMSE score and CDR score (Fig. 11), which increases the AUC value to 0.767 (95% confidence interval 0.654-0.880). The third predictor variable is demographic data, MMSE score, CDR score and 2 biomarkers, namely β-amyloid peptide (Aβ) fluorescence intensity and ^{the percentage of Aβ++} cells in total monocytes (Fig. 11), which makes The AUC value increased slightly to 0.783 (95% confidence interval 0.675-0.892). The final predictor variables are demographic data, MMSE score, CDR score and 4 biomarkers, which further increase the percentage of typical monocytes and intermediate monocytes in total monocytes (Fig. 11), which makes AUC The value increased to 0.824 (95% confidence interval 0.725-0.924), and the optimal threshold was 0.585, sensitivity was 0.744, and specificity was 0.852.

7 Seeking a new way to solve the problem of Alzheimer's disease

The human brain is considered to have the "privilege of immunity", that is, because of the blood-brain barrier, peripheral immune cells are prohibited from entering the brain, and therefore, peripheral immune cells are separated from immune surveillance. We know that this statement is not entirely correct, because microglia, as the main immune cells in the brain, can uniformly act on the central immune defense. However, we do not know to what extent peripheral immune cells participate in the central immune defense in neurodegenerative diseases such as Alzheimer's disease. This study first explored the phagocytic ability of human leukocytes for β-amyloid peptide (Aβ) and determined that intermediate monocytes play a major role in phagocytosing peripheral β-amyloid peptide (Aβ). Second, studies have found that intermediate monocytes are the main subset present in the cerebrospinal fluid, which penetrate the brain parenchyma and carry a large amount of amyloid β peptide (Aβ). Finally, by studying 165 volunteers with monocyte beta amyloid peptide (Aβ), we found that in Alzheimer's disease monocytes have a decreased ability to swallow beta amyloid peptide (Aβ). This new method shows that we have a better understanding of the pathogenesis behind Alzheimer's disease, and may lead to new treatment methods and targets to prevent Alzheimer's disease.

8 In Alzheimer's disease, the phagocytic effect of amyloid β peptide (Aβ) decreases.

The decrease in the fluorescence intensity of β-amyloid peptide (Aβ) in Alzheimer's disease state is attributed to the decrease in the percentage ^{of Aβ++ cells in the total monocytes.} Whether ^{the decrease in the percentage of Aβ++} cells is due to the weakening of β-amyloid peptide (Aβ) phagocytic ability, the decrease in the proportion of intermediate monocytes to the total monocytes, or both, it is questionable. No matter which illusion is correct, it indicates that the peripheral β amyloid peptide (Aβ) is impaired in Alzheimer’s disease, which may reflect the endogenous microglia in the brain during aging. Cell changes. Interestingly, the proportion of intermediate monocytes to the total monocytes in Alzheimer's patients has decreased. An appropriate explanation for this is that intermediate-type monocytes are highly fluid, and they enter the central nervous system under the action of chemotactic factors brought about by the pathological changes of Alzheimer's disease. But we still don't know whether this explanation is correct. In fact, monocytes from subjects with Alzheimer’s disease may be more susceptible to apoptosis than monocytes from healthy subjects. This is obvious from our previous research findings that human monocytes The synthesis of β-amyloid peptide (Aβ) can induce irreversible cytotoxicity. The increase in migration rate or mortality requires a faster turnover rate, which is consistent with the findings of the deviation of bone marrow hematopoiesis in the elderly. The suppression of the bone marrow PU.1 target gene network is related to the delayed onset of Alzheimer's disease.

9 cells pass through the blood-brain barrier

In the case of Alzheimer's disease, whether peripheral white blood cells can penetrate into the brain is controversial. However, according to the latest experimental research on mouse chimera, the importance of the peripheral innate immune system in the pathogenesis of Alzheimer's disease has been updated. Current studies have shown that Aβ ⁺⁺ monocytes have chemotactic receptors CX3CR1 and CCR2, as well as amyloid fibrosis receptors CD85A/D and low-density lipoprotein receptors CD91, which may cross the blood-brain barrier and contain neurotoxicity. The targeted metastasis of the beta amyloid peptide (Aβ) deposited in the brain suffering from Alzheimer's disease is very important. From the results of the study, it is assumed that peripheral monocytes, especially intermediate monocytes, may penetrate into the central nervous system of Alzheimer's patients, and these peripheral immune cells play a phagocytosis of beta amyloid peptide (Aβ). Especially when the average age of our cerebrospinal fluid donors is 77 years old, this assumption may be correct. It is not certain whether these monocytes loaded with beta amyloid peptide (Aβ) remain in the central nervous system or return to the peripheral blood. But we have an evidence from an experimental study of APP/PS1 mice (20 months old) that a part of monocytes loaded with amyloid β peptide (Aβ) will return to the peripheral circulation (Fig. 12).

Claims

Application of intermediate monocytes in preparation of drugs for diagnosis and prediction of AD.
The application according to claim 1, wherein the intermediate monocyte is used as a scavenger of β amyloid peptide for the diagnosis and prediction of AD in the preparation of drugs.
The use according to claim 1 or 2, wherein the intermediate monocytes are intermediate monocyte subtype CD14 and/or intermediate monocyte subtype CD16.