Amyloid-beta uptake by peripheral blood monocytes is reduced by ageing and Alzheimer’s disease

Background: Decits in the clearance of amyloid β-protein (Aβ) play a pivotal role in the pathogenesis of sporadic Alzheimer’s disease (AD). The roles of blood monocytes, the counterparts of microglia in the periphery, in the development of AD remain unclear. In this study, we sought to investigate the alterations in the Aβ phagocytosis function of peripheral monocytes during ageing and in AD patients. Methods: A total of 104 cognitively normal participants aged 22 to 89 years old, 22 AD patients, 22 age- and sex-matched cognitively normal (CN) subjects, 15 Parkinson’s disease patients (PD) and 15 age- and sex-matched CN subjects were recruited. The Aβ uptake by blood monocytes were measured and its alteration during ageing and in AD were investigated. Results: Aβ 1−42 uptake by monocytes was associated with Aβ 1−42 levels in the blood. Aβ 1−42 uptake by monocytes decreased during ageing, and further decreased in AD but not in PD patients. Among the Aβ uptake-related receptors and enzymes, the expression of Toll ‐ like receptor 2 (TLR2) was reduced in monocytes from AD patients.

Amyloid-beta uptake by peripheral blood monocytes is reduced by ageing and Alzheimer's disease Si Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting 35 million elderly individuals (1). Its mechanism remains unclear, and no disease-modifying therapies are currently available. A large amount of evidence suggests that de cit in the clearance of amyloid β-protein (Aβ), which leads to the cerebral accumulation of Aβ, plays a pivotal role in the development of sporadic AD (2).
Recent studies show that a series of AD risk gene mutations are associated with immune responses and endocytosis, suggesting that dysfunctional innate immunity, mainly involving microglia and peripheral myeloid cells, is a critical reason for AD (3)(4)(5)(6)(7). Indeed, studies have suggested that the reduced Aβ uptake capacity of microglia in the brain is a major mechanism underlying the development of AD (8,9).
However, the alterations in the functions and roles of peripheral myeloid cells in AD remains unclear.
While resident microglia play a key role in the clearance of Aβ in the brain, approximately 40-60% of Aβ from the brain is estimated to diffuse into the blood and be cleared in the periphery, indicating that the peripheral system also plays an essential role in clearing Aβ from the brain (10)(11)(12). It remains undetermined how this brain-derived Aβ is cleared in the periphery. Blood monocytes are the counterparts of microglia in the periphery. Some studies have demonstrated that monocytes are more effective at neuroprotection, neuroin ammation regulation and Aβ clearance than microglia in AD (13)(14)(15). In addition, the depletion of blood monocytes exacerbates Aβ accumulation in transgenic AD models (16,17). Therefore, monocytes might play a critical role in the clearance of brain-derived Aβ in the periphery.
In the present study, we aimed to investigate alterations in Aβ uptake by peripheral monocytes during ageing in cognitively normal subjects and in sporadic AD patients and to evaluate the role of peripheral monocytes in Aβ clearance.

Clinical assessment
The clinical evaluation was performed by following the protocol described in our previous studies (18). In brief, demographic data including age, sex, education level, and occupation were collected on admission.
The medical history including current medications, prior head trauma and surgery, prior gas poisoning, schizophrenia, hypothyroidism, coronary heart diseases, atrial brillation, cerebrovascular diseases, chronic obstructive pulmonary disease, chronic hepatitis, chronic renal insu ciency, hypertension, diabetes mellitus, hypercholesterolemia and regular use of non-steroidal anti-in ammatory or prescription drugs, was collected from the medical records and a formal questionnaire. Blood and CSF sampling Blood sampling. To avoid possible circadian rhythm effects, the sampling conditions, including sampling timing and fasting state, were consistent among AD and PD patients and they were matched pairs. A portion of fasting blood samples was aliquoted for measuring complete blood cell counts, and fasting glucose, thyroxin, creatinine, urea, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total cholesterol levels. For another portion of blood, plasma was separated within 30 minutes after sampling and stored at −80°C for the further analysis of Aβ. For Aβ uptake-related assay, the blood samples were applied for the isolation of peripheral blood mononuclear cells (PBMCs) within two hours after blood drawing.
CSF sampling. For a subgroup of patients who underwent urological surgery, fasting blood and cerebrospinal uid (CSF) were sampled at the same time during subdural anaesthesia before surgery. The CSF samples were collected, free from blood contamination, in polypropylene tubes by lumbar puncture, centrifuged at 1800 × g at 4°C for 10 minutes within 1 hour after collection, and stored at −80°C until analysis.

Isolation of blood monocytes
Heparinized blood was diluted with PBS (1:1 ratio; vol/vol). PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque, and mononuclear sections were collected and washed with PBS three times. A portion of the PBMCs was used for the Aβ uptake assay, and the other portion of PBMCs was used for monocyte isolation by CD14 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and passed through a MACS column for the positive selection of CD14 + cells, according to the manufacturer's instructions. The remaining PBMCs were frozen at a concentration of 1-2 × 10 6 cells per ml in 10% DMSO (Sigma-Aldrich, Saint Louis, USA)/90% foetal calf serum (vol/vol Gibco, California, Australia) for future use.
Aβ uptake assay Isolated PBMCs were resuspended in RPMI medium with 10% foetal calf serum and 1% penicillin/streptomycin and adjusted to a concentration of 2×10 6 cells/mL. To test the uptake of Aβ, PBMCs were incubated with FITC-Aβ 1-42 (2 μg/mL) (GL Biochem, Shanghai, China) overnight at 37°C in a 5% CO 2 incubator. Following incubation, the cell suspensions were discarded, and adherent cells were Monocytes were gated using forward and side scatter, and monocyte subsets were identi ed by differential expression of CD14 and CD16, as indicated in Figure 1. The data were analysed by NovoExpress software based on forward and side scatter and the mean uorescence intensity (MFI). To maintain consistent testing conditions, a gating strategy was designed and applied equivalently across all study samples.

Imaging ow cytometry
Imaging ow cytometry (IFC) was performed according to a previous report (20). In brief, the procedures for labelling surface markers were the same as those used for conventional ow cytometry, which is described above. IFC was performed on a two-camera ISX with INSPIRE acquisition software (Amnis, NJ, USA). Excitation lasers used for analysis included a 5 mW 405 nm, a 100 mW 488 nm and a 150 mW 642 nm. A 2.5 mW 785 nm laser was used for internal calibration to provide a scatter signal and measure speed beads. FITC and PE were excited by the 488 nm laser, and the emission was captured in the ranges of 505-560 nm (Ch02) and 560-595 nm (Ch03). APC was excited by the 647 nm laser, and the emission was captured in the wavelength range of 642-745 nm (Ch05). In total, 25,000 events were acquired, and all images were captured with the 20 × objective and a cell classi er (threshold) applied to the bright eld channel (Ch01) to exclude small particles. Monocytes were identi ed using Amnis IDEAS software as shown in Figure 1. Cells with high-intensity labelling of the CD14 marker were chosen as monocytes (R3).
Aβ 1-42 assay Plasma and cerebrospinal uid Aβ 1-42 levels were measured using an ultra-sensitive single molecule array (SIMOA) on the Simoa HD-1 Analyzer (Quanterix, Lexington, Massachusetts), as previously described (21). SIMOA technique implied immunocapture of the target protein on magnetic beads, which are trapped in femto-liter volume wells, followed by the addition of enzyme-labelled detection antibody and accurate digital quanti cation. The high analytical sensitivity of this technique allows for pre-dilution of CSF and plasma samples, thus contributing to reducing matrix interferences. It has been widely used and been validated useful in numerous studies (22).

Statistical analysis
For each statistical analysis, appropriate tests were selected based on whether or not the data were normally distributed. Differences in demographic characteristics were assessed by the Chi-square test.
Statistical comparisons between two groups were made by using a paired t test, the Wilcoxon matchedpairs test or an unpaired t test, where appropriate. Speci cally, a Pearson correlation or covariate correlation analysis was utilized to analyse the association of Aβ 1-42 uptake with ageing or Aβ 1-42, Aβ 1-40 , Aβ 1-42 / Aβ 1-40 levels in the plasma and CSF, respectively. The trajectory of Aβ 1-42 uptake with age was modelled using third-order polynomial (cubic) curves. All statistical analyses were performed with GraphPad Prism v5.0 software. The data are expressed as the mean ± SD, and signi cance was achieved at p<0.05.

Data availability
The data, analytic methods, and study materials that support the ndings of this study will be available from the corresponding authors on request, after the request is submitted and formally reviewed and  (23). We found that all subsets of monocytes could intracellularly take up Aβ 1-42 (Supplemental Figure 1). The CD14 + CD16 + subset had the highest uptake of Aβ 1-42 among the three subsets, with no signi cant difference in Aβ 1-42 uptake between the CD14 + CD16 + and CD14 dim CD16 + subsets ( Figure 1).
Correlation and trajectory of Aβ 1-42 uptake by monocyte subsets relative to age Then, we measured Aβ 1-42 uptake by monocytes in 104 CN subjects aged 22 to 89 years with no differences in sex among the different age groups (Supplemental Table 1). Aβ 1-42 uptake by the total monocyte population was correlated with age; that is, the older the age was, the lower the Aβ 1-42 uptake level (γ=-0.401, P<0.0001). Among the three subsets, Aβ 1-42 uptake was correlated with age in the CD14 + CD16subset (γ=-0.445, P<0.0001) but not in the CD14 + CD16 + (γ=-0.030, P=0.760) and CD14 dim CD16 + subsets (γ=-0.113, P=0.253; Figure 2). Aβ 1-42 uptake by total monocytes and the CD14 + CD16subset decreased rapidly in the 20-40 years of age group, but the reduction rate became relatively slow after 40 years of age. This result suggests that the decrease in Aβ uptake is a life-long process that may have existed prior to the cerebral accumulation of Aβ ( Figure 2).
To investigate whether the alteration in Aβ 1-42 uptake by monocytes is speci c to AD patients, 22 AD patients, 15 age-and sex-matched PD patients and their matched CN controls were enrolled (Supplemental Table 2 and 3). There were no signi cant differences in sex, age, years of education, APOE ε4 carrier, comorbidities including hypertension, hyperlipidaemia, and diabetes mellitus, or medications between the matched groups. We found that Aβ 1-42 uptake by total monocytes and the various subsets was lower in AD patients than in CNs ( Figure 5). There were no signi cant differences in Aβ 1-42 uptake by total monocytes and their subsets between PD patients and CN controls (Supplemental Figure 7). These results suggest that Aβ 1-42 uptake by monocytes might be speci cally decreased in AD patients.

Expression of Aβ 1-42 uptake-related receptors and Aβ-degradingenzymes in monocytes of AD patients
The expression of TLR2 was lower in AD patients than CNs. However, no differences were observed in the expression of receptors, including TREM2, CD36, CD33, and SCARA1, between AD patients and CN ( Figure   6a-k). There were no signi cant differences in the protein levels of Aβ-degrading enzymes, including cathepsin D and cathepsin S, between AD patients and CN controls (Figure 6l-m).

Discussion
Ageing is an important factor for the development of AD (24). We found that Aβ 1-42 uptake by monocytes decreased as patient age increased. Consistently, a previous study showed that the internalization of Aβ 1-42 by aged human blood cell-derived monocytes was lower than that by human umbilical cord blood cellderived monocytes (25). It is worth noting that the decrease in Aβ uptake by monocytes began at the age of 20 years in our study, suggesting that a decrease in Aβ uptake ability is a life-long process. Despite the impact of ageing, the Aβ uptake ability of monocytes is further decreased in AD patients, implying that compromised Aβ uptake by monocytes is involved in AD pathogenesis (26)(27)(28).
In humans, peripheral monocytes can be divided into non-classic, intermediate and classic monocyte subsets (23). These monocyte subsets have different functions in AD, which are not fully understood. As re ected by our ndings, the intermediate subset had the highest Aβ uptake ability, while there was no signi cant difference in Aβ uptake between the classic and non-classic subsets in cognitively normal controls. The Aβ uptake ability was mainly decreased in the classic subset during ageing but decreased in all subsets in AD patients. These results suggest that there is an overall decrease in Aβ uptake by all three monocyte subsets in AD patients, implying that the mechanisms underlying the alteration in Aβ uptake ability by monocytes in AD patients are different from those associated with ageing.
The mechanisms underlying the decreased Aβ uptake ability by monocytes during ageing and AD remain to be investigated. In AD, the decrease could be partially due to de cits in Aβ recognition by monocytes, as re ected by the reduced expression of TRL2 in monocytes. TRL2 is a type I transmembrane pattern recognition receptor and acts as a natural innate immune receptor to clear Aβ 1-42 and delay cognitive decline in a mouse model of AD (29). However, we did not nd any differences in the expression of Aβdegrading enzymes between AD patients and CN controls, suggesting that dysfunctional Aβ recognition could be particularly important for the decrease in Aβ uptake by monocytes in AD patients.
The decrease in Aβ uptake by monocytes seems speci c to AD, as it was not changed in PD patients compared with each matched CN controls in our study. Additional evidence indicates that the Aβ uptake ability is correlated with blood Aβ levels in CN subjects, that is, the greater the Aβ uptake ability is, the lower the blood Aβ levels. These ndings suggest that monocytes might play a critical role in clearing Aβ from the blood. We did not nd a correlation between Aβ uptake by monocytes and Aβ 1-42 levels in the CSF. This might be due to the lack of a correlation between plasma and CSF Aβ 1-42 levels in our CN subjects.

Limitations
There are several limitations in our present study. Although we have made certain that statuses of participants before blood drawing were as consistent as possible, it is still di cult to rule out potential in uence of other conditions, such as insomnia, nutritional status. Besides, it is di cult to exclude some other chronic comorbidities which may affect the status of monocytes of patients, thus causing bias of the results of our study.

Conclusion
In conclusion, our ndings are of signi cance to the understanding of the pathogenesis of sporadic AD. Aβ in the brain can be transported to the peripheral blood (30)(31)(32), and the clearance of Aβ in the periphery has been suggested to substantially contribute to the clearance of Aβ from the brain (10,33). Therefore, the decrease in Aβ uptake by monocytes could play a signi cant role in the development of sporadic AD. The recovery of the Aβ clearance function of blood monocytes may represent a potential strategy for the prevention and treatment of AD.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare no con icts of interests, and approval the contents of this study.

Funding
This study was supported by National Natural Science Foundation of China (81930028, 91749206, 81625007 and 31921003).

Authors' contributions
Si-Han Chen performed the design, acquisition of data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content; Ding-Yuan Tian performed the design, acquisition of data and critical revision of the manuscript for important intellectual content; Ying-Ying Shen performed sample collection, critical revision of the manuscript for important intellectual content; Yuan Cheng studied the concept and design, performed critical revision of the manuscript for important intellectual content; Dong-Yu Fan studied the concept and design, performed critical revision of the manuscript for important intellectual content; Hao-Lun Sun studied the concept and design, performed critical revision of the manuscript for important intellectual content; Chen-Yang He studied the concept and design, performed critical revision of the manuscript for important intellectual content; Pu-Yang Sun studied the concept and design, performed critical revision of the manuscript for important intellectual content; Xian-Le Bu studied the concept and design, performed critical revision of the manuscript for important intellectual content; Fan Zeng studied the concept and design, performed critical revision of the manuscript for important intellectual content; Juan Deng studied the concept and design, performed critical revision of the manuscript for important intellectual content; Zhi-Qiang Xu studied the concept and design, performed critical revision of the manuscript for important intellectual content; Yang Chen studied the concept and design, performed critical revision of the manuscript for important intellectual content; Yan-Jiang Wang studied the concept and design, drafted the manuscript and performed critical revision of the manuscript for important intellectual content. Figure 1 Flow cytometry analysis of peripheral monocyte subsets in a cognitively normal population. For imaging ow cytometry, single cells were selected from debris by gating on cells in focus (a) followed by gating on the area and aspect ratio of the bright eld image (b). Cells with high-intensity labelling of CD14 were chosen as monocytes (c). Monocytes were stained with APC-conjugated anti-CD14 mAb (red) and PEconjugated anti-CD16 mAb (yellow), whereas FITC-conjugated Aβ1-42 is shown in green. Images of FITC-  Correlation and trajectory of Aβ1-42 uptake by monocyte subsets relative to age. Pearson correlation analysis was utilized to investigate the correlation between ageing and the uptake of Aβ1-42 by all monocytes (a), by the CD14+CD16-subset (b), by the CD14+CD16+ subset (c), and by the CD14dimCD16subset (d). The trajectory of Aβ1-42 uptake by total monocytes and the CD14+CD16-subset relative to age was modelled using third-order polynomial (cubic) curves. N=104. MONO = monocytes; Aβ = amyloidβ protein.  Comparison of Aβ1-42 uptake by monocyte subsets between AD patients and CN subjects. Compared with CN controls (n=22), AD patients (n=22) had decreased Aβ1-42 uptake by total monocytes (a), the CD14+CD16-subset (b), the CD14+CD16+ subset (c) and the CD14dimCD16-subset (d). AD = Alzheimer's disease; CN = cognitively normal control; MONO = monocytes; Aβ = amyloid-β protein.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.