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 from Chongqing Daping Hospital between March 2017 and May 2019. Another 25 CN subjects who received epidural anaesthesia were enrolled for CSF collection.
Subjects were not eligible if they had a family history of dementia; had a concomitant neurologic disorder except for AD and PD; were in a state of obvious infection or inflammation potentially affecting the status of blood cells; had severe cardiac, pulmonary, hepatic, renal diseases, or any kinds of tumour; had any potent haematopathy, including acute monocytic leukaemia and myelodysplastic syndrome, during the recovery period of agranulocytosis; had autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus; had an endocrine system disease, including Cushing syndrome and thyroid disorders; and declined to participate in the study.
The study was approved by the ethics committee of Chongqing Daping Hospital. Written consent was obtained from all participants or their legal representatives.
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 fibrillation, cerebrovascular diseases, chronic obstructive pulmonary disease, chronic hepatitis, chronic renal insufficiency, hypertension, diabetes mellitus, hypercholesterolemia and regular use of non-steroidal anti-inflammatory or prescription drugs, was collected from the medical records and a formal questionnaire.
Cognitive status was assessed using a neuropsychological battery that included Minimum Mental State Examination (MMSE), Activities of Daily Living (ADL) and Montreal Cognitive Assessment (MoCA).The subjects with abnormal performance in MMSE or MoCA assessment were further scheduled for neuropsychological tests, including Clinical Dementia Rating (CDR), Pfeiffer Outpatient Disability Questionnaire (POD) and Hachinski Ischemic Score (HIS) for assessing significant vascular disease. Subjects with abnormal cognition were further subjected to a brain CT/MRI investigation and blood tests for thyroxine, vitamin B12, folic acid and HIV/syphilis to rule out metabolic and infectious reasons for cognitive decline.
Dementia was diagnosed based on DSM-IV criteria. The diagnosis of probable AD was made according to the criteria of the National Institute of Neurological and Communicative Diseases and Stroke and the Alzheimer Disease and Related Disorders Association (NINCDS-ADRDA). Idiopathic PD was diagnosed according to the Parkinson’s Disease Society Brain Bank criteria (19).
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 fluid (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 × 106 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 2×106 cells/mL. To test the uptake of Aβ, PBMCs were incubated with FITC-Aβ1-42 (2 μg/mL) (GL Biochem, Shanghai, China) overnight at 37°C in a 5% CO2 incubator. Following incubation, the cell suspensions were discarded, and adherent cells were detached from the well plate by 0.25% trypsin and washed with fluorescence-activated cell sorting (FACS) buffer twice. Then, the cell suspensions were preincubated with Human TruStain FcX (Biolegend, CA, USA) on ice for 20 minutes to avoid producing a high background by the non-specific binding of the Fc receptor expressed on immune cells to the Fc fragment of the fluorophore-labelled antibody. Then, the cell suspensions were washed and stained with the following fluorophore-labelled antibodies according to the corresponding manufacturer’s instructions (BD, NJ, USA): APC-anti human CD14, PE-anti human CD16, APC-mouse IgG2a, κ isotype control, PE-mouse IgG2a, and κ isotype control. Following incubation, the cells were washed twice with FACS buffer and fixed with 1% paraformaldehyde. Flow cytometry was performed on a NovoCyte Flow Cytometer (ACEA Biosciences, CA, USA) after appropriate compensation. Monocytes were gated using forward and side scatter, and monocyte subsets were identified 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 fluorescence intensity (MFI). To maintain consistent testing conditions, a gating strategy was designed and applied equivalently across all study samples.
Imaging flow cytometry
Imaging flow 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 flow 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 classifier (threshold) applied to the bright field channel (Ch01) to exclude small particles. Monocytes were identified using Amnis IDEAS software as shown in Figure 1. Cells with high-intensity labelling of the CD14 marker were chosen as monocytes (R3).
Plasma and cerebrospinal fluid Aβ1-42 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 quantification. 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).
Measurement of Aβ uptake-related receptors in monocytes
The staining of Aβ uptake-related receptors, including Toll‑like receptor 2 (TLR2), triggering receptor expressed on myeloid cells 2 (TREM2), CD36, CD33, and macrophage scavenger receptor 1 (SCARA1), was performed by flow cytometry. Following CD14 positive selection by magnetic activated cell sorting, 1.0×105 monocytes were preincubated with Human TruStain FcX (Biolegend, CA, USA) on ice for 20 minutes. For cell surface staining, cells were incubated with monoclonal antibodies against APC-anti human CD33, BB515-anti human CD282, Percp-CyTM5.5-anti human CD36, BV421-anti human MSR1 (Biolegend, CA, USA), and PE-anti human TREM2 for 15 minutes, washed via centrifugation twice and fixed with 1% paraformaldehyde. Cells were acquired on a FACS Navios (Beckman, CA, USA), and analyses were performed using FlowJo v10 software.
Measurement of Aβ-degradingenzymes in monocytes
Western blotting was performed as previously described. After being thawed, PBMCs were washed in 10 ml of PBS. Cells were lysed in RIPA buffer. Samples (15-30 μg) were subjected to electrophoresis on SDS-PAGE (8-12% acrylamide) gels. The blots were probed with antibodies against cathepsin D (1:1000, monoclonal, Arigobio), cathepsin S (1:1000, monoclonal, Arigobio), and β-actin (1:1000, monoclonal, Sigma-Aldrich). The protein bands were scanned using Odyssey scanner software (Li-COR Bioscience, CA, USA) and quantified by Quantity One 6.0. The band density was normalized to that of β-actin in the same sample.
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 matched-pairs test or an unpaired t test, where appropriate. Specifically, a Pearson correlation or covariate correlation analysis was utilized to analyse the association of Aβ1-42 uptake with ageing or Aβ1-42, Aβ1-40, Aβ1-42/ Aβ1-40 levels in the plasma and CSF, respectively. The trajectory of Aβ1-42 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 significance was achieved at p<0.05.
The data, analytic methods, and study materials that support the findings of this study will be available from the corresponding authors on request, after the request is submitted and formally reviewed and approved by the Ethics Committee of the Institute of Daping Hospital, Third Military Medical University, Chongqing, China.