Studying activity of peripheral blood monocytes and adiponectin receptors expression in association with plasma adiponectin in patients with Alzheimer’s disease

Project: Monitored by Research Administration

Project Details

Grant Program

Collaborative Research Grants Program 2020-2022

Project Description

In the past ten years considerable information has been accumulated on the importance of adiponectin activity (APN) in the pathogenesis of Alzheimer’s disease (AD). However, the published data are controversial, and the role of adiponectin in the etiology of AD is not fully understood. Therefore, more studies are needed to reveal an association between plasma adiponectin leveland AD. Further insights into the various mechanisms through which APN is involved in AD pathology may contribute to deeper understanding of AD pathogenesis and provide clues for development of novel biomarkers and treatment strategies. The overall goal of the proposed research is studying expression of adiponectin receptors AdipoR1 and AdipoR2 on the surface of peripheral blood monocytes, their M1/M2 polarization phenotypes, anti-inflammatory response and phagocytic activity in association with plasmaadiponectin levels in patients with Alzheimer’s disease (AD) in comparison with age- and sex- matched healthy individuals.Specific aims:1. Study of AdipoR1 and AdipoR2 expression levels on the surface of peripheral blood monocytes collected from the patients with AD and age- and sex- matched healthy individuals in association with plasma adiponectin levels.2. Study of the M1/M2 polarization phenotypes of blood monocytes collected from the patients with AD and age- and sex- matched healthy individuals in association with plasma adiponectin levels.3. Study of pro-inflammatory and anti-inflammatory cytokine release by activated blood monocytes collected from the patients with AD and age- and sex- matched healthy individuals in association with plasma adiponectin levels.4. Study the effects of adiponectin treatment on phagocytic activity of Аβ42 in peripheral blood monocytes collected from the patients with AD and age- and sex- matched healthy individuals. The long-range goal of the proposed research is to provide novel information on some mechanisms through which adiponectin and peripheral blood monocytes could be involved in AD. These finding will contribute to deeper understanding of AD pathogenesis and may serve as a basis for the development of prognostic biomarkers of Alzheimer's disease.

Project Impact

Main Results of the project for 2020:1.We initiated recruitment process of the project participants, determined study inclusion and exclusion criteria, recruited a group of 70 individuals with Alzheimer's disease, and selected a control group of 119 healthy individuals without cognitive impairment. 2.Venous blood samples were taken from all recruited individuals, samples were delivered to the laboratory for further processing.3.Serum adiponectin concentration in blood samples of the patients diagnosed with Alzheimer’s disease and healthy age-and sex matched individuals was determined and analyzed. 4.PBMC (peripheral blood mononuclear cells) were isolated from the blood samples of the 70 study participants; monocytes purification and differentiation into macrophages protocol was developed. 5.The expression level of AdipoR1 and AdipoR2 receptors assay protocols were testedHealthy individuals and AD patients recruitmentThe recruiting of participants for the project was carried out with the participation of neurologists, therapists of inpatient and outpatient medical institutions in Nur-Sultan and Almaty city. A differential diagnosis of Alzheimer's disease was made based on the results of the short scale of neuropsychiatric status, the clock drawing test and the assessment of neurological status according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) and the criteria of the National Institute of Neurological and Communication Disorders, Stroke, Alzheimer's disease and other related disorders (NINCDS-ADRDA). For the control group we used blood samples from our biobank, collected in previous years. Collection of blood samples During the first interview at the clinic, the study participants were sampled fasting venous blood by qualified medical personnel in disposable plastic vacuum tubes with K2-EDTA (purple cap, 10 ml) and coagulation activator gel (yellow cap, 8 ml). The procedure for collecting blood from a vein in the arm does not pose a risk and is routine, and the collected blood volume from the study participants corresponds to the volume collected during routine outpatient tests (no more than 20 ml). To prepare serum, blood samples were left to clot at room temperature for 30 min, then centrifuged for 10 min at 4000 rpm. The obtained serum was prepared in 0.5 ml aliquots, which were stored at - 86 ° C until further analysis.Determination of serum adiponectin concentration in blood samplesImmunoenzymatic analysis of serum adiponectin was carried out using Human ELISA Kit (RAB0005, SIGMA) according to manufacturer’s protocol. The final comparative analysis for plasma adiponectin measurement included 58 people with AD and 119 people in the control group (62% of people in the AD group were women, while 58% were women in the control group) . The median serum adiponectin level was tree times higher in AD compared to cognitively normal controls (p<0.001). PBMC (peripheral blood mononuclear cells) isolation and monocytes purificationPeripheral blood mononuclear cells were isolated from 70 blood samples of the study participants. PBMC were collected from blood by Ficoll separation. Before freezing or further manipulations cell viability were measured using Bio-Rad TC20 cell counter. It was observed that cell viability remained higher than 90%. In order to isolate monocytes from PBMC cells were resuspended in RPMI (containing 10% FBS) and seeded on chambered coverslips. Cells were allowed to adhere, after 1-day non-adherent cells were washed. Coverslips with purified primary human monocytes were cultured in RPMI-1640 medium containing 10%FBS and 1% p/s for 6 days. To differentiate monocytes cell medium was supplemented with 50 ng/ml M-CSF. On the day 7 cells had a typical macrophage-like morphology The expression level of AdipoR1 and AdipoR2 receptors assay (Natalie Barteneva)Preliminary experiments of AdipoR1 and AdipoR2 receptors evaluation in PBMC from healthy donors were performed using a conventional 5-laser flow cytometer FACSAria (BD Biosciences, USA) and 4-laser imaging cytometer Imagestream X Mark II system (Amnis-Luminex, USA). Shortly, antibody titration was done and working concentrations were quantitated on conventional flow cytometer for CD45, CD3, CD4, CD56, CD14 antibodies. Then similar parameters were used and modified for Imagestream X Mark II instrument. Firstly, single cell population was gated from PBMC cells using Area/Aspect ratio parameters (BrightField). During next step, we identified focused cells inside single cell population using Rmax Gradient parameter. Furthermore, we designed 4-5-color panels for the evaluation of different PBMC subpopulations by imaging and flow cytometers from available sets of antibodies (Alexa 488, PE-Cy5.5, PE-Cy-7, Alexa 647 etc.). Using single staining controls we optimized spectral overlap from different fluorochromes. Furthermore, a preliminary staining protocol with AdipoR1 and AdipoR2 receptors was developed.
Main Results of the project for 2021:1.      We have finished recruitment process of the project participants. Overall, we have recruited a group of 91 individuals with Alzheimer's disease (including 58 participants in 2020), and a control group of 119 healthy individuals without cognitive impairment (including 58 participants in 2020). All study participants (and/or their guardians) were informed by the coordinator about the purpose of the research project and gave written informed consent to participate in the study. During interviews at the clinic, the study participants were sampled fasting venous blood. Mononuclear mass was isolated and purified from peripheral blood of all participants. Fasting blood samples were taken from all cases and controls for biochemical measurements and were analyzed for routine biochemical parameters immediately after collection.

2.      The expression levels of AdipoR1 and AdipoR2 receptors in the blood of the study participants were measured. We have analyzed the expression levels of AdipoR1 and AdipoR2 receptors in peripheral blood mononuclear cells (PBMC) isolated from 14 AD patients and 12 healthy controls using ELISA kits (MyBiosource, Vancouver, Canada). We observed the median level of ADIPOR1 14.0 (7.87-17.1) ng/ml in the AD patients and 3.61 (2.86 -13.8) in the control. Wilcoxon rank-sum test revealed a statistically significant difference between groups based on a p-value of 0.05 (p= 0.01088). ADIPOR2 levels were not different between study groups (p= 0.5604). The median for ADIPOR2 in AD patients was 9.98 (6.60-12.4) ng/ml and 9.04 (5.83-11.9) in the controls.

3.      Determination of M1/M2 phenotype of isolated monocytes protocols were tested (reported  separately by prof. Barteneva)

4.      The expression levels of pro-inflammatory and anti-inflammatory cytokines in blood samples were assessed.  We simultaneously assayed cytokines in the serum of our AD patients and normal controls using MILLIPLEX Map Human Cytokine/Chemokine Magnetic Beas Panel on the Luminex xMAP technology. The kit included the following 41 human cytokines and chemokines: EGF, IL-12P70,  IL-13, IL-15, IL-17A, IL1RA, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, VEGF, FGF-2, TGF-α, FIT-3L, Fractalkine, GRO, MCP-3, MDC, PDGF-AA, PDGF-AB/BB, sCD40L, IL-1α, IL-1β, TNFα, G-CSF, IFNγ, TNFβ,  IL-10, IL-2, GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, Eotaxin, IFNα2,  and IL-12P40. The levels of IL-12P40, IL-9, IL-7, GM-CSF, IL-10, IFNγ, IL-1α, MCP-3, FIT-3L, IL-17A, IL-13, IL-15, and IL-12P70 were significantly higher in Alzheimer’s patients compared to controls. At the same time, cognitively healthy controls had higher concentrations of PDGF-AB/BB, PDGF-AA, RANTES, and FGF-2 in the peripheral blood. There were significant positive correlations between serum adiponectin level and the following cytokines/chemokines: GM-CSF, IL-2, IL-13, IL-12p70, IL-15, IL-3, and negative correlations with SCD40L, EGF, GRO, MCP-1, MDC, IL-5, FGF-2, TGF-α, IL1RA.

5.      The preliminary results were presented online at 2021 Alzheimer's Association International Conference (July 26-31, 2021, Denver, USA) and the international scientific and practical online conference "Priority directions of development of science and education", dedicated to the 30th anniversary of Kazakhstan's independence (December 7, 2021, Chimkent, Kazakhstan).

StatusActive
Effective start/end date1/1/2012/31/22

Keywords

  • Alzheimers disease
  • Adiponectin
  • Monocytes

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