α-Synuclein-containing erythrocytic extracellular vesicles: essential contributors to hyperactivation of monocytes in Parkinson’s disease

The study was approved by the Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University. In this study, whole blood samples from 15 patients with PD and 9 age- and sex-matched healthy controls were collected from Tiantan Hospital. All subjects underwent evaluations including medical history, physical and neurological examinations, laboratory tests, and neuropsychological assessments. Briefly, all PD patients met UK PD Society Brain Bank clinical diagnostic criteria for PD. Control subjects were in good health without any signs or symptoms suggesting cognitive impairment or neurological disease. All participants underwent detailed informed consent procedures. Demographic information is listed in Table 1 for all subjects.

All animal procedures were approved in accordance with Chinese Guidelines for the ethical review of laboratory animal welfare (LA2020056). PD A53T double transgenic homozygous mice (dbl-PAC-Tg (SNCA^A53T); Snca^−/−) and SNCA-KO α-syn homozygous knockout mice (B6; 129X1-Snca^tm1Rosl/J) were purchased from Jackson laboratory and kept on a 12-h light–dark cycle with ad libitum food and water. Age-matched wild-type mice (WT) were selected as the control group. In this study, 7-month-old, male mice were employed for the isolation of circulating monocytes and RBC-EVs.

RBC-EVs were isolated from cultured human and mouse RBCs according to a previous article with minor modifications [8]. Briefly, human and mouse RBCs were separated from whole blood and cultured in RPMI1640 culture medium containing 25 mM HEPES at 37 °C with a humidified atmosphere of 5% CO₂ for 48 h. Then, culture medium was collected and centrifuged at 1500×g for 10 min to remove RBCs, followed by filtering the supernatant through 0.22-μm filters. Subsequent filtrate was centrifuged in an ultracentrifuge (Beckmann) at 140,000 × g for 2 h to collect EVs. The collected pellets were washed with ice cold PBS and centrifuged again at 140,000×g for 2 h. Finally, EVs were resuspended and collected using ice cold PBS for further experiments.

THP-1 cells (ATCC #TIB-202) were maintained in RPMI1640 medium with 2 mM L-glutamine and 25 mM HEPES, supplemented with 10% fetal bovine serum.

For immune sensitization experiments, THP-1 cells were cultured with 200 μg RBC-EVs from different sources for 24 h (baseline) and then stimulated with 200 ng/mL bacterial lipopolysaccharide (LPS; Sigma, L2630; ≥ 500,000 Endotoxin Units/mg) for 24 h. THP-1 cells were collected for total RNA extraction and culture media were collected for inflammatory cytokines detection, at baseline and after LPS treatment.

For endocytosis inhibitor experiments, THP-1 cells were cultured with inhibitors for 4 h, followed by the addition of RBC-EVs. After 24-h incubation, 200 ng/mL LPS was applied for THP-1 stimulation for 24 h. Total RNA and culture media were collected for inflammatory cytokines detection.

Circulating monocytes in mice and human blood were isolated by peripheral blood monocytes cells separation medium (Solarbio, P5290, P5230, Beijing, China) and maintained in RPMI1640 medium with 2 mM L-glutamine and 25 mM HEPES, supplemented with 10% fetal bovine serum.

For immune sensitization experiments, circulating monocytes were cultured with 200 μg RBC-EVs from different groups for 24 h (baseline) and then stimulated with 200 ng/mL bacterial LPS for 24 h. Monocytes were collected for total RNA extraction and culture media were collected for inflammatory cytokines detection, at baseline and after LPS treatment.

RBC-EVs isolated from different species were dropped onto the grid and then negatively stained with 2% phosphotungstic acid. The morphology of RBC-EVs was acquired by transmission electron microscopy (JEM1400 PLUS).

RBC-EVs isolated from cultured WT mice were resuspended in 1 mL PBS buffer at particle concentration of 2 × 10¹⁰ /mL. Subsequently, 1 mM DiI or DiO stock solution (Coolaber, SL7910, SL7920) was added into the RBC-EV suspension at final concentration of 10 μM to label the RBC-EV for 30 min at room temperature. To remove the excess DiI or DiO dye, the labelled RBC-EV suspension was filtered thrice through Amicon® Ultra-centrifugal filter devices (cutoff MW 30 kDa, Millipore Corporation, Billerica, MA, USA). Finally, the labelled RBC-EVs were collected for further experiments or stored at –80 °C.

The particle concentration and size distribution of RBC-EVs were detected by NTA instrument (NS300; Nanosight). RBC-EVs were first diluted by filtered PBS at 1:100 to optimize the number of particles and injected into the sample sink. The movement of each particle was video captured in triplicate of 60 s. Analysis was performed by NTA 3.2 software (Nanosight, Amesbury, UK).

Total RNA was isolated from THP-1 cells and monocytes by TRIZOL™ reagent (Invitrogen, 15,596,018) according to the manufacturer's instruction. Following the extraction of RNA, cDNA was reverse transcribed from 2 mg total RNA by RT Master Mix Kit with gDNase (MCE, HY-K0511). The mRNA level of target gene in acquired cDNA was determined by real-time PCR with PowerUp SYBR® Green Master Mix (applied biosystem, A25742). ACTB was used as the internal reference for normalization. The primers used are presented in Table 2. All results were analyzed using 2^−△△Ct and were presented as mean ± SEM.

Proteins were extracted from THP-1 cells and monocytes using cell lysis buffer (RIPA, HARVEYBIO, C1503) containing protease inhibitor cocktail (Sigma) and phosphatase inhibitor PhosSTOP™ (Roche, 4,906,837,001). BCA Protein Assay Kit (Applygen, P1511) was applied to determining the protein concentration of RBC-EVs and THP-1 monocytes according to the manufacturer's instruction. A total of 20 μg RBC-EVs or 30 μg THP-1 lysates were loaded to 10% PAGE gels for electrophoresis. Separated proteins were blotted to PVDF membrane (Merck Millipore, 4515). Then the membranes were blocked in TBST buffer (Applygen, B1009) containing 5% BSA (Amresco, A-0332) for 1 h at room temperature and incubated with diluted primary antibody overnight at 4 °C. Antibodies used included Alix (1:1000, Merck, Abc40), TSG101 (1:1000, abcam, ab133586), LRRK2 (1:5000, Abcam, ab133474), p-Rab10 (1:1000, Abcam, ab230261), Rab10 (1:1000, Abcam, ab237703), and β-actin (1:1000, Zsgb-bio, TA-09). The membranes were washed three times by TBST buffer on the second day, followed by incubation with secondary antibody for 1 h at RT. Protein bands were developed by enhanced chemiluminescence reagents (Millipore, WBKLS0100) and detected by Chemi doc™ Imaging System (Bio-Rad, Hercules CA, USA).

THP-1 monocytes pre-treated with or without endocytosis inhibitors for 30 min were incubated with 2 × 10⁹ DiO-labelled RBC-EVs for another 4 h. Then, monocytes were collected and washed for analysis using BD FACS Calibur Flow Cytometer (BD Biosciences, Franklin Lakes NJ, USA) to determine the uptake of RBC-EVs in monocytes. Endocytosis inhibitors included Me-β-CD (200 μM, MCE, HY-101461), Amiloride (1 mM, MCE, HY-B0285A), Dynasore (50 μM, Selleck, S8047), and Nocodazole (40 ng/mL, MCE, HY-13520). The inhibition rate was calculated as the following equation:

2 × 10⁹ Dil labelled RBC-EVs were added into THP-1 monocytes pre-treated with or without endocytosis inhibitors for 30 min and incubated for 4 h. The cell nucleus was stained by Hoechst33258 (Solarbio) for 15 min. Images were acquired using Leica TCS SP8 confocal system.

The detection and quantification for total and aggregated α-syn were based on our previously developed and validated assay [35]. Briefly, antibody against α-syn, MJFR-1 (ab138501, Abcam, only recognizes humanized α-syn) and antibody against conformation specific α-syn filaments, MJFR-14 (ab209538, Abcam, recognizes the structure of α-syn oligomers from both mouse and human species) were, respectively, biotinylated, linker conjugated, and coated onto standard 96-well U-Plex plates (Meso scale discovery). After washing three times with 150 μl Washing Buffer, 50 µl of diluted sample (diluted by Diluent 35 at dilution ratio of 1:5) and calibrator (recombinant α-syn, Sino biological, and oligomeric α-syn, Proteos) were loaded to the immunoassay plate, which was incubated overnight at 4 °C on a shaker at 600 rpm. Next, the plate was washed three times using washing buffer followed by the addition of sulfo-TAG-labelled anti-α-syn antibody (BD42) and incubated at room temperature for 1 h on a shaker at 600 rpm. Finally, 150 μL 2 × read buffer T was added into each well and plates were analyzed in a Quickplex SQ 120 (MSD, USA). Data analysis was performed with the MSD Discovery Workbench 3.0 Data Analysis Toolbox. Total α-syn concentrations were normalized to the total protein levels in RBC-EV, and are shown in unit of pg (total α-syn)/mg (total protein). Oligomeric α-syn concentrations were normalized to the corresponding total α-syn levels, and are shown in units of pg (oligo α-syn)/mg (total protein).

The inflammatory cytokines in culture medium were detected by 10-Vplex plates (containing IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNFα, Meso scale discovery) according to the manufacturer's instruction. Plates were analyzed in a Quickplex SQ 120 (MSD, USA). Data analysis was performed with the MSD Discovery Workbench 3.0 Data Analysis Toolbox.

To investigate the possibility of hyperactivation of monocytes in PD, we started with examining the expression levels of typical inflammatory cytokines of the monocytes isolated from PD patients and healthy controls at baseline and following 24 h LPS stimulation. The purity of CD14 + monocytes was around 84% (Fig. 1A). The mRNA levels of typical pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α, were significantly higher in monocytes of PD patients than healthy controls (Fig. 1B). The protein levels of released inflammatory cytokines (including pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13) were measured by a commonly utilized MSD platform. In the absence of external stimuli, most of the detected inflammatory cytokine levels, except for IL-8, IL-2, IL-4 and IL-13, were higher in the monocytes of PD patients than healthy controls (Fig. 1C, D). To further evaluate the sensitization of monocytes in PD patients, monocytes were stimulated by LPS; following 24 h stimulation, the mRNA levels of IL1b, IL6 and TNF and the protein levels of pro-inflammatory cytokines, especially IL-1β, IL-6, TNF-α and IL-2 became significantly higher in monocytes of PD patients than healthy controls (Fig. 1B, C), indicating the hyperactivation of monocytes in PD patients. The concentration of inflammatory cytokines in monocytes after LPS stimulation was normalized to the concentration of inflammatory cytokines in monocytes at resting state. As shown in Additional file 1: Fig. S1, LPS stimulation can elicit increased production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-2) in monocytes of PD patients, which further indicated increased immune sensitization in PD. To explore the specific mechanism regulating the hyperactivation of monocytes in PD, we analyzed monocytes obtained from the dbl-PAC-Tg (SNCA^A53T) and Snca^−/− double transgenic PD A53T model mice (A53T mice) [41, 42]. The purity of CD14 + mouse monocytes was approximately 75% (Fig. 1E). Subsequently, both mRNA and protein expression levels of typical pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α, were examined in monocytes derived from A53T mice and WT mice. At resting state, the production of all three cytokines was higher in monocytes of A53T mice than WT mice (except for protein level of IL-1β). Following 24 h LPS stimulation, significant increases in all three pro-inflammatory cytokines were observed in the monocytes from A53T mice, compared with WT mice (Fig. 1F, G), indicating that monocytes were hyperactivated in A53T mice.

We previously demonstrated that α-syn containing RBC-EVs isolated from PD patients can pass through the BBB and activate the microglia [8]. To explore potential mechanisms involved in sensitizing monocytes, RBC-EVs were isolated from cultured RBCs of the A53T, SNCA knockout (SNCA-KO) and WT mice and characterized by Western blot, NTA and TEM. As shown in Fig. 2A, the typical markers of EVs, including Alix and TSG101, were enriched in RBC-EVs from A53T, SNCA-KO and WT mice. The size distribution and particle concentration of the RBC-EVs evaluated by NTA were mainly between 70 to 200 nm in diameter (Fig. 2B). Under TEM, RBC-EVs displayed a cup-shaped bilayer membrane structure (Fig. 2C). When the level of total α-syn in EVs was measured with a well-established protocol [35], the concentration of α-syn in WT and SNCA-KO RBC EVs was below detection limit, while the mean concentration of α-syn in A53T RBC-EVs was 487.67 pg/mg. Oligomeric α-syn contained in RBC-EVs was also significantly higher in A53T mice than WT and SNCA-KO mice (A53T: 324.8 pg/mg; WT: 27.35 pg/mg; SNCA-KO: 0 pg/mg).

To explore the possibility of immune regulation of A53T RBC-EVs on monocytes, THP-1, a human leukemia monocytic cell line extensively used to study monocyte functions [43], was selected initially as the experimental model. As shown in Fig. 3A, the mRNA levels of IL1b, IL6 and TNF in THP-1 cells treated with RBC-EVs derived from A53T mice were significantly elevated compared to those measured in THP-1 cells treated with vehicle control, while no significant increase existed in THP-1 cells treated with WT RBC-EVs or SNCA KO RBC-EVs. Alterations in the concentration of pro-inflammatory cytokines released in the culture media paralleled the mRNA results (Fig. 3B, blue bars), suggesting that A53T RBC-EVs significantly activated the THP-1 cells. Furthermore, after pre-treatment with RBC-EVs, the THP-1 cells were stimulated for 24 h with LPS to elicit an additional inflammatory response (Fig. 3A–C, red bars). These results suggest that α-syn might be a key mediator in the immune sensitization of monocytes. The hypothesis was further substantiated by exposing THP-1 cells to free oligomeric α-syn (Fig. 4) or monomeric α-syn (Additional file 1: Fig. S2), demonstrating that oligomeric α-syn significantly and dose-dependently hyperactivated monocytes.

To further probe the mechanisms involved in monocyte sensitization, four types of endocytosis inhibitors, specifically Me-β-CD, Amiloride, Dynasore and Nocodazole [44‐47], were used to test whether internalization of RBC-EVs is necessary. By flow cytometry, we found that the RBC-EVs entered into THP-1 cells readily but this process was significantly inhibited by a 30-min treatment of four endocytosis inhibitors to different extents, with 15.7%, 7.6%, 34.4% and 33.7% inhibition for Me-β-CD, Amiloride, Dynasore and Nocodazole, respectively (Fig. 5A–F). These results were confirmed by confocal microscopy showing that abundant DiI labelled RBC-EVs can be detected in THP-1 cells treated with blank vehicle, while DiI signal was mostly confined to the plasma membrane in Amiloride treated cells, and barely observed in THP-1 cells treated with Dynasore and Nocodazole. For Me-β-CD treatment group, slight DiI signals can be detected in THP-1 cells (Fig. 5H). Next, we analyzed the effect of the four endocytosis inhibitors on monocyte hyperactivation and found that Dynasore itself significantly induced the production of pro-inflammatory cytokines TNF-α and IL-8 (Additional file 1: Fig. S3), likely attributable to the fact that Dynasore can activate the NF-κb pathway and promote the production of some pro-inflammatory cytokines [44]. Thus, Dynasore was excluded in the next series of experiments. To test the hypothesis that endocytosis of RBC-EVs is required to induce sensitization of monocytes, we pre-treated THP-1 cells with A53T RBC-EVs alone, or co-treated with Nocodazole, Me-β-CD or Amiloride for 24 h, followed by LPS stimulation. Measurement of the cytokines released by THP-1 cells revealed that Nocodazole and Me-β-CD co-treatment significantly inhibited the elevated inflammatory response of THP-1 cells induced by A53T RBC-EVs (Fig. 5I, J). To a lesser extent, Amiloride also had an inhibitory effect on THP-1 sensitization (Additional file 1: Fig. S4). To further investigate the role of α-syn in this process, we examined endocytosed oligomeric α-syn levels in THP-1 cells. As observed in Fig. 5G, around 30 pg/ml oligomeric α-syn was detected in THP-1 cells co-treated with A53T RBC-EVs and vehicle control, while no oligomeric α-syn was detectable in THP-1 cells treated with vehicle control alone, indicating the delivery of α-syn oligomers from RBC-EVs to the THP-1 cells. Compared with A53T RBC-EVs and vehicle control co-treatment group, oligomeric α-syn was hardly detected in THP-1 cells co-treated with A53T RBC-EVs and Nocodazole or with A53T RBC-EVs and Me-β-CD, indicating that Nocodazole and Me-β-CD could largely abolish the delivery of α-syn oligomers into the THP-1 cells. Taken together, these results indicate that α-syn-containing RBC-EVs can sensitize monocytes and this process is mediated by endocytosis.

Next, we investigated whether LRRK2, a kinase enzyme known to be critical to immune regulation of PD patients, and its substrate Rab10, are involved in monocyte sensitization induced by A53T RBC-EVs. As shown in Fig. 6A, B, the levels of LRRK2 and phosphorylated Rab10 (p-Rab10) were significantly higher in A53T mouse monocytes compared to WT monocytes. THP-1 cells cultured with A53T RBC-EVs, but not WT RBC-EVs, also resulted in increased levels of LRRK2 and p-Rab10 (Fig. 6C–G). An inhibitor of LRRK2, Mli2 [48, 49], was used to determine if LRRK2 activity is essential in hyperactivation of monocytes. As observed in Fig. 6H–J, the treatment of Mli2 blocked increased mRNA levels of IL1b, IL6, and TNF and protein levels of pro-inflammatory cytokines (such as IL-1β, IL-6, IL-8 and TNF-α) in THP-1 cells treated with A53T RBC-EVs at both baseline and LPS stimulated state. These results indicate that hyperactivation of monocytes induced by A53T RBC-EVs is mediated at least in part by LRRK2 kinase activity.

To confirm the significance of the above results in human disease, RBC-EVs were isolated from cultured RBCs from 5 PD patients and 3 neurologically normal control subjects (Table 1 and Fig. 7A–C). Consistent with earlier observations [30, 31, 34, 35], total and oligomeric α-syn contained in RBC-EVs were significantly higher in PD patients compared to healthy controls (HCs) (Fig. 7D, E). In addition, consistent with A53T mouse experiments, THP-1 cells treated with the RBC-EVs derived from PD patients demonstrated significantly elevated mRNA levels of IL1b, IL6 and  TNF (Fig. 7F), and concentration of inflammatory cytokines (Fig. 7G, H) in THP-1 cells at resting state. Following 24 h LPS stimulation, the mRNA and protein levels of inflammatory cytokines further significantly increased in THP-1 cells pre-treated with PD RBC-EVs (Fig. 7F–H). In addition, LRRK2 and p-Rab10 expression were significantly increased in THP-1 cells cultured with PD RBC-EVs, while LRRK2 and p-Rab10 were only slightly increased in THP-1 cells treated with HC RBC-EVs (Fig. 7I–K). These results indicate that RBC-EVs derived from PD patients are able to induce inflammatory sensitization of monocytes, with a process likely involving LRRK2 activation.

To further confirm that PD RBC-EVs can induce the hyperactivation of monocytes, monocytes from healthy controls were isolated and treated with HC RBC-EVs or PD RBC-EVs. As shown in Fig. 8, PD RBC-EVs can significantly increase the mRNA levels of IL1b, IL6 and TNF, and the protein levels of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IFN-γ in monocytes. Following 24 h LPS stimulation, the mRNA levels of IL1b, IL6 and TNF and the protein level of pro-inflammatory cytokines, especially IL-1β, IL-6 and TNF-α, became significantly higher in monocytes pre-treated with PD RBC-EVs than HC RBC-EVs.