2.1 Parabiosis
All experiments involving mice were approved by the Laboratory Animal Welfare and Ethics Committee of Shenzhen Luohu Hospital Group (SLHG), Shenzhen, China. To exclude the influence of gender on brain Aβ deposition, 6–8 week-old female pathogen-free APP/PS1 mice and C57BL/6 wild-type (Wt) mice were used in the present study. All mice were purchased from Shanghai Model Organisms Center, Inc (SMOC, Shanghai, China). Animals acclimatized for 2–5 days in the pathogen-free animal facility of the SLHG Precision Medicine Research Institute. Mice were housed in a room with a 12- hour light-dark cycle and provided with food and water ad arbitrium. Parabiosis surgery was performed on pairs of mice after they had adapted to each other by living together in a cage for 1 month at the age of 3 months according to the procedure from a previous study 40 (Fig. 1a). Firstly, ketamine (100mg/kg), xylazine (20mg/kg), and acepromazine (3mg/kg) were used to anesthetize animals, then placed animals in a parallel orientation. A left lateral incision was made on one mouse while a right one was made on the partner mouse (from the back of the ear to the back of the thigh). The head end and tail end are threaded using 5 − 0 nylon without sutures respectively. The scapulae and femoral bone of the mice were fixed together with 4 − 0 silk sutures. Then, sutured the corresponding dorsal and ventral skin with 4 − 0 silk. After the surgery, before transferred into the husbandry area, the parabiotic mice were allowed to recover in a warm and clean environment (Fig. 1b). Prophylactic antibiotic treatment (enrofloxacin, 5mg/kg) was started 1 day prior to the surgery and continued for 2 week, and all animals accepted analgesic/anti-inflammatory treatment (acetylsalicylic acid 5mg/kg) for 2 weeks.
Parabiosis was maintained for 4 and 8 months, while age and weight-matched female Wt mice (n = 6, per group) and female Tg mice (n = 6, per group) without parabiosis were used as controls. In order to investigate the effects of short-term stimulation of high plasma Aβ1−42 levels on mononuclear macrophage. Exogenous Aβ1−42 peptide labeled with HiLyte Fluor 488 (AnaSpec, Fremont, CA, USA) 200ul of 100uM in phosphate buffered solution was intravenously injected into 6–8 week-old female Wt mice via the tail vein (n = 6) three times in a week, and the age and weight matched female Wt mice (n = 6) were used as controls. The mice in control group received the same volume of vehicle. Bioluminescent imaging was performed using the IVIS spectrum imaging system (Caliper Life-Sciences, Hopkinton, MA, USA) 1 hour (h) after tail intravenous injection (Fig. 1c).
2.2 Cell Preparation
Parabiotic animals were sacrificed at 7 and 11 month old respectively, namely after 4 and 8 months sustaining stimulation with high plasma levels of Aβ1−42, and 6–8 week-old Wt mice after tail vein with Aβ1−42 peptide one week were also sacrificed. The monocytes and myeloid derived suppressor cells (MDSCs) suspensions were obtained from mice spleen by pressing them through a 70 um Falcon cell strainer, macrophages of abdominal cavity were harvested by washing three times with 10ml of RPMI 1640 medium, and bone marrow cells (BMCs) were isolated by flushing the marrow from mice femoral and tibial bone. All the above cell suspensions were collected and rinsed, centrifuged for 10 min with RPMI 1640. After red blood cells lysis, adjusted the cells concentration to 2×106/ml by using complete medium with penicillin and streptomycin (1%; Hyclone, Logan, UT) and fetal bovine serum (10%; Sigma Aldrich, St Louis, MO), prepared for the following experiments.
2.3 The Proportions Of Mononuclear Macrophage, Mdscs, And Bmcs Using Flow Cytometry Assays
The monocytes, macrophages, pro and anti-inflammatory macrophages, MDSCs, and BMCs were incubated in 0.5ug Fc Block (cat. 51-9006315, BD Biosciences) for 15minutes (mins) at room temperature to block nonspecific responses. All stainings were performed in a dark environment at 4℃. Monocytes and MDSCs from mice spleen were incubated with the following antibodies: BB515-conjugated anti-mouse CD11b (cat. 564454, BD Pharmingen TM), PerCP-Cy5.5-conjugated anti-mouse CD115 (cat. 745906, BD Pharmingen TM), PE-conjugated anti-mouse Ly6G (cat. 560599, BD Pharmingen TM), and APC-conjugated anti-mouse Ly6C (cat. 560592, BD Pharmingen TM) for 30mins. Each monocyte and MDSC samples were stained in three duplicate tubes.
Macrophages from abdominal cavity were incubated with antibodies BB515-conjugated anti-mouse CD11b (cat. 564454, BD Pharmingen TM), BB700-conjugated anti-mouse F4/80 (cat.746070, BD Pharmingen TM), BV510-conjugated anti-mouse CD40 (cat. 745041, BD Pharmingen TM) for 30mins at 4°C to detect extracellular molecules. Then cells were permeabilized with the Fixation/Permeabilization solution Kit (cat. 554714, BD Pharmingen TM) for 20mins. The intracellular molecules were stained with antibodies BV421-conjugated anti-mouse CD206 (cat. 141717, BioLegend), APC-conjugated anti-mouse arginase-1 (Arg-1) (cat. 5868A, R&D systems), and PE-conjugated anti-mouse inducible nitric oxide synthase (iNOS) (cat. 12-5929-82, Invitrogen) for 30mins at 4°C. Flow cytometric analysis was performed by using FLOWJO 7.6.1 (Treestar).
BMCs were stained with the BD Stemflow TM mouse Hematopoietic Stem Cell (HSC) Isolation Kit (cat. 560492, BD Pharmingen TM) which containing 7-amino-actinomycin D, FITC-conjugated anti-mouse CD34, PE-conjugated anti-mouse c-Kit, PE-Cy7-conjugated anti-mouse Sca-1, and APC-conjugated anti-mouse Lineage antibody Cocktail. BV421-conjugated anti-mouse CD135 (cat. 562898, BD Pharmingen TM) and BV510-conjugated anti-mouse CD16/32 (cat. 740111, BD Pharmingen TM) were also used to stain BMCs. All the antibodies were incubated for 1h at 4℃.
2.4 Analysis Of Cytokines And Aβ Levels
Cytokines levels including IL-6 (cat. 558301), IL-12p70 (cat. 558303), TNF-α (cat. 558299), IL-10 (cat. 558300), and IL-1β (cat. 560232) were evaluated in the serum of mice levels using Cytometric Beads Array (CBA) Flex Set (BD-Pharmingen, La Jolla, CA, USA). Briefly, mice soluble protein flex set capture beads were mixed and washed with 0.5ml wash buffer and centrifuged at 200g for 5mins. The supernatant was carefully discarded by aspiration without disturbing the bead pellet. The beads were then resuspended in capture bead diluent to a final concentration of 50ul/test and incubated for 15mins at room temperature. Afterward, 50ul capture beads were mixed with 50ul sample or standard and incubated at room temperature for 1h. Subsequently, 50ul mixed phycoerythrin (PE) detection reagent was added to each assay tube and incubated at room temperature for 2h. Next, 1ml wash buffer was added to each assay tube and centrifuged at 270g for 5mins. After carefully aspirating and discarding the supernatant from each assay tube, 300ul buffer was added to each assay tube to resuspend the beads. Samples were analyzed by flow cytometry (excitation wavelength 565nm and emission wavelength 578nm) and the data was acquired. Data were analyzed using FCAP Array software.
Plasma levels of Aβ1−42 (cat. CEA947Hu, Cloud-Clone Corp, Wuhan, China) and inflammatory cytokines transforming growth factor-β (TGF-β) (cat. SEA124Mu, Cloud-Clone Corp, Wuhan, China) were measured by enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. The minimum detectable dose of hAβ42 kit was typically 1.44 pg/mL and the minimum detectable dose of TGF-β kit was typically 15.6 pg/mL. The plasma hAβ42 levels of Tg mice and paWt(Wt-Tg) mice were run with 1:2 serial dilutions.
2.5 Analysis Of Peritoneal Macrophage Phagocytosis Assays
1×105 macrophages from abdominal cavity were plated in 24-well plates incubated with HiLyte Fluor 488-labeled Aβ1−42 (cat. AS-60479, AnaSpec, Fremont, CA, USA) 1ug/mL for 24h. After incubation, the macrophages washed with phosphate buffer saline (PBS) to remove excess Aβ1−42 and stained with APC-Cy7-conjugated anti-mouse CD11b (cat. 557657, BD Pharmingen TM) and BB700-conjugated anti-mouse F4/80 (cat. 746070, BD Pharmingen TM) for 30mins at 4℃. Flow cytometric analysis was performed by using FLOWJO 7.6.1 (Treestar).
2.6 Analysis Of Proliferative Function Of Bmc
BMC proliferation was measured by CCK-8 kits (cat. FC101, TransDetect, Beijing, China). A total of 1×104 cells in a volume of 100uL per well were cultured in a 96-well plate medium containing 10% FBS for 2, 5, 6, and 7 days respectively and each sample had three replicate wells. The CCK-8 reagent (10uL) was added to 90uL RPMI 1640 to generate a working solution, of which 100uL was added per well and incubated for 4h. The optical density (OD) was measured at 450nm.
2.7 Aβ Deposition And Bone Marrow-derived Macrophages In Mice Brain Using Immunohistochemical And Immunofluorescence Assays
The brain slices were deparaffinized and dehydrated with xylene and ethanol. Tissues were then incubated in 3% H2O2 for 5–10 mins to eliminate endogenous peroxidase activity. The tissues were incubated with primary antibody working solution and incubated at 4 ℃ overnight. We used 6E10 (cat. 803007, biolgend) to label Aβ and IBA-1+ (cat. 019-19741, WAKO), CD68+ (cat. MCA1957T, biorad), P2Y12− (cat. NBP2-33870, novus) to label BMDMs. After blocking in PBS containing 30% BSA for 30mins at room temperature. Next HRP conjugated secondary antibody was added and incubated for 2h at room temperature. Finally, the samples were fixed with PBS containing 10% DAPI for 2mins. The samples were observed using a confocal microscope (Zeiss LSM 800, Carl Zeiss Microimaging Inc., NY, USA) under a 20 X objective.
2.8 Statistical Analysis
Differences among groups were tested by the rank-sum test. Differences of categorical data were tested by Chi-square. For all statistical tests, two-sided P-values less than 0.05 were defined as statistically significant. All analyses were carried out using Statistical Package for Social Sciences (SPSS) version 23.0 software (IBM, West Grove, PA, USA).