Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

Podsumowanie

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Streszczenie

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

Wprowadzenie

Since the discovery of the blood-brain barrier by Paul Erhlich in the late 19^th century, the brain has been considered virtually separated from the other organs and the bloodstream. Yet, this last decade has seen the emergence of the concept that brain function is shaped by various biological factors, such as gut microbiota and systemic immune cells and signals^1,2,3,4. In parallel, other brain borders such as meninges and choroid plexuses (CP) have been identified as interfaces of active immune-brain cross talk rather than inert barrier tissues^5,6,7,8.

The CP constitute the blood-cerebrospinal fluid barrier, one of the borders separating the brain and the periphery. They are located in each of the four ventricles of the brain, i.e., the third, the fourth, and both lateral ventricles, and are adjacent to areas involved in neurogenesis such as the subventricular zone and subgranular zone of the hippocampus³. Structurally, the CP are composed of a network of fenestrated blood capillaries enclosed by a monolayer of epithelial cells, which are interconnected by tight and adherens junctions^9,10. Major physiological roles of the CP epithelium involve the production of cerebrospinal fluid, which flushes the brain from waste metabolites and protein aggregates, and the production and controlled blood-to-brain passage of various signaling molecules including hormones and neurotrophic factors^11,12,13. Secreted molecules from CP shape brain's activity, i.e., by modulating neurogenesis and microglial function^{14,15,16,17,18,19}, which makes CP crucial for brain homeostasis. CP also engage in various immune activities; whereas the main immune cell type in the brain parenchyma under non-pathological conditions is microglia, the diversity of CP immune cell populations is as broad as in peripheral organs^3,7, suggesting that various channels of immune regulation and signaling are at work at the CP.

The space between the endothelial and epithelial cells, the CP stroma, is mainly populated by border-associated macrophages (BAM), which express pro-inflammatory cytokines and molecules related to antigen presentation in response to inflammatory signals³. Another subtype of macrophages, Kolmer's epiplexus cells, are present on the apical surface of the CP epithelium²⁰. CP stroma is also a niche for dendritic cells, B cells, mast cells, basophils, neutrophils, innate lymphoid cells, and T cells which are mostly effector memory T cells able to recognize central nervous system antigens^{7,21,22,23,24}. In addition, the composition and activity of immune cell populations at the CP changes upon systemic or brain perturbation, for example, during aging^{10,14,15,21,25}, microbiota perturbation⁷, stress²⁶, and disease^27,28. Notably, these changes were suggested to indirectly shape brain function, i.e., a shift of CP CD4⁺ T cells towards Th2 inflammation occurs in brain aging and triggers immune signaling from the CP that may shape aging-associated cognitive decline^{14,15,21,25,29}. Illuminating the properties of the CP immune cells would thus be crucial to better understand their regulatory function on CP epithelium physiology and secretion and thereby decipher their indirect impact on brain function under healthy and disease conditions.

CP are small structures that contain only a few immune cells. Their isolation requires microdissection after a preliminary step of perfusion; immune cells in the bloodstream would otherwise constitute major contaminants. This protocol aims to characterize the myeloid and T cell subsets of the CP using flow cytometry. This method identifies about 90% of the immune cell populations that compose mouse CP under non-inflammatory conditions, in accordance with recently published works using other methods to dissect immune CP heterogeneity^7,10,28. This protocol could be applied to characterize changes in the CP immune cell compartment with disease and other experimental paradigms in vivo.

Protokół

All the procedures agreed with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC. They were approved by the ethical committees No. 59, by the CETEA/CEEA No. 089, under the number dap210067 and APAFIS #32382-2021070917055505 v1.

1. Preparation of the materials

1. Store all antibodies (Table of Materials) at 4 °C, protected from light exposure.
2. DAPI stock solution (1 mg/mL): Resuspend the powder in PBS^-/- (Table of Materials), aliquot, and store at -20 °C.
3. DAPI working solution (0.1 mg/mL): Dilute DAPI stock solution with PBS^-/- at a ratio of 1:10.
4. Magnetic-activated cell sorting (MACS) buffer: Prepare 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) (Table of Materials) in PBS^-/-.
5. Collagenase IV stock solution (20 U/μL): Resuspend the powder in PBS^+/+ (Table of Materials), aliquot, and store at -20 °C.
   NOTE: Collagenase IV requires MgCl₂ to be fully active; do not freeze-thaw Collagenase IV solution.
6. Anesthetic-analgesic solution: Mix 150 μL of ketamine (150 mg/kg), 25 μl of xylazine (5 mg/kg), and 330 μl of buprecare (0.1 mg/kg) in 1 mL of PBS^-/-.
   NOTE: Prepare anesthetic-analgesic solution extemporaneously and do not keep it longer than a day.
7. Heparin solution (100 U/mL): Resuspend the powder in PBS^-/-.
8. Prepare the infusion inset system connecting a tube to a decanter Erlenmeyer filled with PBS^-/-. Connect a 23 G needle to the extremity of the infusion tube. Open the infusion inset and let the PBS run until there are no bubbles in the tube.
9. Prepare the binocular loupe equipped with a light.

2. Housing of C57BL/6 mice

1. For the analysis of CP myeloid or T cells, use four mice for each and pool the four CP (two from each lateral ventricle, the third ventricle and the fourth ventricle CP; for eight mice in total). Not pooling CP carries the risk of failing to detect the rare immune populations in CP due to their low abundance.
2. Let the mice acclimatize for at least 7 days prior to any experimentation. Keep the mice under pathogen-free conditions at constant temperature and humidity, in a 12/12 h or 14/10 h light/dark cycle, with water and standard pellet food ad libitum.

3. PBS perfusion and brain dissection

1. Weight each mouse and inject 10 μL/g of the anesthetic-analgesic mix solution (step 1.6) intraperitoneally.
2. Wait around 30 min for efficient analgesia (check for the depth of anesthesia by pinching the mouse's digits).
3. Position the anesthetized mouse flat on its back, on the dissection support, and tape its palms to the dissection support.
4. Pinching the skin of the animal with forceps and using scissors, open the abdomen, the diaphragm, and the thorax to expose the heart.
   NOTE: When the thorax is opened, the brain becomes anoxic. One must proceed precisely and swiftly through the next steps of the perfusion.
5. Inject 20 μL of 100 U/mL heparin solution directly into the left ventricle.
6. With fine scissors, make an incision of at least 3 mm in the right atrium to allow the blood to flow out of the body.
7. Immediately after, insert the 23 G needle placed at the extremity of the infusion tube through the tip of the left ventricle.
8. Open the infusion system at the maximum and wait for complete perfusion: Using a 23 G needle, at a flow rate of about 6 mL/min, the perfusion is complete in 3 min.
   NOTE: The even discoloration of organs such as the liver attests for perfusion efficacy.
9. Close the infusion system and remove the needle from the heart ventricle.
10. Remove the tape from the mouse's palms. Place the mouse in a ventral position.
11. Pinching the skin of the animal with forceps and using scissors, remove the skin of the top of the head from the eyes to the ears.
12. With the help of scissors, cut the skull first between the eyes and then laterally from each eye to the spinal cord just above the masseter muscles. For this, use the extremity of scissors and proceed gently to avoid damaging the brain with the scissors.
13. Open the skull with forceps by pinching it from the extremity between the eyes.
14. Use scissors to cut the spinal cord and extract the brain with forceps, placing their two points on the lateral side of the brain to tilt it and place it in a Petri dish filled with ice-cold PBS^+/+. The CP are then immediately collected.
    NOTE: Check for discoloration of the brain to verify perfusion efficacy.

4. Dissection of the Choroid Plexus from the brain

1. Position the brain dorsal side up in the Petri dish and below the objectives of the binocular loupes.
2. With the help of forceps to maintain the brain in place, insert the two ends of another forceps down through the midline between the hemispheres.
3. Use the forceps to pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle.
4. Identify the lateral CP as a long veil lining the lateral ventricle that is flaring at both ends. Use the two ends of a thin forceps to catch the lateral CP. Be careful to collect the triangular posterior part that may be hidden by the posterior fold of the hippocampus.
5. Pull the cortex with the corpus callosum and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle.
6. Collect with fine forceps the third CP, which can be identified as a short structure with a granular surface aspect.
7. Collect the other lateral CP.
8. Insert the two ends of a forceps down between the cerebellum and midbrain. Detach the cerebellum from the pons and medulla to expose the fourth ventricle.
9. Identify the fourth CP as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and the medulla. Collect the fourth CP with fine forceps.
   NOTE: Silane-base coating of forceps may prevent stickiness of CP on forceps.
10. Repeat steps 3.1-4.9 for each of the other seven mice. In the meantime, the collected CP can be kept temporally in a tube placed on ice.

5. Preparation of samples for flow cytometry analysis

1. Fill the tube containing all dissected CP to 750 μL of PBS^+/+.
   NOTE: The deposition of dissected CP with thin forceps in the tube brings a small volume of PBS^+/+. Rather complete the existing volume to 750 μL than remove and replace it with fresh 750 μL of PBS^+/+ to avoid a possible loss of the CP tissue.
2. Add 15 μL of 20 U/μL Collagenase IV stock solution (see step 1.5) for a 400 U/mL final concentration.
   NOTE: DNAse I (150 μg/mL) may prevent excessive cell clumping.
3. Incubate CP with Collagenase IV at 37 °C under mild agitation (300 RPM) for 45 min.
4. Gently pipette up and down around 10 times to finalize the CP dissociation.
5. Fill CP tube to 1.5 mL with MACS buffer (see recipe step 1.4) to stop collagenase IV activity.
   NOTE: Collagenase IV activity is stopped at low temperature and inhibited by serum albumin.
6. Centrifuge the cells at 500 x g, for 5 min, at 4 °C. Discard the supernatant and wash the cells with 1.5 mL of MACS buffer.
7. Centrifuge the cells at 500 x g for 5 min, at 4 °C. Discard the supernatant and resuspend cells in 220 μL of MACS buffer.
8. Separate an aliquot of 10 μL from the cell suspension to be used as unstained control (next step 5.18).
9. Separate an aliquot of 10 μL from the cell suspension to be used as DAPI-mono-stained control (next step 5.12).
10. Preincubate the remaining 200 μL with anti-mouse CD16/CD32 blocking antibody (1:100) for 20 min, at 4 °C, to block nonspecific Fc-mediated interactions.
11. Split the cells into two 100 μL tubes (one for myeloid cells analysis, the other for T cells analysis) (next step 5.14).
12. Take the sample with 10 μL of cells for the DAPI-mono-stained control (step 5.9) and fill it up to 100 μL with MACS buffer (next step 5.14).
13. Prepare eleven new tubes containing 100 μL of MACS for the antibody mono-stained (step 5.14.4) and all-stained controls (step 5.14.5) and add a drop of compensation beads into each.
    NOTE: The compensation controls will allow setting up the voltage of fluorescent detection laser and assess the potential unspecific signal detection of fluorescent dye in the other channels that will be further compensated.
14. Antibody incubation of samples:
    1. Myeloid sample: Add 0.1 mg/mL DAPI (1:100) (see step 1.3), FITC anti-mouse CD45 (1:100), PE-Dazzle 594 anti-mouse CD11b (1:100), APC anti-mouse CX₃CR1 (1:100), BV711 anti-mouse Ly6C (1:100), PE anti-mouse F4/80 (1:100), and APC-Cy7 anti-mouse IA-IE (1:100).
    2. T cells sample: Add 0.1 mg/mL DAPI (1:100), FITC anti-mouse CD45 (1:100), PE-Dazzle 594 anti-mouse CD11b (1:100), APC anti-mouse TCRβ (1:100), PE anti-mouse CD8a (1:100), and APC-Cy7 anti-mouse CD4 (1:50).
    3. DAPI-mono-stained control (from step 5.12): Add 0.1 mg/mL DAPI (1:100).
    4. Antibody mono-stained compensation beads: Add each of the nine previously used antibodies for myeloid and T cells samples (same dilution), separately (one for each tube) to the nine tubes containing the beads.
       NOTE: The mono-stained compensation controls will allow the determination of positive peaks of fluorescence for each of the used fluorescent markers during the compensation step. The analyzer will compare them to the negative signal observed in the unstained control CP cells.
    5. Antibody all-stained compensation beads: Add all the antibodies used for myeloid staining to one tube and those used for T cells staining to another.
       NOTE: The all-stained compensation controls will allow setting up the voltage of each fluorescent laser detection and assess the potential unspecific signal detection of fluorescent dye in the other channels.
    6. Incubate for 30-45 min on ice, protected from light exposure.
15. Fill all tubes up to 1.5 mL with MACS buffer. Centrifuge the cells and compensation beads at 500 x g, for 5 min, at 4 °C.
16. Discard the supernatant and wash the cells and compensation beads in 1.5 mL of MACS buffer.
17. Centrifuge the cells and compensation beads at 500 x g, for 5 min, at 4 °C. Discard the supernatant.
18. Resuspend the cells and compensation beads in 500 μL of MACS buffer. Fill unstained control (step 5.8) up to 500 μL with MACS buffer.
19. Filter each tube with CP cells through a 70 μm strainer (unstained CP, DAPI mono-stained control, and myeloid and T cells samples).

6. Flow cytometry

NOTE: The flow cytometer used in this protocol is equipped with the following 5 lasers: a 355 nm UV laser, a 405 nm Violet laser, a 488 nm Blue laser, a 561 nm Yellow-Green laser and a 637 nm Red laser.

1. Perform the daily quality control checks on the cytometer using the cytometer setup, tracking (CST) interface, and the CST control beads to ensure consistency between analyses, instrument quality, and consistent target fluorescence intensities.
2. To set up the flow cytometry experiment, create a new experiment with bivariate plots and histograms. Ensure inclusion of a forward scatter area (FSC-A) and side scatter area (SSC-A) plots as well as histogram plots for each color to monitor the acquisition.
3. Define the cell morphology by setting up the PMT voltage for FSC-A and SSC-A parameters on unstained control CP cells.
4. Define the voltages of each fluorescent marker by setting up the negative peaks of the fluorochromes on unstained control CP cells and the positive peaks of the fluorochromes using compensation beads stained with all antibodies and ensuring that the detector signals are not off the scale and are not too strongly cross-detected in other channels.
5. Create a compensation matrix to analyze each of the single-color compensation controls. After gating on appropriate FSC-A/SSC-A populations, check single-stained controls on histogram plots and record compensation controls. After recording all single-stained samples, calculate the compensation matrix.
6. Record all events detected for both myeloid and T cell populations.

7. CP myeloid cells gating

1. Select FSC-A vs. SSC-A and gate for cells (based on size).
2. Create a daughter gate for single cells with FSC-A vs. forward scatter height (FSC-H).
3. Create a daughter gate for live cells with DAPI vs. FSC-A to exclude DAPI⁺ cells.
4. Create a daughter gate for CD45⁺ immune cells with CD45 vs. FSC-A.
5. Create a daughter gate from the CD45⁺ cells and select CD11b vs. F4/80, identify the following two groups: the CD11b⁺ F4/80⁺ and the CD11b⁺ F4/80^- populations (next step 7.8).
6. Create a daughter gate for CD11b⁺ F4/80⁺ cells and select CX₃CR1 vs. IA-IE. Identify both CX₃CR1⁺ IA-IE⁺ BAM that are further denominated as CD11b⁺ F4/80^high CX₃CR1⁺ IA-IE⁺ BAM, and CX₃CR1⁺ IA-IE^- macrophages.
7. Create a daughter gate for CD11b⁺ F4/80⁺ CX₃CR1⁺ IA-IE^- macrophages and select F4/80 vs. CD11b. Identify both the CD11b^high F4/80^intermediate and the CD11b⁺ F4/80^high populations that are further called CD11b^high F4/80^intermediate CX₃CR1⁺ IA-IE^- Kolmer's epiplexus macrophages and CD11b⁺ F4/80^high CX₃CR1⁺ IA-IE^- BAM, respectively.
   NOTE: CD11b^high F4/80^intermediate CX₃CR1⁺ IA-IE^- and CD11b⁺ F4/80^high CX₃CR1⁺ IA-IE^- cells appeared a bit intermixed between F4/80^intermediate-F4/80^high and CD11b^high-CD11b⁺ levels.
8. From the CD11b⁺ F4/80^- population (step 7.5), gate for Ly6C vs. IA-IE. Select both IA-IE⁺ Ly6C^- population and IA-IE^- Ly6C⁺ cells that mainly correspond to monocytes/neutrophils.
   NOTE: A high presence of IA-IE^- Ly6C⁺ cells likely reflects problems in the perfusion efficacy of animals without an inflammatory condition; their abundance should be low.
9. Create a daughter gate for CD11b⁺ F4/80^- IA-IE⁺ Ly6C^- population and select CD11b vs. CX₃CR1, identify both CD11b^high CX₃CR1^low and CD11b⁺ CX₃CR1^- populations.

8. CP T cells gating

1. Select FSC-A vs. SSC-A and gate for cells (based on size).
2. Create a daughter gate for single cells with FSC-A vs. forward scatter height (FSC-H).
3. Create a daughter gate for live cells with DAPI vs. FSC-A to exclude DAPI⁺ cells.
4. Create a daughter gate for CD45⁺ immune cells with CD45 vs. FSC-A.
5. Create a daughter gate from the CD45⁺ cells and select TCRβ vs. CD11b. Exclude CD11b⁺ myeloid cells and select TCRβ⁺ CD11b^- T cells population.
6. Create a daughter gate for TCRβ⁺ CD11b^- population and select CD4 vs. CD8a. Identify both CD8^- CD4⁺ T cells and CD8⁺ CD4^- T cells.

Wyniki

The flow cytometry analyses presented here successfully revealed the major subsets of myeloid and T cells (Figure 1 and Figure 2, respectively), and their relative total number per mouse in a highly reproducible manner (Figure 3).

The flow cytometry analysis of myeloid cells showed that CP are populated by CD11b⁺ CX₃CR1⁺ F4/80^high BAM, representing almost 80% of...

Dyskusje

Studies aiming to understand the immunological contributions to brain homeostasis and disease have mainly focused on cells residing within the brain parenchyma, neglecting brain borders such as CP, which are nevertheless crucial contributors to brain function^2,3. The analysis of immune cell populations at CP is challenging due to the small size of CP, low numbers of resident immune cells, and complicated access to this tissue. Flow cytometry performed on total br...

Materiały

Name	Company	Catalog Number	Comments
anti-mouse CD16/CD32	BD Biosciences	553142	Flow cytometry antibody
Albumin, bovine	MP Biomedicals	160069	Blocking reagent
APC anti-mouse CX3CR1	BioLegend	149008	Flow cytometry antibody
APC anti-mouse TCRb	BioLegend	109212	Flow cytometry antibody
APC-Cy7 anti-mouse CD4	BioLegend	100414	Flow cytometry antibody
APC-Cy7 anti-mouse IA-IE	BioLegend	107628	Flow cytometry antibody
BD FACSymphony A5 Cell Analyzer	BD Biosciences		Flow cytometry analyzer
BV711 anti-mouse Ly6C	BioLegend	128037	Flow cytometry antibody
Collagenase IV	Gibco	17104-019	Enzyme to dissociate CP tissue
DAPI	Thermo Scientific	62248	Live/dead marker
EDTA			Ion chelator
fine scissors	FST	14058-11	Dissection tool
FITC anti-mouse CD45	BioLegend	103108	Flow cytometry antibody
Flow controller infusion inset	CareFusion	RG-3-C	Blood perfusion inset
FlowJo software	BD Biosciences		Analysis software
forceps	FST	11018-12	Dissection tool
Heparin	Sigma-Aldrich	H3149-10KU	Anticoagulant
Imalgene	Boehringer Ingelheim		Ketamine, anesthesic
OneComp eBeads	Invitrogen	01-1111-42	Control beads to realize compensation
PBS-/-	Gibco	14190-094	Buffer
PBS+/+	Gibco	14040-091	Buffer
PE anti-mouse CD8a	BioLegend	100708	Flow cytometry antibody
PE anti-mouse F4/80	BioLegend	123110	Flow cytometry antibody
PE-Dazzle 594 anti-mouse CD11b	BioLegend	101256	Flow cytometry antibody
Rompun	Bayer		Xylazine, anesthesic
thin forceps	Dumoxel Biology	11242-40	Dissection tool
Vetergesic	Ceva		Buprenorphin, analgesic