Isolation, Transfection, and Culture of Primary Human Monocytes

Podsumowanie

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Streszczenie

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4⁺ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14⁺ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Wprowadzenie

Human immunodeficiency virus-1 (HIV-1) infection causes severe immune dysfunction, which can lead to opportunistic infections and acquired immunodeficiency syndrome (AIDS). Although HIV-infected patients undergoing cART are characterized by low viral loads and normal CD4⁺ T cell counts, functioning of the immune system can be compromised in these individuals, leading to a dysfunctional immune response that has been linked to an increased risk of developing cancer¹. The mechanisms of immune dysfunction in HIV patients on cART remain largely unknown. Therefore, characterizing patient-derived immune cells and investigating their biology and function is a critical component of current HIV research.

Monocytes and macrophages are key regulators of immune responses and play fundamental roles in HIV infection^2,3,4,5. Heterogeneous and plastic in nature, macrophages can be broadly classified into classically activated (M1) or alternatively activated (M2). While this general classification is necessary when setting up experimental conditions, the polarization status of macrophages may be reversed by a variety of cytokines^6,7,8,9. Although several studies have investigated the effects of HIV infection on monocytes and dendritic cells, molecular details of monocyte-mediated responses are largely unknown^{6,7,10,11,12,13,14,15,16,17,18,19}. Among the factors involved in immune cell regulation and function, microRNAs (miRNAs), short non-coding RNAs that post-transcriptionally regulate gene expression, have been shown to play an important role in the context of major cellular pathways (i.e., growth, differentiation, development, and apoptosis)²⁰. These molecules have been described as important regulators of transcription factors essential for dictating the functional polarization of macrophages²¹. The potential role of miRNAs in monocytes from HIV-infected individuals undergoing cART has been investigated, but progress in the field requires much more work^{22,23,24,25,26}. This paper discusses an optimized method to transfect miRNAs and siRNAs into primary human monocytes from HIV-infected patients and controls.

This protocol relies on commercially available reagents and kits, as continuity in the technical procedure helps eliminate unnecessary experimental variables when working with clinical samples. Nonetheless, the method provides important tips (i.e., the number of cells plated or brief incubation with serum-free media to promote the adherence of cells to the plate). Additionally, the polarization conditions used in this protocol are derived from published work^27,28,29.

Protokół

All methods described below have been approved by the Louisiana State University Health Sciences Center New Orleans Institutional Review Board. All blood was collected after obtaining informed consent.

NOTE: The entire procedure is performed under sterile conditions in a biosafety level 2 (BSL2) facility so that caution is used to handle biological materials. In particular, each step is performed using sterile techniques under a biosafety cabinet. After each step involving blood, blood products, cells, or cell product pipetting, it is important to rinse all plastic material (i.e., serological pipettes, pipette tips, and tubes) with 10% bleach from a waste container inside the hood prior to proper disposal.

1. Isolation of primary human monocytes by immunomagnetic negative selection

1. Collect 40 mL of fresh, human whole blood (from either an HIV⁺ patient or healthy control) in four 10 mL ethylenediaminetetraacetic acid (EDTA) vacuum tubes (10 mL of blood per tube). Using sterile techniques under a biosafety cabinet, transfer all 40 mL of blood into one 50 mL conical propylene tube.
2. Following the manufacturer's protocol for the selected human monocyte isolation kit (Table of Materials), add 2 mL of monocyte isolation cocktail, provided in the kit, to the tube of blood. Vortex magnetic beads, also provided in the kit, for 30 s, and add 2 mL to the tube of blood.
   1. If less than 40 mL of blood is available, scale down the reagents added. To mix the solution, pipette up and down with a plastic 25 mL serological pipette and incubate for 5 min at room temperature (RT).
3. Separate the blood mixture equally into four 50 mL tubes and add 30 mL of sterile phosphate-buffered saline (PBS) containing 1 mM EDTA to each tube. Mix by pipetting up and down with a plastic 25 mL serological pipette.
4. Place the tubes in magnet holders for 10 min to remove the antibody-conjugated magnetic beads. Use four magnet holders simultaneously, one for each tube, to allow consistent incubation and isolation times for each blood sample.
5. Draw up the contents from the center of each tube, using a pipette, while they are still in the magnet holders. Be careful not to draw up red blood cells (no more than 10% of the 10 mL starting volume), and place the contents into one of four new 50 mL tubes.
6. Add 500 µL of vortexed magnetic beads to each 50 mL tube. Pipette up and down with a 25 mL pipette and incubate at RT for 5 min. Then, place the tubes into magnet holders for 5 min.
7. Carefully transfer the contents from the center of each tube while still in magnet holders into one of four new 50 mL tubes. Directly place each new 50 mL tube in the magnet holders for 5 min.
8. Carefully transfer contents from the center of each tube into one of four new 50 mL tubes. Spin all new 50 mL tubes at 300 x g for 5 min. Aspirate the supernatant and resuspend all four cell pellets in a total of 10 mL of sterile PBS.
9. Count cells by trypan blue exclusion using a hemocytometer.
   NOTE: 8-20 x 10⁶ cells are generally obtained from 40 mL of whole blood.

2. Culturing of primary human monocytes

1. Using a 37 °C water bath, warm serum-free RPMI 1640 media supplemented with 1% penicillin-streptomycin (pen/strep), and (while continuing to use sterile techniques under a biosafety cabinet) resuspend the isolated monocytes in this media at a concentration of 1 x 10⁶ cells/mL.
2. Add 1 mL of resuspended cells to each well of a 6 well plate or into a 35 mm dish (the final number of cells should be 1 x 10⁶ cells/plate), and place in a 37 °C incubator with 5% CO₂. Wait 0.5-1.0 h for cells to adhere.
3. Using a 37 °C water bath, warm heat-inactivated (HI) fetal bovine serum (FBS). Add 100 µL (10% final concentration) of FBS to each plate. Add growth factors to the cells to promote macrophage differentiation.
   1. Prime macrophages for an M1-like phenotype by adding 25 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) to media. Prime macrophages for an M2-like phenotype by adding 50 ng/mL of macrophage colony-stimulating factor (M-CSF) to media²⁸.
      NOTE: Both GM-CSF and M-CSF allow monocyte differentiation to a general macrophage phenotype (M0) while priming cells for M1 or M2, respectively^7,28,30.

3. Transfecting primary human monocytes in culture

1. Transfect monocytes with miRNA mimics or inhibitors or siRNA using a kit containing a polymer-based transfection reagent (Table of Materials).
   1. Following the manufacturer's protocol for the transfection kit (and continuing the use of sterile techniques under a biosafety cabinet), first dilute the selected miRNA mimics/inhibitors or siRNAs in the buffer to a final concentration of 1.83 µM. Prepare 10 µL of diluted mimic/inhibitor per transfection of 1 x 10⁶ cells.
2. Prepare the transfection reagent by adding 1 µL of the provided polymer to a fresh 1.5 mL microcentrifuge tube, followed immediately by adding 90 µL of provided buffer (for a total of 91 µL of reagent per transfection). Vortex for 3-5 s.
3. Pipette 90 µL of transfection solution into the tube containing 10 µL of diluted miRNA mimic/inhibitor or siRNA. Mix by gentle pipetting and incubate for 15 min at RT.
4. Add 100 µL of transfection complex to one well (or dish) of 1 x 10⁶ plated monocytes. Incubate cells for 4 h at 37 °C, then replace the medium with 3 mL of complete media (RPMI 1640 supplemented with 1% penicillin-streptomycin and 10% heat-inactivated FBS) containing either GM-CSF or M-CSF.

4. M1/M2 differentiation and activation

1. Monocytes immediately begin differentiating to broad M0 macrophages upon plating in culture. On the third day after plating, continue the using sterile techniques under a biosafety cabinet and replace media with 3 mL of fresh RMPI 1640 media (supplemented with 1% penicillin-streptomycin, 10% heat-inactivated FBS, and either 25 ng/mL GM-CSF to promote M1-like polarization or 50 ng/mL M-CSF to promote M2-like polarization). Culture the cells in these conditions for a total of 6 days from initial plating in an incubator at 37 °C, 5% CO₂.
2. To advance polarization of primed cells to the M1-macrophage phenotype, activate cells on day 6 of incubation by replacing cell media with new media containing 5% heat-inactivated FBS, 1% pen/strep, 100 ng/mL E. coli-derived lipopolysaccharide (LPS), and 20 ng/mL interferon gamma (IFN-γ).
3. To advance polarization of primed cells to the M2-macrophage phenotype, activate cells on day 6 of incubation by replacing cell media with new media containing 5% heat-inactivated FBS, 1% pen/strep, 10 ng/mL M-CSF, and 20 ng/mL interleukin 4 (IL-4).
4. After 24 h, harvest the cells for RNA, protein, or flow cytometry analyses.
   1. When cells are ready for collection, wash cells in the dish 2x with PBS (at RT for RNA extraction or chilled on ice for protein extraction). Because the differentiated macrophages are now firmly attached to the plates, lyse the cells in plates directly to obtain material for RNA and protein analyses.
   2. For collection of material for flow cytometry, add PBS containing 2 mM EDTA to the dish, incubate the cells for 10 min at 37 °C, gently scrape the cells from the dish, and collect the contents in a 1.5 mL microcentrifuge tube before proceeding with standard protocols.

5. Flow cytometry

1. Rinse cells in PBS to remove the culturing medium. Spin down 100,000 cells (per each flow cytometry condition) and resuspend the pellet in 100 µL of PBS containing 2 µL of HuFcR binding inhibitor. Incubate at RT for 15 min.
2. Add 50 µL of staining buffer and the desired antibodies (here, CD80, CD83, CD163, and CD209 were used) in the recommended amounts.
3. Mix gently and incubate at 4 °C in the dark for 30 min.
4. Wash 2x with PBS and resuspend the stained cells in 150 µL of PBS before running the sample on a cytofluorimeter.

Wyniki

Using the procedure described, primary human monocytes from HIV-infected individuals and healthy donors were isolated. All data presented here were obtained from HIV⁺ subjects undergoing cART with low (<20 copies/mL) or undetectable viral loads and normal CD4⁺ T cell counts. Immediately after isolation, cells were stained, and flow cytometry was performed to confirm the purity of cell populations. Results showed that >97% of cells stained positive for CD14 (d...

Dyskusje

The presented protocol demonstrates the use of primary cells from HIV-infected subjects as a model for studying monocytes and macrophages. HIV⁺ patients undergoing cART live with infection for multiple years and can also have other co-infections related a compromised immune system. To study immunomodulation in the presence of HIV chronic infection, cells were harvested from patients directly. As miRNAs have been shown to play major roles in cell development and differentiation, the protocol focuses on the abil...

Materiały

Name	Company	Catalog Number	Comments
0.5M EDTA	Invitrogen	AM9260G
BD Vacutainer Plastic Blood Collection Tubes with K2EDTA	BD Biosciences	366643
Brilliant Stain Buffer	BD Horizon	563794	Flow cytometry
CD14 PerCP	Invitrogen	46-0149-42	Flow cytometry- conjugated antibody
CD163 BV711	BD Horizon	563889	Flow cytometry- conjugated antibody
CD209 BV421	BD Horizon	564127	Flow cytometry- conjugated antibody
CD80 FITC	BD Horizon	557226	Flow cytometry- conjugated antibody
CD83 APC	BD Horizon	551073	Flow cytometry- conjugated antibody
Easy 50 EasySep Magnet	StemCell Technologies	18002
Easy Sep Direct Human Monocyte Isolation Kit	StemCell Technologies	19669
EIF4EBP1 mAb	Cell Signaling	9644	Monoclonal antibody for Western blot
EIF4EBP1 siRNA	Santa Cruz	sc-29594
Fetal Bovin Serum Defined Heat Inactivated	Hyclone	SH30070.03HI
Gallios Flow Cytometer	Beckman Coulter	B43618
GAPDH mAb	Santa Cruz	SC-47724	Monoclonal antibody for Western blot
HuFcR Binding Inhibitor	eBiosciences	14-9161-73	Flow cytometry- blocking buffer
Kaluza Analysis Software	Beckman Coulter	B16406	Software to analyze flow cytometry data
Lipopolysaccharides from Escherichia coli O55:B5	Sigma	L4524
miRCURY LNA microRNA Mimic hsa-miR-146a-5p	Qiagen	YM00472124
MISSION miRNA Negative Control	Sigma	HMC0002	Scrambled miRNA conjugated with a near infrared dye
Nunc 35mm Cell Culture Dish	Thermo Scientific	150318
PBS	Gibco	20012027
Penicillin-Streptomycin	Gibco	15140122
Recombinant Human GM-CSF	R&D Systems	215-GM-050
Recombinant Human IFN-γ	R&D Systems	285-IF-100
Recombinant Human IL-4	R&D Systems	204-IL-010
Recombinant Human M-CSF	R&D Systems	216-MC-025
RPMI 1640 with L-Glutamine	Corning	10040CVMP
Scrambled Control siRNA	Santa Cruz	sc-37007
Viromer Blue Transfection Reagent Kit	Lipocalyx	VB-01LB-01
WST-1 Cell Proliferation Reagent	Roche	5015944001	Colorimetric assay to assess cell viability