Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

Abstract

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Introduction

Tissue-resident macrophages (Mφ) are a heterogeneous population of phagocytic immune cells that sense and respond to invading pathogens^1,2. In addition, they play an essential role in tissue development, remodeling, and maintaining homeostasis^1,3. Many tissue Mφ derive from yolk sac progenitors during embryogenesis and persist in the tissue throughout the life^4,5. The phenotype and functions of these cells are dictated by collaborative and hierarchical interactions of specific transcription factors and the local microenvironment^6,7,8,9. A growing understanding of this dependency increases the need for effective in vivo methods for gene manipulation of Mφ within their physiologically relevant niche.

Lentiviral vectors are a frequently employed tool for the manipulation of nucleic acids in specific cell populations in vivo^10,11,12, particularly due to their ability to infect both dividing and non-dividing cells and to stably integrate into host genome^13,14. Over the last two decades, lentivirus delivery technology has been optimized, and alternative envelopes and synthetic promoters have been investigated to increase lineage-specific targeting^8,15. Owing to its broad cell tropism, vesicular stomatitis virus envelop glycoprotein (VSV-G)^16,17 has become the "gold-standard" envelope used in lentivirus technology.

In this protocol¹⁸, VSV-G pseudotyped lentiviral particles are employed to demonstrate targeted and effective delivery of short hairpin RNA (shRNA) and microRNA (miR) to mouse peritoneal Mφ (pMφ) in vivo, at steady state¹⁹. Transgene expression was driven by the spleen focus forming virus (SFFV) promoter. Productive infection of cells was defined by expression of lentivirus-derived enhanced green fluorescent protein (GFP). Utilization of this approach allowed easy readout for in vivo lentivirus experiments to define the optimal dose and the experimental timeframe. Finally, in vivo lentiviral challenge of mice during thioglycolate-induced inflammation revealed the natural propensity for selective pMφ infection.

Protocol

All animal work was conducted in accordance with Institutional and UK Home Office guidelines.

NOTE: All in vivo studies with lentivirus should be performed according to local and national guidelines on the ethical use of animals in research, as well as adhering to all regulations associated with the use of category II infectious materials. Animal welfare should also be monitored in accordance with local regulations. In this step of the protocol, extreme care needs to be taken when working with lentiviral particles and sharps.

1. Preparation of HEK293T cells for transfection

NOTE: Perform these steps under a sterile tissue culture biological safety cabinet.

1. Prepare complete Dulbecco's Modified Eagle Medium (cDMEM) for HEK293T cells by combining the 450 mL of DMEM with 10% (v/v) fetal calf serum (50 mL) and a final concentration of 100 U/mL penicillin/streptomycin.
2. Defrost HEK293T cells at least 1 week prior to planned transfection. Maintain healthy growing conditions and passage every 2-3 days using trypsin + EDTA to detach the cells from the flask.
3. One day prior to transfection, carefully aspirate the growth medium from the cells and gently wash cells with sterile DPBS. Add 1-5 mL of trypsin to the flask and incubate at 37 °C in a 5% CO₂ incubator for 2-5 min.
4. Add 5-10 mL of cDMEM and spin the cells at 350 x g for 5 min. Remove the liquid and resuspend the cell pellet in 10 mL of cDMEM. Count the cells and seed 10-11 x 10⁶ viable HEK293T cells per T175 flask in 20 mL of cDMEM.
5. Incubate at 37 °C in a 5% CO₂ incubator overnight to allow the HEK293T cells to reach 70%-80% confluency.
6. The following day, confirm the confluency of cells under a light microscope.
7. Remove the media from the flask with a 25 mL serological stripette without disturbing the cell monolayer.
8. Gently add 10 mL of DPBS and rock the plate to wash the cells, taking care not to disturb the cell monolayer.
9. Remove DPBS with a 25 mL serological stripette.
10. Add 15 mL of new cDMEM on the wall per T175 flask so as not to disturb the cell monolayer, and return the plate to the incubator at 37 °C.

2. Transfection of HEK293T cells using non-liposomal lipid transfection reagent

1. In a 15 mL centrifuge tube, prepare the lentiviral components: 2 µg lentiviral plasmid, 1.5 µg pΔ8.91 (pCMV-Δ8.91), and 1 µg pMD2.G (pCMV-MD2.G) with Buffer EC (Transfection reagent kit) to a final volume of 600 µL.
2. Add 36 µL of enhancer solution. Mix the components using a 1 mL pipette by repeatedly sucking up a part of the liquid and putting it back drop-by-drop directly on the remaining solution. Leave for 5 min at room temperature (RT).
3. Add 120 µL of transfection reagent and mix as above approximately 20 times. Incubate for 10 min at RT.
4. Add 5.2 mL of cDMEM and mix as above with a 5 mL serological stripette.
5. Using a disposable transfer pipette, add the transfection mix dropwise directly onto the HEK293T cells monolayer (avoiding the flask walls), spreading the mix at different spots in the flask.
6. Distribute the plasmid by gently rocking the flask from side to side. Avoid getting any liquid in the flask lid.
7. Incubate the flask at 37 °C in a 5 % CO₂ incubator for 48 h. Confirm effective transfection of HEK293T cells by the appearance of a fluorescent marker, here GFP, within 24 h of transfection monitored under a cell imaging fluorescent microscope.
   NOTE: For constructs without the fluorescent marker, HEK293T cells must be tested for the presence of the used marker gene or by expression change in the gene/protein of interest by appropriate techniques, e.g., qPCR. Alternatively, successful transfection can be indirectly verified during testing of the lentivirus collection on Jurkat cells at the later stages of the protocol by the above techniques.
   ​CAUTION: Transfected HEK293T cells are considered infectious, and appropriate safety precautions should be implemented when handling these cells. Local rules should be followed, which could typically include working under a category II biology safety cabinet, the use of double gloves, and appropriate decontamination solution for disinfection, for example, increased concentration of waste bleach (2,000 ppm) or equivalent solution according to institutional biosafety guidelines. All solutions and plastics coming in contact with lentivirus preparation should be disinfected according to institutional biosafety procedures.

3. Collection of lentiviral particles

1. Prepare decontamination solution, a high concentration (2,000 ppm) bleach (sodium hypochlorite) solution, or equivalent agent according to institutional biosafety guidelines in a bucket and place it in the biology safety cabinet.
2. Prepare the biology safety cabinet by removing any excess items, such as empty tube racks etc.
3. Pre-label a 50 mL centrifuge tube per T175 HEK293T cells flask and remove the lid from the centrifuge tube. Place the tubes in a stable rack.
4. Retrieve the flask from the incubator and confirm GFP expression using a fluorescent microscope with a 488 nm laser (Figure 1A). The intensity of the signal depends on the transfection efficiency of the procedure and used constructs.
5. Collect the media from the cells into the pre-labeled 50 mL centrifuge tube. Tilt the T175 HEK293T flask with transfected cells so the medium collects in the bottom corner of the flask. Using a 25 mL serological stripette, collect the medium without disturbing the cell monolayer. Some floating cells might be visible at this point. Transfer the medium to a clean 50 mL centrifuge tube and close the lid.
   1. Using a 25 mL serological stripette, add 25 mL of fresh, warm cDMEM to the flask. Make sure to direct the medium flow on the walls of the flask not to disturb the cell monolayer. Return the flask with transfected HEK293T cells to the incubator (37 °C, 5% CO₂).
      NOTE: A second collection of lentivirus and further purification can be performed after additional 24 h following the procedure outlined in the steps above.
6. When lentivirus collection is completed after 24 h or 48 h, add decontamination solution to the cells, ensuring it covers the cell monolayer. Keep the flask horizontal for the next 24 h, then discard it following appropriate category II regulations.

4. Purification of lentivirus

1. Filter collect medium from step 3.5.
   1. Remove the plunger from the 50 mL syringe and insert a low protein binding polyether sulfone or polyvinylidene fluoride 0.45 µm sterile filter into the syringe end. Using a 25 mL serological stripette, add the collected medium from step 3.5.1 to the syringe and gently return the plunger.
   2. Push the plug slowly to pass the medium through the filter into a fresh 50 mL centrifuge tube. If the flow reduces significantly, position the syringe with the filter pointing upwards and pull out the plunger enough to clear the medium from the filter.
   3. Carefully, replace the filter on the syringe with a new one and dispose of the used filter in the decontamination solution. Continue medium filtration. When finished, decontaminate the filter and the syringe by submerging the filter and filling the syringe with the decontamination solution.
2. Pre-cool the ultracentrifuge by setting the temperature to 4 °C and shut the lid to allow the temperature to acclimate.
3. Add 3 mL of 20 % sucrose solution (20% w/v in ultrapure water, filter sterilized using a 0.22 µm filter) into the bottom of the conical ultracentrifuge tube.
4. Using a 25 mL serological stripette, overlay filtered medium on top of the sucrose layer, being careful not to disturb the layers.
   1. Use the lowest speed setting on the pipette boy. Angle the conical ultracentrifuge tube to about 45° and slowly add the filtered lentivirus-containing medium from step 4.1 to the tube wall. Ensure that the medium and sucrose do not mix, and clear separation of the layers is visible (Figure 1B). As the volume of the medium layer increases, slowly tilt the ultracentrifuge tube back to an upright position.
5. If the total volume, including the sucrose and the medium, in the ultracentrifuge tube is less than 29 mL, top up with fresh cDMEM to avoid the tube collapsing during the spin. For multiple T175 mL collections, mix filtered medium preparations and use multiple ultracentrifuge tubes. Top up the final ultracentrifuge tube with the medium as required.
6. Ensure the ultracentrifuge buckets are clean; there is no medium residue on the bottom of the bucket or the caps. If required, wipe out with ~70 % alcohol. Put appropriate tube adapters in the bottom of each bucket (Figure 1C) and gently lower the ultracentrifuge tubes into the buckets.
7. Close the buckets securely and hang them on the allocated spaces on the spin-out rotor.
8. Ensure that the buckets are balanced with the same volumes of sucrose and medium. A spin-out rotor must never be run without buckets, although opposing buckets may be left empty²⁰ (Figure 1D).
9. Carefully insert the rotor into the ultracentrifuge and turn on the vacuum. Set the ultracentrifuge acceleration and deceleration rate to the lowest setting, and run the lentivirus preps at 85,000 x g for 90 min at 4 °C.
10. Wait for the ultracentrifuge to reach the required speed (about 3-5 min) before walking away to ensure the rotor is inserted properly and the spin will not terminate.
11. While the ultracentrifuge is running, clean up the workspace according to category II safety requirements and prepare the space for the next steps.
12. When the spin finishes, disable the vacuum and carefully remove the rotor so as not to disturb the samples.
13. Investigate the ultracentrifuge for any spills and confirm no leaks from the buckets. In case of spillage, double glove and decontaminate the centrifuge and external surface of buckets with antiviral products.
14. Remove the buckets from the ultracentrifuge and carefully transport the buckets to the category II biology safety cabinet.
    NOTE: Lentivirus particles collect at the bottom of the ultracentrifuge tube. The pellet is not visible to the naked eye.
15. Pour the sucrose and medium with one smooth motion directly into the waste container with the decontamination solution.
16. Keeping the ultracentrifuge tube inverted, transfer it to the double layer of tissue and leave it to dry for 10 min. Meantime, wipe the inside of the buckets with water-soaked tissue and air-dry upside down.
17. Carefully dry any remaining liquid from the rim of the ultracentrifuge tube with tissue before turning it upright. Disinfect the tissue and the surface underneath.
18. Resuspend the virus pellet in 1 mL of serum-free media or solution required for further experimentation by gently pipetting the solution up and down. Leave it for 15 min at RT inside the category II biology safety cabinet.
19. Gently mix the lentivirus preparations using 1 mL pipette before aliquoting into 1.5 mL screw top tubes (e.g., cryotubes). Prepare the aliquots for the in vivo work (multiplicity of 100 µL) and for lentivirus titer in Jurkat T cells (1 x 20 µL) (see step 5).
20. Avoid freeze-thaw cycles; lentiviral preparations can be stored at -80 °C for up to 6 months without a negative effect on infectivity.

5. Titration of lentivirus production in Jurkat T cells

1. Prepare complete Roswell Park Memorial Institute 1640 medium (cRPMI-1640) for Jurkat T cells by supplementing 500 mL of RPMI-1640 with 10% (v/v) fetal calf serum and final concentration of 100 U/mL penicillin/streptomycin.
2. To ensure a healthy culture of Jurkat T cells, maintain the cells in culture for up to 1 week before infection.
3. On the day of the lentivirus titration, plate out viable Jurkat T cells in a 24-well plate at 2 x 10⁵ cells/well in 200 µL per well of cRPMI-1640.
4. Use freshly collected lentivirus or thaw the lentivirus vial containing 20 µL on ice and mix gently by pipetting up and down.
5. Using serial dilution in cRPMI-1640, infect Jurkat T cells with 0.25 µL, 0.5 µL, 1 µL, 2.5 µL, 5 µL, and 10 µL of lentivirus stock. Gently rock the plate to ensure equal distribution of the lentivirus. Non-infected Jurkat T cells serve as a negative control.
6. Incubate the plate at 37 °C. After 4 h, top up each well with cRPMI to a total volume of 400 µL. Return the plate to the incubator at 37 °C for 3 days.
7. After 48 h post-infection, confirm GFP expression under a fluorescent imaging system.
8. 3 days after infection, collect each well of the 24-well plate into separate 1.5 mL collection tubes and centrifuge the cells at 350 x g at 4 °C for 5 min.
9. Discard the supernatant. Resuspend the pellet in 300 µL of 2% paraformaldehyde (PFA) prepared to 2% (w/v) in DPBS and leave it for 15 min on ice in the dark.
10. Centrifuge the cells at 350 x g at 4 °C for 5 min and resuspend the pellet in fluorescence-activated cell sorting (FACS) buffer (sterile DPBS + 4% FCS + 1 mM sterile EDTA).
11. Analyze the GFP expression frequency and mean fluorescent intensity of the lentivirus infected and its control cells using flow cytometry (Figure 2).

6. In vivo lentivirus infection of tissue-resident peritoneal macrophages

1. In a category II biological safety cabinet, load insulin needles with a total volume 200 µL of serum-free media medium containing the required amount of lentivirus (use single-use needles, 30 G for animal welfare and to avoid fluid loss in the needle).
2. Place the needle sheath back on the needle using the one-hand scoop technique. Keep the needle on ice and inject within 30 min.
3. In the animal facility, set up a category II biological safety cabinet prior to injections (Figure 1E).
   1. Lay down a sheet of clean tissue. Loosen the sheath on the insulin needle containing lentivirus.
   2. Prepare a Petri dish containing small pieces of tissue and chlorhexidine gluconate-based disinfectant, a 50 mL tube containing decontamination solution (2,000 ppm bleach solution), and a sharp safe container.
4. Restrain the mouse by grasping the skin at the nape of the neck²¹. Properly restrained mouse is immobile, and this is required for safety.
5. With the abdomen facing up, point the animal's head slightly down. Inject the lentivirus Intraperitoneally into the lower right quadrant of the abdominal cavity. This is to avoid injection into any peritoneal cavity organs.
6. Before releasing the mouse, fill the syringe with decontamination solution and safely dispose of it in the sharp safe box.
7. Wipe the injection site on the abdomen of the mouse with tissue soaked in disinfectant and return the animal to the cage.
8. House the lentivirus-injected mouse in category II scantainer or individually ventilated cages (IVC) (isolated cages with high-efficiency air filtration) for a minimum of 72 h after injection. Place an appropriate information card on the front of the cage.
9. Move the mouse to the new category I holding cage after 72 h post-infection, depending on local safety approvals. Monitor mice daily for a total of 3 days from injection.

7. Collection of peritoneal cells from lentivirus-infected mouse

CAUTION: For collections within 72 h post lentivirus injection, follow institutional category II biological safety rules. Bedding and holding cage where infected animals were kept in the first 72 h post lentivirus injection must be decontaminated according to institutional category II biological safety rules.

1. Euthanize mice following institutional regulations, for example, by inhalation of increasing concentration of CO₂, followed by confirmation of death by cervical dislocation.
2. Clean the abdomen of the mouse with 70% isopropanol and carefully cut the skin to expose the peritoneal cavity membrane.
3. Lavage the peritoneal cavity with 6 mL of ice-cold FACS buffer using a 10 mL syringe and 23 G needle. Avoid puncturing any organs.
4. Gently massage the peritoneum to dislodge cells to the FACS buffer.
5. Collect the peritoneal fluid using the same syringe and needle.
6. Remove the needle and transfer the cells to a 15 mL centrifuge tube.
7. Keep the peritoneal lavages on ice.
8. Collect other required organs and store them as appropriate to the study.
9. Dispose of animal carcasses according to local animal and safety guidelines.

8. Peritoneal cell staining and analysis

1. Centrifuge the peritoneal lavage at 350 x g at 4 °C for 5 min, discard the supernatant in the decontamination solution, and resuspend the cells in 1 mL of FACS buffer.
2. Count the collected cells and plate 4 x 10⁵ cells per well in a V-bottom 96-well plate.
3. Perform viability staining using a fixable reagent (e.g., Fixable Near-IR Dead Cell Stain Kit used here) according to manufacturer instructions.
4. If cells were infected within the last 72 h, fix the cells in 2% PFA for 15 min on ice. Add an equal volume of cold DPBS and centrifuge again at 350 x g at 4 °C for 5 min.
5. Prepare the blocking buffer: Mix 4 µg/mL 2.4G2 antibody in 10% (v/v) rat serum in FACS buffer for surface staining or permeabilization buffer for intracellular staining.
6. Resuspend each well containing cell pellet in 50 µL of blocking buffer and incubate at 4 °C for 15 min.
7. Prepare antibody mix in either 50 µL of FACS buffer (for surface staining) or permeabilization buffer (for intracellular staining) per each sample. The final staining volume for each sample will be 100 µL, including 50 µL of blocking buffer. Calculate the antibody concentrations accordingly (Table 1).
8. Add 50 µL of antibody mix to samples and 50 µL of isotype controls and control buffer to control samples. Incubate the samples for 30 min on ice in the dark. Include unstained cells and isotype controls as required.
9. Wash each well with 100-200 µL of ice-cold DPBS and centrifuge the plate at 350 x g at 4 °C for 5 min. Repeat this step to wash away any unbound antibodies. Analyze the samples on a flow cytometer.

9. Extraction of cells from organs

1. Prepare 1 mL of digestion mix per organ: Mix Hank's balanced salt solution (HBSS), 2 mg/mL collagenase type IV, and 0.03 mg/mL DNase I (include 1.5 mg/mL of hyaluronidase for lung digestion).
2. Transfer the collected organ to 1 mL of digestion mix and mince it with scissors.
3. Filter the cells through a 40 µm strainer and centrifuge at 350 x g at 4 °C for 5 min.
4. If isolating cells from the lung, spleen, or liver, lyse red blood cells using ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA pH = 7.4). Filter the cells through a 40 µm strainer and centrifuge at 350 x g at 4 °C for 5 min.
5. Stain and analyze the cells as described in section 8.

Representative Results

When followed fully and correctly, this protocol yields a total of 1.5 mL of high-quality lentivirus stock per single preparation, sufficient for twelve in vivo injections at the optimal volume determined in this study¹⁸. The success of the transfection can be evaluated early in the protocol. Healthy and confluent HEK293T cells should display, if present in the plasmids, an easily detectable marker signal (e.g., GFP used in this study) after 48 h post plasmid transfection (Figure 1A). The low intensity of the signal, excessive cell detachment, and low confluency at early steps of the protocol could indicate cell death and will result in a low yield of the lentivirus preparation. Some of the cell detachment prior to optional collection II (step 3.5.1.) is visible and expected.

Three plasmids are used in this protocol for generation of lentivirus particles: pCMV-ΔR8.91 packaging plasmid encoding structural HIV-1 protein (Gag), accessory proteins Tat and Rev, and reverse transcriptase polymerase (Pol)²²; pMD2.G encoding VSV-G envelop under CMV promoter¹³; and pHR'SIN-cPPT-SEW plasmid (encoding enhanced GFP marker)¹⁹ modified accordingly for the expression of shRNA or microRNA.

Lentivirus preparations are titrated in the Jurkat T cell line due to their high infectivity²³. Successful lentivirus preparation will achieve an infection rate of over 95% with a dose as small as 5 µL (Figure 2A). The mean fluorescent intensity of the infected cells continues to increase with the higher doses (Figure 2B,C). If required, viral titers can be measured using real-time PCR of integrated viral components, e.g., the SFFV promoter, which correlated linearly with percentage GFP-expressing cells and logarithmically with GFP MFI (Figure 2D). Depending on the construct, particularly those with a large insert²⁴, some lentivirus preparations can display reduced infectivity in Jurkat T cells, as demonstrated for Cre-GFP construct used in this study (Figure 2E,F). In such a case, multiple preparations of lentivirus particles could be combined and resuspended in 1 mL. We recommend validating immune responses to those preparations in vivo prior to experimentation. Naturally, lentivirus preparations with alternative entry receptors to VSV-G used here might display different infection efficiency in the Jurkat T cell line, depending on the receptor expression on the cells. Cell lines used for the titration should be selected so they express the receptor used by the lentivirus particles to enter the cells and would preferentially lack or have very low expression of the restriction factors²⁵.

Successful production of lentiviral particles is further evidenced by infection of pMφ (defined as CD11b⁺ F4/80⁺ and Tim4⁺ population)¹⁸ (Figure 3A). We determined that intraperitoneal injection of 100 µL lentivirus preparation (in total volume of 200 µL of serum-free media) yields the highest percentage and intensity of the GFP signal in these cells (Figure 3B,C). Injections of higher doses (150 µL and 200 µL of lentivirus preparation) had no beneficial impact on GFP expression in the pMφ. Time course experiments at 4 h, 3 days, 7 days, and 14 days post intraperitoneal (i.p) injection with 100 µL lentivirus revealed a significant percentage of GFP-expressing resident pMφ at days 3 and 7, followed by the disappearance of the infected population at day 14 (Figure 3D, E). Interestingly, GFP-expressing pMφ mostly disappears by day 14 post-infection, due in part to immune recognition of the GFP marker. Indeed, lentivirus experiments with GFP marker in T-Reg selective, Foxp3-DTR-eGFP mice prevent rejection of infected resident pMφ until at least day 21 (Figure 3F). For lentivirus preparations with diminished effectiveness in Jurkat cells, a higher amount would be required to achieve the expected infection rate in vivo. However, as demonstrated with Cre-GFP lentivirus preparation, depending on the construct design, even a dose of 300 µL at 7 days post i.p injection might yield a poor outcome (Figure 3G). We have previously demonstrated successful use of this protocol for overexpression and knockdown of genes in vivo in mouse pMφ, including lentiviral shRNA-mediated Map3k8 and Gata6 knockdown, and Gata6 overexpression¹⁹. Here, we show that this protocol can be also successfully employed for overexpression of murine microRNA 146b (mmu-miR-146b) and for knockdown of intercellular adhesion molecule 1 (ICAM1, CD54) using lentiviral derived shRNA in resident pMφ (Figure 3H,I).

Infection of primary cells, such as macrophages, requires higher lentivirus input, presumably due to the presence of restriction factors in these cells. Restriction factors are natural protective mechanisms of cells that interfere with viruses' life-cycle steps, such as reverse transcription or integration, leading to inhibition of gene expression from the constructs.

Further in vivo validation of the protocol demonstrated no effect of the i.p lentivirus delivery on peritoneal immune cell viability (Figure 4A) and indicated distinct infectivity of resident pMφ subpopulations (defined by the expression of CD73 and Tim4 markers) (Figure 4B,C). Importantly, productive lentivirus infection was limited to resident Mφ at the site of injection as evidenced by lack of significant GFP expression in mesenteric lymph node (mLN), lung, liver, or spleen Mφ after 7 days post-challenge (Figure 4D). Considering that in many cases, genetic targeting of Mφ is required to be performed under inflammatory conditions, we investigated the effectiveness of this protocol in mice challenged intraperitoneally with 0.1 mL of 4% thioglycolate. Thioglycolate injection triggers an influx of inflammatory monocyte-derived Mφ and monocytic cells that can be divided into 5 distinct populations (Figure 4E). Flow cytometry analysis revealed a refractory phenotype of monocytic-like cells (Ly6C^hi populations 1 and 2) to lentivirus infection, in line with previous findings in human cells²⁶. In contrast, resident Mφ and monocytes (groups 3-5) remained most susceptible to infection (Figure 4F).

Detailed flow cytometric analysis detected GFP expression predominantly in resident peritoneal Mφ and major histocompatibility complex (MHC) class II⁺ resident pMφ (MHCII⁺ F4/80⁺ Tim4⁺) (up to 60 % at day 3 after injection) (Figure 5A). Little to no GFP signal was detected in other peritoneal cell populations, including bone marrow-derived peritoneal Mφs/DCs (MHCII⁺, CD11b⁺, CD11c⁺), B cells (CD19⁺), T cells (CD3⁺), mast cells (CD11b^-, FcεR1⁺), eosinophils (Siglec-F⁺), NK cells (CD19^-, NK1.1⁺) and neutrophils (Ly6G⁺) (Figure 5A,B). GFP expression longevity (Figure 5C) in all populations followed this of resident peritoneal Mφ (Figure 1D). A transient increase in neutrophil frequency was recorded after 4 h post i.p injection of 100 µL (in a total volume of 200 µL of serum-free media) of lentivirus (Figure 5D), indicating early mild inflammation present in challenged animals. Finally, resident pMφ experienced a major drop in the frequency between days 7 and 14 post-infection (Figure 5E), suggesting the best experimental window between days 3 and 7 post-injection.

Figure 1: Lentivirus production in HEK293T cells. (A) Representative immunofluorescence pictures of HEK293T cells 48h after successful transfection (fluorescent microscope [488 nm excitation peak, 510 nm emission peak], 20x magnification, scale bar = 400 µm). (B) Photograph of ultracentrifuge conical tube containing a layer of 20% sucrose (bottom, clear) and a layer of medium collected from transfected HEK293T cells (top, red). (C) Photograph of correct insertion of the conical ultracentrifuge in the bucket, including adaptor. (D) Photographs showing correct and incorrect balancing of the ultracentrifuge rotor. (E) Photograph of the optimal setup of the Cat II cabinet and materials for in vivo injections for a right-handed individual. This figure has been modified with permission from Ipseiz N et al.¹⁸. Please click here to view a larger version of this figure.

Figure 2: Lentivirus titration in Jurkat T cells. (A) Representative flow cytometric analysis of Jurkat T cells 72 h after infection with increasing doses of lentivirus containing a GFP plasmid showing the percentage of GFP⁺ cells (GFP⁺), (B) mean fluorescence intensity (MFI) of GFP⁺ cells and (C) a representative histogram of GFP expression. (D) Scatter plot showing MFI (left Y) and the percentage of cells infected with a GFP-expressing lentivirus (right Y) versus the copy number of virus detected per pg of DNA (x-axis). (E) A representative histogram of Jurkat T cell infection with suboptimal Cre-GFP lentivirus (Cre-GFP LV) preparation and control GFP lentivirus (GFP LV) and (F) summary data showing the percentage of Cre-GFP lentivirus infected Jurkat T cells (GFP⁺). This figure has been modified with permission from Ipseiz N et al.¹⁸. Please click here to view a larger version of this figure.

Figure 3: Infection efficiency of the resident pMФ. (A) Gating strategy of resident pMφ (CD11b⁺, Tim4⁺, F4/80⁺) and Tim4^-, F4/80⁺ cells. Cells were gated on singlets, followed by CD11b⁺. (B) Representative dot plots, and (C) a summary of infection frequency (% GFP+ cells) and intensity (MFI) of GFP⁺ cells isolated 3 days after in vivo infection with different amounts of GFP lentivirus preparation. (D) Representative dot plot, and (E) a summary of infection frequency (% GFP+ cells) and intensity (MFI) of GFP+ cells isolated at different time points after in vivo infection with 100 µL of lentivirus in a total volume of 200 µL of serum-free media medium. (F) A summary of cell numbers at days 7, 14, and 21 after intra-peritoneal delivery of GFP-expressing lentivirus in Foxp3-DTR-eGFP mice showing the number of GFP-expressing pMφ and inflammatory macrophages and dendritic cells (InfMØs/DCs [F480^low]). (n= 1-2 per group). (G) Representative dot plot of suboptimal in vivo infection of Gata6-KO^{mye 19} resident pMφ with 300 µL of Cre-GFP lentivirus 7 days post i.p injection. (H) RT-qPCR quantification of mmu-miR-146b-5p expression of resident peritoneal Mφ challenged in vivo with 100 µL lentivirus encoding murine microRNA-146b (miR-146b) or control (C). Resident pMφ (white circles) and Tim4^-, F4/80⁺ cells (grey circles). (I) A representative dot plot of successful downregulation of ICAM1 on pMφ in female 129S6 mice, 7 days after i.p injection of lentivirus containing targeting shRNA. Control shRNA is shown. The overlay shows isotype control. Data expressed as mean ± SEM, n≥2 mice. This figure has been modified with permission from Ipseiz N et al.¹⁸. Please click here to view a larger version of this figure.

Figure 4: Infection efficiency of the resident pMφ subpopulations. (A) Percentage of total single cells and resident Mφ viable 7 days after 100 µL serum-free media (-) or GFP lentivirus (+) i.p injection. (B) Gating strategy showing four major populations of pMφ found in vivo: CD73⁺Tim4⁺, CD73^-Tim4^-, CD73⁺Tim4^-, CD73^-Tim4⁺, and (C) corresponding infection frequency (% GFP+ cells) and intensity (MFI of GFP+ cells) of these populations. (D) Percentage of GFP+ cells in multiple organs 7 days after 100 µL lentivirus i.p injection. Abbreviations: mLN, mesenteric lymph node. (E) Mice were injected i.p with 0.1 mL of 4% thioglycolate for 5 days followed by i.p injection with lentivirus. Gating strategy of pMφ and monocytes post 3 days after lentivirus i.p injection. (F) Infection frequency, MFI, and cell number analysis of GFP+ monocytes (Ly6C⁺) and Mφ (Ly6C^-). Data expressed as mean ± SEM, n≥3 mice. This figure has been modified with permission from Ipseiz N et al.¹⁸. Please click here to view a larger version of this figure.

Figure 5: Impact of lentivirus injection on peritoneal inflammation. Mice were injected in vivo i.p with GFP expressing lentivirus. (A) Infection frequency of cell populations in the peritoneal cavity following various amounts of lentivirus injections (50 µL, 100 µL, 150 µL, or 200 µL). (B) Intensity of GFP expression in productively infected cell populations at 7 days post i.p injection. (C) Infection frequency of cell populations in the peritoneal cavity at various time points after i.p injection. (D) and (E) Percentage of cells at various time points after i.p injection. All lentivirus injections were performed with the same total volume of 200 µL completed by serum-free media medium. Unless specified otherwise, 100 µL lentivirus dose was used. Control mice ("C") received 200 µL of neat serum-free media medium. Data expressed as mean ± SEM, n≥3 mice. This figure has been modified with permission from Ipseiz N et al.¹⁸. Please click here to view a larger version of this figure.

Antibody target	Fluorophore	Clone	Dilution used	final concentration [μg/mL]
HIV-1 Core antigen	RD1	FH190-1-1	1/100	1
I-A/I-E	PerCpCy5.5	M5/114.15.2	1/400	0.5
Ly6G	PerCpCy5.5	1A8	1/400	0.5
CD3e	PerCpCy5.5	17A2	1/200	1
CD3e	PE/Cy7	500A2	1/400	0.5
CD11c	PE/Cy7	N418	1/800	0.25
CD11c	BV605	N418	1/400	0.5
CD226	AF647	10 E 5	1/400	1.25
Tim4	AF647	RTM4-54	1/600	0.83
CD4	APC	GK1.5	1/400	0.5
CD11b	AF700	M1/70	1/700	0.71
CD11b	PerCpCy5.5	M1/70	1/400	0.5
F4/80	Pacific Blue	BM8	1/700	0.71
F4/80	BV605	BM8	1/400	0.25
F4/80	BV711	BM8	1/400	0.5
CD73	eFluor450	TY/11.8	1/400	2.5
CD19	V450	1D3	1/400	0.5
CD19	APC	1D3	1/400	0.5
CD8a	eFluor450	53-6.7	1/400	0.5
SiglecF	BV421	E50-2440	1/400	0.5
NK1.1	APC/Cy7	PK136	1/400	0.5
FceR1	eFluor450	MAR-1	1/400	0.5
ICAM1	PE	1A29	1/100	2
Rat IgG1, κ isotype control	PE		1/100	2

Table 1: List of antibodies

Discussion

Tissue-resident macrophages perform a range of homeostatic and inflammatory tissue-specific functions^1,2 dictated by their physiological environment^6,7,8,9. In this protocol, an effective method¹⁸ for manipulation of peritoneal resident macrophages in vivo using lentivirus particles was introduced to investigate macrophage function in their biological microenvironment.

It is essential for the success of the protocol to use healthy HEK293T cells. It is the best practice to defrost the cells at least a week prior to the start of this protocol to ensure cell recovery and good numbers. Cells should be seeded in an appropriate volume of medium one day before planned transfection and should reach about 80% confluency on the day of transfection. Under- or over-confluent cell preparations will result in reduced lentivirus yield. We recommend transfecting HEK293T cells with the transfection reagent according to the manufacturer's instructions for the best results. Essential steps of the transfection include appropriate mixing of the plasmids and reagents and dropwise addition of the mix directly to the HEK293T cell monolayer. Calcium phosphate transfection of HEK293T cells^27,28 could be employed in this protocol. However, the user should be aware that the effectiveness of this method can vary, and it can result in diminished transfection efficiency.

Safety precautions should be considered in the protocol from the day of HEK293T cell transfection. These include working in a category II safety cabinet, wearing double gloves when handling contaminated material, and appropriate bleaching of the contaminated material (with decontamination solution, for example, 2,000 ppm bleach solution) for minimum 4 h. Users should refer to their institutional regulations regarding work with category II pathogens and waste.

In this protocol, the lentivirus preparation is first purified using a 0.45 µm filter to remove HEK293T cell debris. The use of smaller filters (0.22 µm) and cellulose ester membranes should be avoided as it will result in the loss of lentivirus particles. It is recommended to use low protein binding polyether sulfone or polyvinylidene fluoride filters²⁹. The second purification step is performed on the single 20% sucrose layer in an ultracentrifuge to remove remaining impurities, which is particularly important for the consequent in vivo administration of the lentivirus preparation. In the institutions where an ultracentrifuge is not available, others³⁰ have described sucrose-based lentivirus purification using a standard laboratory centrifuge. This could be implemented in this protocol as an alternative. Lentivirus preparation is titrated in Jurkat T cells on the expression of the marker signal (e.g., GFP used in this protocol). For constructs without markers, physical lentivirus particles could be evaluated by quantification of HIV-1 p24gag protein by ELISA kit³¹, flow cytometry analysis of HIV-1 core antigen¹⁸, or measurement of the changes to the targeted gene (preferentially using the methods allowing measurement of changes in individual cells, e.g., flow cytometry or microscopy). If titration of the control and lentivirus differ significantly in Jurkat T cells, the volumes used for the in vivo studies can be adjusted to reach the most comparable infection between lentivirus preparations.

The major limitation of the protocol is the observed disappearance of GFP⁺ resident peritoneal Mφ within 14 days of i.p lentivirus injection. For long-term studies, mouse lines with stable genetic alteration in a specific cell type or tissues should be considered³². For example, as peritoneal macrophages do not express Foxp3 (a T-Reg protein), we utilized Foxp3-DTR-eGFP mice³³ and confirmed that expression of GFP is maintained in peritoneal Mφ at 21 days post-infection (Figure 3F). However, it is important to note that the lentiviruses contain other foreign components, and this extension in the persistence of infected cells in the Foxp3-DTR-GFP mice may not be permanent. An additional weakness of this method is a low level of inflammation that can be observed in the peritoneal cavity following lentivirus injection, as witnessed by influx of neutrophils at 4 h post injection (Figure 5D), which may mean repeated infections would accelerate the inflammation-associated loss of GFP-expressing resident peritoneal Mφ. Although, we have not detected type I interferons (IFNs) in the peritoneal cavity, VSV-G pseudotyped lentivirus particles were previously demonstrated to induce some of the IFN-stimulated genes in human Mφ in the absence of detectable IFNs³⁴. This should be considered when using this protocol for experiments investigating antiviral immune responses. The occurrence of acute and sustained inflammation after i.p injection of lentivirus preparation might indicate contamination of the preparation.

Areas of troubleshooting include: 1) for low lentivirus titer, ensure the health of HEK293T cells and effective transfection (e.g., marker expression, if present, in HEK293T cells). If the infection rate remains low, consider the size of the construct. Constructs with larger inserts or more complex secondary structures can affect the final lentivirus titer and infectivity²⁴. Therefore, each construct should be tested independently and in parallel to its respective control vector; 2) If a high amount of the lentivirus is required, it is recommended to prepare multiple T175 flasks of HEK293T cells and to scale up the production accordingly. To avoid variation of the lentivirus preparations, it is the best practice to merge the final collections prior to aliquoting and storage. For such larger production, plasmids mix (section 2) should be prepared in 50 mL tubes to ensure effective mixing of the components.

Despite its broad tropism, VSV-G pseudotyped lentiviral particles predominantly target tissue macrophages as demonstrated previously for alveolar³⁵, and here¹⁸ for pMФ when administered by the respective routes. Alterations to the envelope on lentivirus particles can, in some cases, result in diminished transduction of macrophages in vivo³⁶ and are unnecessary for this protocol. Compared to other viral approaches for in vivo gene manipulation in macrophages (reviewed in³⁷), the use of lentiviral vectors offers stable integration of the transgene in tissue macrophages³⁵, efficient transgene expression, and the largest vector size limit (approximately 8 Kb).

Peritoneal Mφ play an important role in the prevention, onset, progression, and resolution of various diseases, including abdominal cancers, pancreatitis, and peritonitis³⁸. This protocol describes an effective tool for gene modification in murine pMφ, allowing investigation of the biological processes behind these pathologies within a physiologically relevant microenvironment. While lentiviral vectors themselves are becoming a tool of interest for clinical interventions³⁹, due to the origin from the immunodeficiency virus and a stable genome integration, the safety concerns impede their therapeutic implementation. Further understanding of the macrophage responses to lentiviral gene modulation could advance the application of this highly effective tool in a clinical setting.

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA (1x) (Trypsin 500 mg/L or 0.02 mM)	Thermo Fisher Scientific	25300054
0.22 μm sterile millex GP filter	Merck	SLGS033SS
0.45 μm sterile millex GP filter	Merck	SLHP033RS
0.5 mL U-100 insulin syringe with needle, 0.33 mm x 12.7 mm (29 G)	BD	324892
1 Litre Sharps Container	N/A	N/A
2.4G2 antibody (TruStain FcX anti-mouse CD16/32)	Biolegend	101320
40 μm strainer	Thermo Fisher Scientific	22363547
AimV medium (research grade), AlbuMax Supplement	Thermo Fisher Scientific	31035025
Blocking buffer	prepared in house
Brewer thioglycolate medium	Sigma-Aldrich	B2551	4% stock solution prepared in water, autoclaved and kept frozen.
CD11b	Biolegend	101222	Refer to Table 1 for the dilution and concentration
CD11b	BD	550993	Refer to Table 1 for the dilution and concentration
CD11c	Biolegend	117317	Refer to Table 1 for the dilution and concentration
CD11c	Biolegend	117333	Refer to Table 1 for the dilution and concentration
CD19	BD	560375	Refer to Table 1 for the dilution and concentration
CD19	Biolegend	152410	Refer to Table 1 for the dilution and concentration
CD226	Biolegend	128808	Refer to Table 1 for the dilution and concentration
CD3e	BD	560527	Refer to Table 1 for the dilution and concentration
CD3e	Biolegend	152313	Refer to Table 1 for the dilution and concentration
CD4	Biolegend	100412	Refer to Table 1 for the dilution and concentration
CD73	eBioscience	16-0731-82	Refer to Table 1 for the dilution and concentration
CD8a	eBioscience	48-0081-82	Refer to Table 1 for the dilution and concentration
Cell culture flask (T175 fask, 175 cm2, 550 mL)	Greiner Bio One	658175
Centrifuge tubes, conical bottom tubes 25 mm x 89 mm	Beckman Coulter	358126
Centrifuges	Beckman Coulter		Ultracentrifuge and TC centrifuge
Collagenase type IV	Sigma-Aldrich	C5138
Conical centrifuge tubes (15 mL and 50 mL)	Greiner Bio One	11512303 & 11849650
Cryotubes	Greiner Bio One	123277	or cryotubes
DMEM medium (1x) + 4.5g/L D-glucose, 400 µM L-glutamine	Thermo Fisher Scientific	41965-062
Dnase I	Sigma-Aldrich	11284932001
Effectene transfection reagent	Qiagen	301425
F4/80	Biolegend	123123	Refer to Table 1 for the dilution and concentration
F4/80	Biolegend	123133	Refer to Table 1 for the dilution and concentration
F4/80	Biolegend	123147	Refer to Table 1 for the dilution and concentration
FceR1	eBioscience	48-5898-80	Refer to Table 1 for the dilution and concentration
Fetal calf serum (FCS)	Thermo Fisher Scientific	10270-106	heat inactivated for 30 min at 56 °C and sterile filtered through 0.22 μm filter
Flow cytometer	Thermo Fisher Scientific	Attune NxT
Flow cytometry (FACS) buffer	prepared in house
Fluorescent tissue culture microscope	Thermo Fisher Scientific	EVOS FL
Forceps	N/A	N/A	User preference
Hank's balanced salt solution (HBSS)	Gibco, Life Technologies	14175-053
HEK293T cell line			grown for at least a week prior transfection. Mycoplasma free
HIV-1 Core antigen	Beckman Coulter	6604667
Hyaluronidase	Sigma-Aldrich	H3506
Hydrex surgical scrub, chlorhexiding gluconate 4% w/v skin cleanser	Ecolab	3037170
I-A/I-E	Biolegend	107625	Refer to Table 1 for the dilution and concentration
ICAM1	Becton Dickinson	554970	Refer to Table 1 for the dilution and concentration
Jurkat T cell line			grown for at least a week prior use. Mycoplasma free
LIVE/DEAD fixable near-IR dead cell stain kit	Thermo Fisher Scientific	L34975
Ly6G	Biolegend	127615	Refer to Table 1 for the dilution and concentration
Mice			here used C57BL/6 females, aged 8-12 weeks (Charles Rivers), unless specified differently
Microcapillary pipettes (volume range 0.5-1,000 μL)	Fisher Scientific & Starlab	11963466 & 11943466 & 11973466 & S1111-3700
NK1.1	Biolegend	108724	Refer to Table 1 for the dilution and concentration
Paraformaldehyde	Sigma-Aldrich	P6148-500G	prepared to 2% w/v in PBS
pCMV-ΔR8.91 packaging plasmid		Zuffrey, R., et al. 1997	encodes Gag-Pol HIV protein driven by cytomegalovirus promoter. Ampicilin resistance.
Penicillin/Streptomycin (100x, 10,000 U/mL)	Thermo Fisher Scientific	15140122
Petri dish	Greiner Bio One	664160
pHR'SIN-cPPT-SEW plasmid		Rosas, M. et al. 2014	modified for shRNA and miR expression studies. Encodes EGFP marker downstream SFFV promoter and upstream of the Woodchuck hepatitiv virus enhancer. Ampicilin resistance.
pMD2.G plasmid		Naldini, L. et al. 1996	encodes vesicular stomatis virus g-glycoprotein (VSV-G) envelope. Ampicilin resistance.
Rat IgG1, κ isotype control	Becton Dickinson	550617	Refer to Table 1 for the dilution and concentration
Rat serum	Sigma-Aldrich	R9759-10ML
Red blood ACK lysis buffer	prepared in house
RPMI 1640 medium (1x) + 400 uM L-glutamine	Thermo Fisher Scientific	21875-091
Saponin	Sigma-Aldrich	S4521
SiglecF	BD	562681	Refer to Table 1 for the dilution and concentration
Sodium hypochlorite Tablets (bleach, 2,000 ppm)	Guest Medical	H8818
Sterile 24-well cell culture plate	Greiner Bio One	662160
Sterile Dulbecco's PBS (DPBS) (1x) Mg++ and Ca2+ - free	Thermo Fisher Scientific	14190144
Sterile EDTA	Thermo Fisher Scientific	15575020
Sterile VWR disposable transfer pipets (23.0 mL, 30 cm)	VWR	612-4515
Sucrose	Thermo Fisher Scientific	15503022
surgical scissors	N/A	N/A	User preference
Syringes (50 mL and 1 0mL)	Fisher Scientific	10084450 & 768160
Tim4	Biolegend	130007	Refer to Table 1 for the dilution and concentration
U-bottom 96-well cell culture plate	Greiner Bio One	650180