Rapid Depletion of Renal Macrophages using Human CD59/Intermedilysin Cell Ablation Tool

A protocol is reported here for the selective ablation of renal macrophages to study their regeneration using the human CD59/intermedilysin cell ablation tool. This method is also applicable for studying the function and regeneration of the other cell populations in the kidney, liver, and fatty tissue.

Renal macrophages (RMs) are essential for kidney health, orchestrating immune surveillance, tissue homeostasis, and responses to injury. Previously, we reported the use of a human CD59 (hCD59)/intermedilysin (ILY) cell ablation tool to study the distinct fate, dynamics, and niches of RMs of bone marrow or embryonic origin. RMs originate from yolk sac-derived macrophages, fetal liver monocytes, and bone marrow-derived monocytes and are maintained in adulthood through local proliferation and recruitment of circulating monocytes. Here, we report a detailed protocol for the selective ablation of RMs to study their regeneration, including 1) generation and characterization of the Cre-inducible expression of hCD59 in mouse RMs, 2) purification of ILY and characterization of ILY activity, 3) induction of hCD59 expression on RMs in compound mice, and 4) characterization of regeneration after ILY-mediated RM ablation. ILY specifically and rapidly depletes RMs in compound mice, with efficient macrophage ablation within 1 day of ILY administration. Renal macrophage regeneration began by day 3 post-ablation, with ~88% recovery by day 7. This model offers a powerful tool for studying macrophage biology and can be used for selectively ablating other cell populations in the kidney, liver, and fatty tissues to investigate their function and regeneration.

Renal macrophages (RMs) are essential immune cells that maintain kidney homeostasis, regulate immune responses, and promote tissue repair following injury. They perform a variety of functions, including phagocytosis, antigen presentation, and the orchestration of both inflammatory and anti-inflammatory responses^1,2,3. Depending on the local environment, RMs can polarize into either pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, either exacerbating injury or facilitating healing^4,5,6. Dysregulation of RMs has been implicated in the onset and progression of acute kidney injury (AKI) and chronic kidney disease (CKD), making them critical players in kidney health and pathology^7,8. RMs arise from various sources, including yolk sac-derived macrophages during embryogenesis, fetal liver monocytes, and bone marrow-derived monocytes in adulthood. RMs expand and mature in parallel with renal growth postnatally, primarily originating from fetal liver monocytes before birth, with self-maintenance through adulthood supplemented by peripheral monocytes⁹. In adults, circulating monocytes are recruited to the kidney by homeostatic or injury signals, differentiating into macrophages under local microenvironmental influences. RM maintenance is sustained through local proliferation and periodic replenishment from circulating monocytes^9,10,11,12.

We previously demonstrated the use of the human CD59 (hCD59)/intermedilysin (ILY) cell ablation system as a tool^13,14 to investigate the distinct fates, dynamics, and microenvironmental niches of RMs derived from either bone marrow or embryonic origins⁹. ILY selectively lyses human cells within seconds by forming pores in targeted cell membranes¹⁵. This specificity arises from ILY's exclusive binding affinity for human CD59 (hCD59), with no interaction or lytic effect on cells from other species lacking this receptor¹⁵. Based on this concept, we engineered a tool allowing the rapid, conditional, and targeted ablation of human CD59-expressing cells in transgenic mouse models through the application of intermedilysin (ILY)¹⁴. To facilitate the use of this tool, we developed a model of conditional and targeted cell ablation by generating floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination¹³. Previously, it was found the CX3CR1cre-EFP gene exclusively expressed on CD11bintF480hi, defined as renal macrophages⁹. Therefore, we used CX3CR1CreER2^+/+ lines to cross with ihCD59 mice to express hCD59 on renal macrophages. We successfully generated compound mice with the genotype ihCD59^+/-/CX3CR1CreER^+/-9 (+/-, +/+ and -/- indicate the homozygote, hemizygote, and noncarrier transgenic mice, respectively). Tamoxifen administration in ihCD59^+/-/CX3CR1CreER^+/- compound mice conditionally labeled and marked RMs by hCD59 expression⁹. Following the depletion of the RM niche through ILY treatment, peripheral monocytes promptly differentiated into bone marrow-derived RMs, effectively repopulating the niche as previously described in⁹. This regeneration was critically dependent on the CX3CR1/CX3CL1 signaling axis, underscoring its essential role in both the maintenance and restoration of the RM population⁹. We also show that due to their distinct glycolytic capacities, embryonic-origin RMs have a higher capacity for scavenging immune complexes and are more sensitive to immune challenges than bone marrow-derived RMs⁹.

We present a detailed protocol for the selective ablation of RMs to investigate their regeneration using Cre-inducible hCD59 (ihCD59)-mediated rapid cell ablation following administration of ILY. Injection of ILY caused the targeted ablation of RMs that conditionally and specifically express hCD59 in CX3CR1CreER^+/-/ihCD59^+/- compound mice. After ablation, we monitored macrophage dynamic changes using flow cytometry and found rapid depletion of macrophages followed by the regeneration of RMs. Recombinant ILY was expressed in E. coli BL21(DE3) cells and purified using nickel-NTA affinity chromatography. The purity and functionality of the recombinant ILY were confirmed by SDS-PAGE, spectrophotometry, and a hemolysis assay, demonstrating its characteristic cholesterol-dependent cytolysin activity. We used ILY to deplete the RMs in the mice, achieving efficient macrophage ablation within 1 day of ILY administration. Renal macrophage regeneration began on day 3 post-ablation, with ~85% recovery by day 7. The data suggest that regeneration is primarily driven by monocyte recruitment. This model offers a powerful tool for studying macrophage biology and has therapeutic potential for the targeted manipulation of macrophage populations in kidney diseases. The ihCD59/ILY cell ablation tool can be used to study cell function and regeneration in the kidney, liver, fatty tissue, and other organs.

The animal study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Tulane University, School of Medicine (Protocol number 1482). The experimental mice, Cx3cr1CreER^+/+ and ihCD59^+/+, aged 10-12 weeks and weighing 25-30 g, were housed under specific pathogen-free (SPF) conditions at the university's animal facility. All procedures involving kidney isolation were conducted in a sterile environment, with researchers wearing gloves and face masks to prevent contamination. Details regarding the reagents and equipment utilized in the study can be found in the Table of Materials.

1. Animal preparation

1. Obtain CX3CR1CreER^+/+mice from The Jackson Laboratory. House the mice at the Tulane University School of Medicine (SOM) in a specific pathogen-free (SPF) facility. Maintain the animals under a 12 h light/dark cycle with controlled environmental conditions.
2. Use previously generated ihCD59+/+ mice with a C57BL/6 genetic background for breeding. Cross homozygous CX3CR1CreER^+/+ mice (deficient in CX3CR1 expression) with ihCD59^+/- mice to produce the following offspring genotypes: CX3CR1CreER^+/-/ihCD59^-/-and CX3CR1CreER^+/-/ihCD59^+/-.
3. Ensure that heterozygous CX3CR1CreER^+/- mice retain functional CX3CR1 expression, while homozygous CX3CR1CreER^+/+ mice are CX3CR1-deficient by flow cytometry analysis with Anti-CX3CR1 antibody⁹.

2. ILY production (derived from^14,15 )

1. Day 0
   1. Retrieve frozen stock of the ILY bacterial strain BL21-DE3-RIPL carrying His-tagged ILY sequences in pTrcHis-A plasmid (Supplementary Figure 1) stored at -80 °C. Inoculate 10 µL of the bacterial stock into 40 µL of Luria broth (LB) medium containing ampicillin at a concentration of 1 µg/mL.
   2. Spread the inoculated mixture onto an LB agar plate supplemented with ampicillin (1 µg/mL). Incubate the plate overnight at 37 °C in a bacterial incubator.
2. Day 1
   1. Select six individual colonies from the LB agar plate. Inoculate each colony into 3.5 mL of LB medium containing ampicillin. Incubate the cultures overnight at 37 °C.
3. Day 2
   1. Transfer each 3.5 mL starter culture into 250 mL of LB medium supplemented with ampicillin. Incubate the cultures at 37 °C for 3 h with shaking at 230 x g to allow bacterial growth.
   2. After 3 h, add isopropyl β-D-1-thiogalactopyranoside (IPTG) from a 1 M stock solution (stored at -20 °C) at a 1:1000 dilution to achieve a final concentration of 1 mM. Continue incubation for an additional 4 h to induce protein expression.
   3. Weigh an empty centrifuge bottle and record the weight. Transfer approximately 250 mL of the induced culture into the centrifuge bottle.
   4. Pellet the cells by centrifuging at 10,000 x g for 15 min at 4 °C. Discard the supernatant, then weigh the bottle containing the pellet. Calculate the pellet weight by subtracting the empty bottle weight from the total weight after centrifugation.
   5. Store the bacterial pellet at -20 °C for subsequent extraction and purification of ILY.
4. Day 3 : Extraction and purification of ILY
   1. Prepare the lysis buffer by combining 30 mL of Bug Buster Protein Extraction Reagent with 30 µL of benzonase nuclease (1200 Kunitz units) and 0.9 µL of lysozyme (10,000 units of enzymatic activity) in a 50 mL centrifuge tube.
   2. Mix the solution gently to ensure the lysis buffer is homogeneous. Add the prepared lysis buffer to the bacterial pellet (2-5 g). Vortex the mixture thoroughly for 30-60 s to fully resuspend the bacterial cells.
   3. Divide the resuspended solution equally between two 50 mL centrifuge tubes. Place the tubes on ice and incubate for 30-90 min. During incubation, gently shake the tubes using an orbital shaker and vortex briefly every 5-10 min for 10-15 s to enhance cell lysis.
   4. After incubation, centrifuge the lysates at 4,900 x g for 30 min at 4 °C.
   5. Wash the column thoroughly with tap water using a 1 mL pipette tip to remove any remaining resin from previous use.
   6. Rinse the column with 70% ethanol to sanitize it. Wash the column 2x-3x with cold ddH₂O to remove any residual ethanol. Place the column in the holder.
   7. Add 6-10 mL of cold ddH₂O to further wash and equilibrate the column for purification. Add approximately 3 mL of resin beads to the column. Connect the column to a peristaltic pump set to a speed of 30.
   8. Add 10 mL of cold ddH₂O to fully suspend the resin and prepare it for protein binding. Wash the resin beads with 15 mL of 1x Charging Buffer to prepare the resin for protein binding.
   9. Collect the bacterial lysate supernatant after centrifugation in a fresh tube, ensuring the pellet is discarded. Filter the supernatant using a 0.45 µm filter with a syringe to remove any remaining cellular debris.
   10. Pass 20-25 mL of the filtered bacterial lysate through the column in three separate rounds. After each pass, collect 20 µL of the flow-through and label it as P.E. for later optical density (OD) measurement.
   11. Wash the column with 20-30 mL of Binding Buffer to remove unbound proteins. Wash the column with 20-30 mL of Wash Buffer. Collect 20 µL of the wash fraction for OD measurement.
   12. Elute the bound protein by adding 10-20 mL of Elution Buffer. Start collecting the elution after 3-4 min using microcentrifuge tubes. Collect approximately 1 mL per tube, filling 15-17 tubes.
   13. Measure the absorbance of each elution fraction at A280 using a spectrophotometer. Use the Elution Buffer as the blank during A280 readings. Collect the tubes with dramatically increased absorbance at A280 since they contain the His-tagged ILY protein that is typically found in fractions around tube 13.
   14. Store the elution fractions (or tubes) containing ILY protein at 4 °C. Proceed with further steps within the following day to ensure optimal protein stability.
       NOTE: Perform all steps on ice or at 4 °C where possible to maintain protein integrity.
5. Day 4: Endotoxin removal from the elution
   1. Rinse the endotoxin removing resin column with 6-10 mL of ultra-pure water. Gently press the column to regulate the flow.
   2. Rinse the column with 10 mL of 1% sodium deoxycholate solution (freshly made or no older than 1 month) to remove bound endotoxins. Rinse the column again with 6-10 mL of ultra-pure water to remove any residual detergent.
   3. Apply the prepared protein sample to the resin column. Incubate the sample in the column for 1 h at room temperature (RT) to allow optimal endotoxin binding.
   4. Collect the initial flow-through fraction. Add ddH₂O to the column for further elution and collect eluted fractions in approximately 10 tubes (1 mL per tube).
   5. Store the resin column in 25% ethanol at 4 °C for future use. Measure the optical density (O.D.) of the eluted protein samples to quantify the yield. Expect to recover approximately 50%-70% of the total protein from the first elution batch.
6. Day 5-6: Dialysis
   1. Prepare 2 L of dialysis buffer by autoclaving 1600 mL of distilled, deionized water (ddH₂O). Add 200 mL of 10x PBS, 200 mL of glycerol, and 3.5115 g of MES (2-(N-morpholino) ethanesulfonic acid) to the autoclaved water.
   2. Stir the mixture with a magnetic stir bar until all reagents are completely dissolved. Store the resulting buffer at 4 °C.
   3. Add the stored elution fractions to the dialysis kit, then remove air from the dialysis kit with a needle. Dialyze the protein solution using the dialysis kit for 24 h at 4 °C with an agitation speed set to approximately 2.
   4. Configure the dialysis kit to ensure it is fully submerged in the buffer. Replace the dialysis buffer in the dialysis kit with fresh dialysis buffer in a 2 L container the next day. Continue the dialysis for an additional 24 h.
   5. Carefully transfer the entire solution, including any white precipitates, into a sterile tube to preserve the ILY protein, which may aggregate. Concentrate the ILY protein using a centrifugal filter unit, following the manufacturer's instructions for centrifugation.
   6. Aliquot the concentrated ILY protein into 200 µL portions in sterile tubes. Label each tube with the date and name. Store the aliquots at -80 °C for future use in gel electrophoresis and hemolysis assays.

3. ILY characterization by SDS-PAGE electrophoresis

1. Prepare the MOPS SDS running buffer (1x) by diluting running buffer (20x) with ddH₂O.
2. Prepare the sample buffer in a fume hood by mixing 90 µL of 6x loading buffer with 10 µL of β-mercaptoethanol (β-ME). Mix the solution thoroughly to ensure complete homogenization.
3. Combine the prepared sample with the loading buffer to achieve a final volume of 30 µL. Thaw the samples and vortex them vigorously to ensure complete mixing.
4. Heat the samples at 95 °C for 5 min to denature the proteins. Load the samples onto a 4%-20% SDS-PAGE gel. Run the electrophoresis at a constant voltage of 80 V for 90 min.
5. Carefully open the gel cassette and immerse the gel in ddH₂O for 5 min. Repeat the washing step 2x using a shaker.
6. Stain the gel with Coomassie Brilliant Blue for 1 h at room temperature (RT). Wash the gel for 30 min in ddH₂O. Replace the water with fresh ddH₂O and incubate the gel on a shaker for a period ranging from overnight to 3 days for complete destaining.
7. Capture an image of the stained gel using a camera (Figure 1A). Analyze the gel using ImageJ software for quantitative and qualitative assessments.

4. Hemolytic assay for ILY activity

1. Thaw the stored ILY (Intermedilysin) samples completely at room temperature. Vortex the thawed samples thoroughly to ensure homogeneity, as ILY can aggregate upon thawing.
2. Set up a 96-well plate for serial dilutions. Add 160 µL of the ILY stock solution (26.7 ng/µL) to the first well. Add 80 µL of 1x PBS to each of the subsequent wells in the same row or column.
3. Transfer 80 µL of the ILY solution from the first well to the second well. Mix thoroughly by pipetting up and down to ensure the solution is homogeneous. Transfer 80 µL from the second well to the third well and mix thoroughly.
4. Repeat this process for each well, transferring 80 µL from the previous well to the next until the desired number of dilutions is prepared. After transferring 80 µL to the last well, discard 80 µL from that well to ensure consistent volumes across the plate.

5. Preparation of human red blood cells (RBCs)

1. Draw 1-2 mL of fresh human blood into a tube containing an anticoagulant, such as EDTA. Centrifuge the blood at room temperature at 3,000 x g for 5 min.
2. Discard the supernatant and wash the human RBC pellet with PBS 2x-3x until the supernatant is clear. Dilute the washed human RBCs by adding 10 µL of RBCs to 900 µL of 1x PBS to achieve a 1:100 dilution.
3. Add 10 µL of the diluted human RBCs to each well. Add 30 µL of the serially diluted ILY solution from the dilution plate to each well. Add 160 µL of 1x PBS to bring the final volume to 200 µL per well (starting concentration: 4 ng/µL).
4. Add 10 µL of the diluted human RBCs to the control wells. Add 190 µL of water or 1x PBS to the control wells for negative controls. Mix the plates by gently tapping them.
5. Incubate the plates at 37 °C for 30 min to allow hemolysis to occur. Centrifuge the plates at 3,000 x g for 5 min to pellet any intact human RBCs.
6. Carefully transfer 100 µL of the supernatant from each well to a new flat-bottom 96-well plate. Measure the absorbance of the supernatants at 405 nm using a microplate reader.
7. Calculate the hemolytic activity of ILY by comparing the absorbance values of the samples to the control¹⁶.

6.Tamoxifen treatment, RM depletion, and regeneration

1. Preheat corn oil to 42 °C. Dissolve 100 mg of tamoxifen in 5 mL of preheated corn oil to prepare a stock solution with a concentration of 20 mg/mL.
2. Administer tamoxifen intraperitoneally (i.p.) to 10-12-week-old male CX3CR1CreER^+/-/ihCD59^+/- mice at a dose of 100 µg/g body weight. Repeat the tamoxifen injection for 3 consecutive days.
3. Wait for 15 days after the final tamoxifen injection to allow for Cre-mediated hCD59 expression.
4. Inject a single dose of intermedilysin (ILY) intravenously into CX3CR1CreER^+/-/ihCD59^+/- mice and CX3CR1CreER^+/-/ihCD59^-/- littermate controls at a dose of 120 ng/g body weight.
5. Monitor and confirm RM ablation and subsequent regeneration by performing flow cytometry at 1, 3, and 7 days post-ILY administration¹⁷.

7. Mouse euthanasia

1. Place the animal in an appropriate chamber and expose it to isoflurane in accordance with IACUC protocols. Verify anesthesia by checking for the absence of reflexes, such as the pedal withdrawal reflex.
2. Once the mouse is fully anesthetized and unresponsive, perform cervical dislocation to ensure euthanasia. Confirm euthanasia by verifying the absence of reflexes, such as the pedal withdrawal reflex.
3. Make a midline incision of approximately 2 cm on the abdomen using a sterile scalpel and gently retract the skin to expose the thoracic cavity. Open the rib cage along the sternum using blunt-tipped surgical scissors to expose the heart.
4. Insert a 21G-23G needle into the left ventricle. Begin perfusion with 10-20 mL of cold PBS to flush out blood.
5. Cut the right atrium open to allow blood to drain from circulation. Continue perfusion until the kidneys appear pale, indicating effective blood clearance.
6. Locate the kidneys against the dorsal body wall. Gently separate the kidneys from surrounding tissues using forceps and scissors. Excise the kidney capsules and immediately place the kidneys in cold PBS to prevent degradation.

8. Kidney digestion

1. Prepare the digestion enzyme cocktail by mixing 9 mL of HBSS (Ca/Mg free), 1 mL of collagenase IV (5 mg/mL), and 20 µL of DNase I (10 mg/mL). Prepare the enzyme solution fresh and keep it on ice until use.
2. Mince the kidneys into tiny fragments (ideally ≤ 1mm) using sterile scissors in a Petri dish containing 5 mL of HBSS. Transfer the kidney tissue fragments into 15 mL tubes containing 10 mL of the prepared enzyme solution.
3. Incubate the tubes at 37 °C for 30 min with gentle agitation to dissociate the tissue. Triturate the tissue gently using a pipette or syringe plunger to further dissociate cell clumps.
4. Remove tissue debris by passing the cell suspension through a 40 µm cell strainer. Centrifuge the cell suspension at 650 x g for 10 min at 18 °C.
5. Carefully decant the buffer after centrifugation. Resuspend the pellet in 5 mL of lysis buffer and incubate for 5 min at room temperature to lyse red blood cells.
6. Wash the cells with PBS to remove the lysis buffer. Maintain the resulting single-cell suspension on ice until further use.

9.Density gradient centrifugation

1. To enrich hematopoietic cells, resuspend the cell pellet from the previous wash in 10 mL of 30% density gradient solution. Carefully layer the solution over 3 mL of 70% density gradient solution in a centrifuge tube.
2. Centrifuge the gradient at 500 x g for 30 min without applying a brake. Following centrifugation, carefully collect the interphase layer between the 30% and 70% solutions; this layer contains the enriched single cells.
3. Wash the collected cells 2x with 10 mL of PBS by centrifuging at 500 x g for 10 min each time. Resuspend the final cell pellet in 1 mL of FACS buffer (1x PBS with 2% FBS) in a 1.5 mL tube.
4. Count the cells using a hemocytometer under a bright-field microscope. Adjust the cell concentration to 2 x 10⁶ cells/mL.

10. Flow cytometry staining, acquisition

1. Adjust the cell concentration from single-cell suspensions prepared from mouse kidney tissue to 2-3 x 10⁶ cells/mL for flow cytometric analysis.
2. Pellet the cells in 1.5 mL tubes by centrifuging at 650 x g for 5 min. Resuspend the cell pellet in 1 mL of FACS buffer.
3. Add anti-CD16/32 antibody at a 1:200 dilution to block non-specific Fc receptor binding. Incubate the cells for 15 min at room temperature.
4. Use Aqua Live/Dead dye to distinguish live from dead cells, following the manufacturer's instructions.
5. Stain the cells with pre-conjugated antibodies at a 1:100 dilution, including CD45-e450, CD11b-PE-Cy7, hCD59-PE, and F4/80-BV605. Add the antibody cocktail to the cell suspension.
6. Incubate the sample for 30 min at 4 °C in the dark to protect fluorophores from photobleaching. Wash the stained cells 2x with FACS buffer (PBS with 2% fetal bovine serum) by centrifuging at 650 x g for 5 min.
7. Fix the cells in 1% paraformaldehyde (PFA) for 30 min on ice. Wash the fixed cells 2x with FACS buffer to remove residual PFA.
8. Acquire stained and fixed cells using a flow cytometer. Analyze the data using the software.
9. Identify kidney and other tissue-resident macrophages and microglia using markers CD45, CD11b, and F4/80 as described in⁹ .
10. Perform initial gating to select live cells based on forward scatter (FSC) and side scatter (SSC) profiles. Eliminate doublets by gating on forward scatter area (FSC-A) versus forward scatter height (FSC-H). Exclude dead cells using the LIVE/DEAD viability dye.
11. Enrich immune cells by gating on CD45+ populations. Define kidney-resident macrophages as CD11b+F4/80++ cells within the CD45+ population, as shown in the gating strategy (Figure 2A).
12. Define microglia populations as CD11b+CD45ⁱⁿᵗ cells (Figure 2B). Perform data acquisition on the flow cytometer. Conduct the final analysis using the software to complete the flow cytometric study.

The purification of His-Tag recombinant ILY followed the same protocol as previously described in¹⁴. ILY was successfully expressed in E. coli BL21(DE3) cells, transformed with a plasmid encoding the ILY gene from Streptococcus intermedius. Upon induction with IPTG, overexpression of the target protein was evident. Post-expression, ILY was purified using nickel-NTA affinity chromatography, exploiting a C-terminal His-tag for specific binding. The purity and molecular weight of IL...

The successful expression, purification, and functional validation of His-tagged recombinant ILY in this study followed a well-established protocol¹⁵. However, the process involved several critical steps that ensured high protein yield, purity, and biological activity. The induction of E. coli BL21 (DE3) cells with IPTG was optimized to balance protein expression levels while minimizing inclusion body formation. Despite the robustness of this protocol, cer...

The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript. No conflicts of interest, including financial, non-financial, professional, or personal affiliations, have influenced the design, conduct, interpretation, or presentation of the study.

We extend our gratitude to both past and current members of the Qin Lab for their contributions to the development and refinement of the protocols used in this study. We also thank Dr. R. K. Tweten's group at the University of Oklahoma Health Sciences Center for generously providing the recombinant ILY plasmid, which was instrumental in this research. This study was supported by the National Institutes of Health (NIH) through grant NIH 5 P51OD011104-58, R01DK129881 (X.Q.), and R21OD024931 (X.Q.).