Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

Summary

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

Abstract

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-α, IL-1β, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a diffuse pulmonary inflammatory disease caused by many factors¹. In the cytokine microenvironment of the Th1 and Th2 immune response, macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2). Lipopolysaccharides (LPS) or the cytokine IFN- γ induce M1 macrophages to polarize and produce pro-inflammatory cytokines, including iNOS, IL-1, IL-6, TNF-α, and IL-12. In contrast, the type II cytokines IL-4 and IL-13 drive the polarization of M2 macrophages, which can produce different fibroblast growth-promoting factors, such as TGF-β and PDGF, that promote pulmonary fibrosis². The pathological process of IPF is accompanied by macrophage activation and infiltration. IPF mediates injury repair, inflammation, and fibrosis through the release of cytokines³. As only limited therapeutic options are available, exploring the molecular pathological mechanisms of IPF holds great significance for developing new strategies for IPF prevention and treatment. Previous studies by our group and other researchers^4,5 have confirmed the increased release of IL-33 in IPF patients and in mouse models with bleomycin (BLM)-induced IPF. IL-33 is released by the epithelial and endothelial cells during fibrosis and is involved in macrophage activation, resulting in the abnormal proliferation of fibroblasts, leukocyte infiltration, and the eventual loss of lung function⁵. The current protocol describes the adoptive transfer of IL-33-stimulated interstitial macrophages (IMs) into the alveoli as a means to study IPF development in mouse models. Here, IMs were isolated from the lung tissue of host mice, cultured in vitro, stimulated with IL-33 for 24 h, and then adoptively transferred into the alveoli of recipient mice by tracheal injection. The direct collection of stimulated mouse macrophages and their adoptive transfer into the recipient alveoli was found to aggravate the degree of pulmonary fibrosis and can more clearly illustrate the influence of stimulating factors on fibrosis compared to the previous studies⁶. The technique described in this paper can enable researchers to explore the function of macrophages stimulated by potential cytokines in the development of IPF.

Protocol

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. All the animal experiments were approved by the Experimental Animal Welfare Ethics committee of Jiangnan University (JN No. 20211¹³⁰m1720615[501]).

NOTE: In total, 10 male C57BL/6 mice aged 6-8 weeks old and weighing 20-25 g were used in this study. The three experimental groups in the study included three recipient mice each, and one host mouse was used for the IM isolation.

1. Depletion of mouse pulmonary macrophages

1. Take out the vial of clodronate liposomes (see Table of Materials) from the refrigerator 30 min in advance, warm it to room temperature, and invert it several times to ensure uniform mixing.
   NOTE: Clodronate liposomes encapsulate the hydrophilic dichloromethylene bisphosphonate molecules. When these liposomes are consumed by the macrophages, the clodronate is gradually released by the action of the lysosome phosphatase, and its accumulation consequently triggers cell apoptosis and the depletion of macrophages⁷.
2. Anesthetize mice with 3% isoflurane. Check the depth of anesthesia by pinching one foot to check consciousness. Apply veterinary ointment to both eyes to prevent dryness under anesthesia.
3. Aspirate 60 µL of clodronate liposomes using a pipette with a sterile suction tip. Administer the drug dropwise into the nasal cavity of the anesthetized mouse, such that when the mouse inhales, the drug is inhaled into the trachea. After each drop, make sure the mouse inhales the drug completely and is breathing evenly. Administer liposomes containing PBS alone as a control⁸ (Figure 2).
4. Place the mice on a 38 °C constant temperature table for rewarming to speed up their recovery. Monitor the mice until they wake up. After the mice recover well and achieve sternal recumbency, transfer them to the cage.
5. Use mice with depleted alveolar macrophages 2 days after the treatment with clodronate liposomes as the recipient mice for the transfer experiment.
   ​NOTE: In this study, the depletion of alveolar macrophages was confirmed by checking the expression of macrophage markers as described earlier^8,9 following the administration of clodronate liposomes by inhalation (see Supplementary Figure 1).

2. Isolation and culture of IMs

1. Anesthetize the host mouse with an intraperitoneal injection of Ketamine (120 mg/kg) and Xylazine (16 mg/kg). Confirm the depth of anesthesia via loss of the toe pinch reflex. Apply veterinary ointment to both eyes to prevent dryness under anesthesia. Then, disinfect the skin of the anesthetized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue
2. Aspirate 10 mL of 1x PBS in a syringe with a 20 G needle and insert the tip of the needle into the right atrium of the mouse (Figure 3A). Cut the inferior vena cava of the mouse with surgical scissors, and then manually perfuse the mouse with PBS at a constant speed (10-20 mL/min) until the lung tissue turns white. Excise the lung tissue and transfer it into ice-cold PBS in a culture dish (Figure 3B).
3. Cut the lung tissue into fragments of approximately 2 mm x 2 mm x 2 mm. Then, add 15 mL of Dulbecco's modified Eagle's medium (DMEM) containing 1% collagenase A to the lung tissue to isolate the macrophages. Incubate at 100 rpm for 30 min on a 37 °C shaking table.
4. Aspirate the lung tissue suspension through a 10 mL syringe at least 20x to make it as fine as possible. Filter this suspension through a 40 µm cell strainer. Centrifuge the filtrate at 400 x g for 10 min and discard the supernatant.
5. Lyse the red blood cells in the pellet by adding 3 mL of RBC lysis buffer (see Table of Materials) on ice for 2-3 min.
6. After centrifugation at 150 x g for 5 min, resuspend the cell pellet with 10 mL of DMEM (containing 100 U/mL penicillin and 100 µg/mL streptomycin). Count the cells using a hemocytometer.
7. Seed the cells into 10 cm adherent cell culture dishes at 2 x 10⁷ cells/dish, and incubate for 1 h at 37 °C and 5% CO₂. This allows the lung IMs to adhere to the dish⁵. After 1 h, aspirate the supernatant and floating cells, and add 10 mL of fresh complete culture medium (DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin). Incubate for more than 8 h or overnight.
8. Determine the purity of the obtained IMs using flow cytometry with staining for F4/80 and CD11c markers, as described earlier⁵ (see Supplementary Figure 1).

3. Adoptive transfer of IMs into the lung alveoli

1. Replace the culture medium from the plate containing the isolated pulmonary IMs (step 2.7) with a complete culture medium containing 10 ng/mL IL-33 for stimulating the IMs. Incubate for 24 h at 37 °C and 5% CO₂. Use 1x PBS in place of IL-33 as a control.
2. Dissociate the pulmonary IMs by treating them with 1 mL of 0.25% trypsin for 5 min. Then, quench the digestion by adding 3 mL of fresh culture complete medium, and harvest the pulmonary macrophages by centrifuging the cell suspension at 150 x g and 4 °C for 5 min. Count the cells using a hemocytometer, and resuspend the cells in PBS to a final concentration of 5 x 10⁵ cells/50 µL.
3. Anesthetize the recipient mice with 3% isoflurane gas. Wait for the mouse to become unconscious (as in step 1.2), and then fix the limbs of the anesthetized mouse on a vertical plate with medical tape. Gently pull the mouse's tongue to one side using a cotton swab.
4. Turn on the intubation lamp and shine it on the throat of the mouse so that the trachea can be seen. Check that the trachea is close to the base of the tongue, the esophagus is near the back of the neck, but the opening of the esophagus is not visible during the operation.
5. Use the intubation lamp (22 G) to help intubate the mouse. Guide the indwelling needle cannula into the trachea with a guide wire (0.4 mm diameter). Remove the guide wire and push the cannula into the mouse trachea to complete the intubation.
6. After the cannula is observed to enter the trachea, inject 50 µL of the cell suspension (containing 5 x 10⁵ IMs) into the recipient mouse through the trachea.
7. Place the mice on a 38°C constant warming pad for rewarming to speed up the recovery. Monitor the mice until they wake up. After they recover well and achieve sternal recumbency, transfer them to the cage.
8. After 24 h, administer bleomycin (BLM) via the trachea (using the procedure in steps 3.3-3.5) at a dose of 1.4 U/kg body weight to induce IPF. Administer an equal volume of saline to the control group.
9. After 21 days, determine the degree of severity of pulmonary fibrosis for the different groups of mice that were adoptively transferred with PBS or the IL-33-stimulated IM suspension by evaluating the expression of markers for fibrosis and the Ashcroft scores¹⁰.
   1. Extract the total RNA from the lung tissue using an RNA extraction reagent (see Table of Materials).
      NOTE: Quantitative analysis was carried out with a microplate reader. If the OD260/OD280 ratio is 1.8-2.0, the RNA purity is high. All the samples were stored at −80 ° C.
   2. Perform fluorescence quantitative PCR to evaluate the expression of α-smooth muscle actin (SMA) and fibronectin using specific primers.
      ​NOTE: The primers used for α-SMA were forward 5'- GACGCTGAAGTATCCGATAGAACACG-3' and reverse 5'-CACCATCTCCAGAGTCCAGCACAAT-3', and the primers used for fibronectin were forward 5'-TCTGGGAAATGGAAAAGGGGAATGG-3' and reverse 5'-CACTGAAGCAGGTTTCCTCGGTTGT-3'.
10. Perform immunohistochemistry as described below.
    1. Dehydrate the left lung, embed it, and slice it with a paraffin slicer to a thickness of 4 µm.
    2. Place the tissue sections onto slides and bake the slides in an oven at 65-70 ° C for 30 min to 1 h. Dewax the slides with a decreasing percentage of alcohol (i.e., 100%, 95%, 90%, 80%, and 70% alcohol) for 5 min.
    3. Stain the slides in hematoxylin dye solution (analytically pure) for 5 min. Then, wash the slides with tap water. Soak the slides in 1% hydrochloric acid alcohol for 3 s, wash off the floating color, and soak in flowing water for 5 min. The sections will turn blue.
    4. Immerse in eosin solution for 10 s to 1 min (determine the dyeing time according to the color), wash with tap water, and place in 70%, 80%, 95%, 100%, and 100% alcohol, respectively, for 5 min each. Then place, the slides in xylene I and xylene II solutions for 3 min. After drying, observe and take photos under the vertical microscope with neutral adhesive sealing film.
    5. Perform Ashcroft scoring on the sections to indicate the severity of pulmonary fibrosis¹⁰. Perform statistical analysis of the data using a t-test of two independent samples.

Representative Results

The protocol used here is summarized in the flowchart in Figure 1. The inhalation of clodronate liposomes through the nose (Figure 2) was used to deplete the pulmonary macrophages of adult C57BL/6 mice, and this produced a good recipient mouse model. Pulmonary IMs were isolated from another untreated (host) mouse (Figure 3A,B) and cultured in vitro. The isolated macrophages were stimulated with IL-33 for 24 h and then intratracheally instilled into the recipient mice (Figure 3C), with unstimulated macrophages used as a control. After 24 h, BLM was administered to the recipient mice to induce a pulmonary fibrosis model. The extent of pulmonary fibrosis after the adoptive transfer of IMs with or without IL-33 stimulation was compared 21 days after BLM administration. Hematoxylin and eosin (H&E) staining of the pathological tissue sections showed that the typical pathological changes of fibrosis were observed after BLM administration: the lung tissue structure of the mice was destroyed, the aggregation of fibroblasts was observed, and the normal alveoli disappeared or decreased. The adoptive transfer of IL-33-stimulated macrophages exacerbated the degree of lung tissue destruction and increased fibroblast aggregation in the BLM-stimulated mice (Figure 4A). The increase in the Ashcroft score further illustrated the degree of pulmonary fibrosis (Figure 4B). The pathological process of pulmonary fibrosis is associated with the increased aggregation of myofibroblasts, which secrete α-smooth muscle actin (α-SMA) and fibronectin. The determination of the expression levels of these markers showed that the mRNA levels of α-SMA (Figure 5B) and fibronectin (Figure 5A) in the lung tissues of the recipient mice containing adoptively transferred IL-33-stimulated IMs and treated with BLM were further increased compared with those of the BLM-treated wild-type mice.

Figure 1: Schematic diagram of the establishment of the macrophage adoptive transfer experiment. Please click here to view a larger version of this figure.

Figure 2: Depletion of macrophages in the recipient mice. The clodronate liposomes were dropped into the nasal cavity of the mice. Please click here to view a larger version of this figure.

Figure 3: Isolation and adoptive transfer of interstitial macrophages. (A) The inferior vena cava of the host mouse was cut to facilitate perfusion. (B) The lung tissue from the host mouse was cut into small pieces. (C) The interstitial macrophages were purified to 94% using flow cytometry with F4/80 and CD11c markers. (D) IL-33-stimulated IMs were administered to the recipient mice through the trachea. Please click here to view a larger version of this figure.

Figure 4: The effect of the adoptive transfer of IL-33-stimulated macrophages in the BLM-stimulated mice. (A) H&E staining of lung tissue sections from the recipient mice. Scale bar = 100 µm. (B) Ashcroft scores determined from the lung tissue sections of the recipient mice. Data are shown as mean ± SEM (n = 3). *p < 0.05, **p < 0.01. Please click here to view a larger version of this figure.

Figure 5: Expression levels of pulmonary fibrosis marker genes in the recipient mice. (A) The mRNA level of fibronectin. (B) The mRNA level of α-SMA. Data are shown as mean ± SEM (n = 3). *p < 0.05, **p < 0.01. Please click here to view a larger version of this figure.

Supplementary Figure 1: Depletion of alveolar macrophages and purity of IMs. (A) The depletion of alveolar macrophages was confirmed by checking the expression of F4/80 and CD11b markers. (B) The purity of the obtained IMs was assessed using flow cytometry with staining for F4/80 and CD11c markers, and the purity of the isolated IMs was determined to be 94.4% using flow cytometry. Please click here to download this File.

Discussion

This study provides an effective method to deplete, isolate, culture, and transfer macrophages, which can help in studying the mechanisms of pulmonary fibrosis in mice. There are many methods for mouse macrophage depletion, such as tracheal administration, tail vein injection, and nasal inhalation¹¹. This study optimized the nasal inhalation method, which is simple to operate and can effectively deplete pulmonary macrophages^8,9. After the IL-33 stimulation of IMs in culture for 24 h, the IMs were adoptively transferred to the lungs of the recipient mice using a non-invasive tracheal administration route, which caused the least trauma to the mice. During intubation, an anesthesia duration of >20 min was employed to ensure the smooth progress of the intubation, which greatly improved the survival rate of the mice. The initial tracheal administration of a 50 µL IM suspension requires a micropipette to ensure the accuracy of the administration. After the tracheal administration, 500 µL of air should be immediately added into the lungs with a 1 mL syringe to ensure that the cell suspension in the catheter can completely enter the lung tissue. Although this method clears the alveolar macrophages, macrophages from other parts of the mouse may also migrate to the alveoli over the course of the disease, thus diluting the proportion of cells transferred into the alveoli. Therefore, experiments with strict requirements for the proportion of cells transferred need to further explore other methods.

Pulmonary macrophages play an important role in the defensive function of the lungs^12,13. Of these, alveolar macrophages mainly express the F4/80 and CD11b markers, while interstitial macrophages mainly express F4/80 and CD11c¹². IMs were chosen for adoptive transfer in this study, as the process required 5 x 10⁵ cells, and IMs constitute a major fraction of the pulmonary macrophages, which makes them easier to obtain. A large number of studies have shown that macrophages regulate the inflammatory response by releasing inflammatory factors and play an important role in the pathological process of pulmonary fibrosis¹⁴. Studies have shown that IL-33 regulates TGF-β and other cytokines to regulate the function of macrophages, which promotes BLM-induced pulmonary fibrosis⁴. Therefore, induced changes in macrophage function play an important role in the pathogenesis of pulmonary fibrosis. This study provides a method for the adoptive transfer of IMs that can help researchers further explore the role of macrophages in lung diseases such as asthma and IPF. There is currently no effective therapeutic drug for IPF, but this study indicates that the factor IL-33 may be relevant for the research and treatment of idiopathic pulmonary fibrosis and, thus, provides a direction for the further exploration of pulmonary fibrosis drug research. For example, a neutralizing antibody against IL-33 can be prepared to explore its effect and feasibility as an experimental IPF drug, thereby providing an important direction for the treatment of idiopathic pulmonary fibrosis.

Materials

Name	Company	Catalog Number	Comments
DMEM	Life technologies Biotechnology,USA	1508012
Arterial indwelling needle	B Braun Melsingen AG,Germany	21G15G8393
BD Accuri C6 Plus	Becton Dickinson,USA
Bleomycin	Biotang, USA	Ab9465
Carbon dioxide incubator	Thermo Forma, USA	Thermo Forma370
CD11b	R&D Systems,USA	1124F
CD11c	R&D Systems,USA	N418
Cell culture dish	Thermo Forma, USA	174926
Clodronate liposomes	Clodronate liposomes,Netherlands	CI-150-150
Collagenase A	Sigma-Aldrich, USA	10103578001
F4/80	R&D Systems,USA	521204
Falcon Cell Strainer	Becton,Dickinson and Company, USA	352340
Fetal bovine serum (FBS)	Life technologies,USA	1047571
Hematoxylin Eosin	Nanjing Jiancheng Technology,China	06-570
LightCycler 480 PCR detection system	Roche, USA
Murine recombinant factor IL-33	Peprotech, USA	210-33
Nikon microscope	Nikon Corporation, Japan	941185
Penicillin, streptomycin	Life technologies,USA	877113
Phosphate buffer (PBS)	Guangdong Huankai Microbial Technology ,China	1535882
RBC lysis buffer	Beyotime Biotechnology Company,China	C3702
RNA Isolater	Vazyme company,China	R401-01-AA	Total RNA extraction reagent
RWD Inhalation Anesthesia Machine	Shenzhen Rayward Life Technology ,China	R500
Semi-automatic paraffin slicer	Leica, Germany	LeicaRM2245
SYBR Premix Ex Taq	Takara, Japan	410800
Trypsin 0.25%	Life Technologies, USA	1627172