Name: adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo
Uploaded: 2023-05-05T13:30:00.000+00:00
Duration: 6 min 29 s
Description: Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

The authors acknowledge the Special Topic of Laboratory Management of Jiangnan University: Construction of Digital Slice Library Based on Pathological Specimens (JDSYS202223) and the National Natural Science Foundation of China (81800065).

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Aspirate 60 &#181;L 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).</li>
	<li>Place the mice on a 38 &#176;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.</li>
	<li>Use mice with depleted alveolar macrophages 2 days after the treatment with clodronate liposomes as the recipient mice for the transfer experiment. 
	&#8203;NOTE: In this study, the depletion of alveolar macrophages was confirmed by checking the expression of macrophage markers as described earlier8,9 following the administration of clodronate liposomes by inhalation (see Supplementary Figure 1).</li>
</ol>
2. Isolation and culture of IMs
<ol>
	<li>Anesthetize the host mouse&#160;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</li>
	<li>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).</li>
	<li>Cut the lung tissue into fragments of approximately 2 mm x 2 mm x 2 mm. Then, add 15 mL of Dulbecco&#39;s modified Eagle&#39;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 &#176;C shaking table.</li>
	<li>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 &#181;m cell strainer. Centrifuge the filtrate at 400 x g for 10 min and discard the supernatant.</li>
	<li>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.</li>
	<li>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 &#181;g/mL streptomycin). Count the cells using a hemocytometer.</li>
	<li>Seed the cells into 10 cm adherent cell culture dishes at 2 x 107 cells/dish, and incubate for 1 h at 37 &#176;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 &#181;g/mL streptomycin). Incubate for more than 8 h or overnight.</li>
	<li>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).</li>
</ol>
3. Adoptive transfer of IMs into the lung alveoli
<ol>
	<li>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 &#176;C and 5% CO2. Use 1x PBS in place of IL-33 as a control.</li>
	<li>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 &#176;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 &#181;L.</li>
	<li>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&#39;s tongue to one side using a cotton swab.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After the cannula is observed to enter the trachea, inject 50 &#181;L of the cell suspension (containing 5 x 105 IMs) into the recipient mouse through the trachea.</li>
	<li>Place the mice on a 38&#176;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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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 &#8722;80 &#176; C.</li>
		<li>Perform fluorescence quantitative PCR to evaluate the expression of &#945;-smooth muscle actin (SMA) and fibronectin using specific primers. 
		&#8203;NOTE: The primers used for &#945;-SMA were forward 5&#39;- GACGCTGAAGTATCCGATAGAACACG-3&#39; and reverse 5&#39;-CACCATCTCCAGAGTCCAGCACAAT-3&#39;, and the primers used for fibronectin were forward 5&#39;-TCTGGGAAATGGAAAAGGGGAATGG-3&#39; and reverse 5&#39;-CACTGAAGCAGGTTTCCTCGGTTGT-3&#39;.</li>
	</ol>
	</li>
	<li>Perform immunohistochemistry as described below.
	<ol>
		<li>Dehydrate the left lung, embed it, and slice it with a paraffin slicer to a thickness of 4 &#181;m.</li>
		<li>Place the tissue sections onto slides and bake the slides in an oven at 65-70 &#176; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

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 macrophages8,9. 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 &#62;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 &#181;L IM suspension requires a micropipette to ensure the accuracy of the administration. After the tracheal administration, 500 &#181;L 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 lungs12,13. 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-&#946; 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.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.
Step 2.1 of the Protocol was updated from:
Anesthetize the host mouse with 3% isoflurane, and then euthanize it by cervical dislocation. Disinfect the skin of the euthanized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue
to:
Anesthetize the host mouse&#160;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

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.

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

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- &#947; induce M1 macrophages to polarize and produce pro-inflammatory cytokines, including iNOS, IL-1, IL-6, TNF-&#945;, 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-&#946; 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 researchers4,5 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.

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

adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo

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-&#945;, IL-1&#946;, 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.

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&#38;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 &#945;-smooth muscle actin (&#945;-SMA) and fibronectin. The determination of the expression levels of these markers showed that the mRNA levels of &#945;-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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64742/64742fig01.jpg" /> 
Figure 1: Schematic diagram of the establishment of the macrophage adoptive transfer experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/64742fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64742/64742fig02.jpg" /> 
Figure 2: Depletion of macrophages in the recipient mice. The clodronate liposomes were dropped into the nasal cavity of the mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/64742fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64742/64742fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/64742fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64742/64742fig04.jpg" /> 
Figure 4: The effect of the adoptive transfer of IL-33-stimulated macrophages in the BLM-stimulated mice. (A) H&#38;E staining of lung tissue sections from the recipient mice. Scale bar = 100 &#181;m. (B) Ashcroft scores determined from the lung tissue sections of the recipient mice. Data are shown as mean &#177; SEM (n = 3). *p &#60; 0.05, **p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/64742fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64742/64742fig05.jpg" /> 
Figure 5: Expression levels of pulmonary fibrosis marker genes in the recipient mice. (A) The mRNA level of fibronectin. (B) The mRNA level of &#945;-SMA. Data are shown as mean &#177; SEM (n = 3). *p &#60; 0.05, **p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/64742fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64742/supplementary.png" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

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

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).

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

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

adoptive-transfer-il-33-stimulated-macrophages-into-bleomycin-induced

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.1K Views.  Jiangnan University. Het protocol beschrijft de isolatie van pulmonale IMS en adoptieve overdracht na IL-33-bepaling in een muismodel, wat de in vivo studie van idiopathische longfibrose kan vergemakkelijken. De techniek stelt de onderzoekers in staat om de functie van macrofagen te onderzoeken, gesimuleerd door traditionele cytokines bij het ontwikkelen van IPF. Haal om te beginnen de injectieflacon met clodronate liposomen uit de koelkast.Laat het 30 minuten opwarmen bij kamertemperatuur en keer meerdere...

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Medicine

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

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

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