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
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.
...

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+macrophages+in+the+development+of+acute+and+chronic+inflammatory+lung+diseases.">The role of macrophages in the development of acute and chronic inflammatory lung diseases.</a> Cells. 10 (4), 897 (2021).</li><li>Luzina, I. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-33+potentiates+bleomycin-induced+lung+injury.">Interleukin-33 potentiates bleomycin-induced lung injury.</a> American Journal of Respiratory Cell and Molecular Biology. 49 (6), 999-1008 (2013).</li><li>Nie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AKT2+regulates+pulmonary+inflammation+and+fibrosis+via+modulating+macrophage+activation.">AKT2 regulates pulmonary inflammation and fibrosis via modulating macrophage activation.</a> Journal of Immunology. 198 (11), 4470-4480 (2017).</li><li>Qian, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+PU.1+promotes+alternative+macrophage+polarization+and+asthmatic+airway+inflammation.">The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation.</a> Journal of Molecular Cell Biology. 7 (6), 557-567 (2015).</li><li>Shah, S., Dhawan, V., Holm, R., Nagarsenker, M. 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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 ...

An erratum was issued for:&#160;<a href="https://www.jove.com/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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;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&#160;Accuri&#160;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&#38;D Systems,USA</td>
			<td>1124F</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>R&#38;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&#160;</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&#38;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&#160;</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...

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 ...

The protocol describes the isolation of pulmonary IMS and adoptive transfer after IL-33 determination in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis. The technique enables the researchers to explore the function of macrophages, simulated by traditional cytokines in developing IPF. To begin, take out the vial of clodronate liposomes from the refrigerator.Allow it to warm for 30 minutes at room temperature and invert several times to ensure uniform mixing. Aspirate 60 microliters of clodronate liposomes using a pipette with a sterile suction tip. Administer the control and drug drop-wise into the nasal cavity of the anesthetized mouse.After each drop, ensure the mouse inhales the drug completely and breathes evenly. After the depletion of alveolar macrophages in the recipient mouse, begin by disinfecting the skin of the anesthetized host mouse with 75%alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue.Aspirate 10 milliliters of PBS in a syringe equipped with a 20-gauge needle, and insert the tip of the needle into the right atrium of the mouse. Then cut the inferior vena cava of the mouse with surgical scissors and manually perfuse the mouse with PBS at a constant speed of 10 to 20 milliliters per minute until the lung tissue turns white. Further, excise the lung tissue and transfer it into ice cold PBS in a culture dish.Cut the lung tissue into fragments and add 15 milliliters of DMEM medium containing 1%collagenase A to isolate the macrophages, then incubate it at 100 RPM for 30 minutes on a 37 degree Celsius shaking table. Aspirate the lung tissue suspension approximately 20 times through a 10-milliliter syringe to make it fine. Filter the suspension through a 40-micrometer cell strainer.Centrifuge the filtrate at 400 G for 10 minutes. After discarding the supernatant, lyse the red blood with three milliliters of cell lysis buffer on ice for two to three minutes. Centrifuge at 150 G for five minutes and resuspend the cell pellet in 10 milliliters of DMEM.Then count the cells using a hemocytometer. Seed the cells into a 10-centimeter adherent cell culture dish and incubate for one hour at 37 degrees Celsius and 5%carbon dioxide to adhere the lung interstitial macrophages to the dishes. After one hour, aspirate the supernatant and floating cells.Add 10 milliliters of the fresh, complete culture medium and incubate for over eight hours or overnight. To stimulate the isolated interstitial macrophages, replace the spent culture medium with a complete medium interleukins 33. Also prepare a control plate by adding PBS instead of interleukins 33.Incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. To dissociate the pulmonary interstitial macrophages, treat them with one milliliter of 0.25%trypsin for five minutes. Then add three milliliters of fresh culture complete medium to quench the digestion and centrifuge the cell suspension to harvest the pulmonary macrophages.After counting the cells, resuspend the cells in PBS to a final concentration of five times 10 to the fifth cells per 50 microliters. To begin the transfer of interstitial macrophages, fix the limbs of the anesthetized recipient mouse on a vertical plate with medical tape and gently pull the mouse's tongue to one side, using a cotton swab. After turning the intubation lamp on, shine it on the throat of the mouse to see the trachea, which should be close to the base of the tongue.Insert the indwelling needle cannula into the trachea using the 22-gauge intubation lamp and 0.4 millimeter diameter guide wire. To finish the intubation, remove the guide wire and push the cannula into the mouse trachea. Now inject 50 microliters of the cell suspension into the recipient mouse through the trachea when the cannula is seen to enter the trachea.After 24 hours, administer bleomycin via the trachea to induce idiopathic pulmonary fibrosis. Also administer an equal volume of saline to the control group. After 21 days, determine the degree of severity of pulmonary fibrosis by evaluating the expression of markers for fibrosis and the Ashcroft scores.The HE staining showed that the adoptive transfer of interleukins 33 stimulated macrophages exacerbated the degree of lung tissue destruction and increased fibroblast aggregation in the bleomycin-stimulated mice, compared to control. The increase in the Ashcroft score also illustrates the degree of pulmonary fibrosis. The expression levels of mRNA of alpha SMA and fibronectin in lung tissues of the recipient mouse containing adoptively transferred interleukins 33-stimulated interstitial macrophages and treated with bleomycin were higher, compared with those of the BLM-treated wild-type mice.500 microliters of air should be immediately entered into the lungs with a one-millimeter syringe to ensure that the cell suspension in the cannula can completely enter the lung tissue.

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