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).
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.
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 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.
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
2. Isolation and culture of IMs
3. Adoptive transfer of IMs into the lung alveoli
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.
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 >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 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-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.
The authors have nothing to disclose.
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).
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 |