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 ....

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

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.......

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 .......

Materials

List of materials used in this article

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