Isolation, cultivation, identification, and induction of M1/M2 polarization in bone marrow-derived macrophages from C57BL/6 mice

Abstract

BACKGROUND: Macrophage polarization demonstrates significant potential in disease treatment, particularly in areas such as cancer, inflammation, and autoimmune diseases. Establishing standardized in vitro models can lay the groundwork for in-depth research into the mechanisms of macrophage polarization. OBJECTIVE: To observe the in vitro growth characteristics of bone marrow-derived macrophages from C57BL/6 mice and to establish a standardized in vitro model for M1 and M2 macrophage polarization. METHODS: Femurs and tibias of C57BL/6 mice were aseptically separated, and the contents of the bone marrow cavity were collected. After filtering through a mesh and lysing erythrocytes, the contents were resuspended in high-glucose DMEM containing 20 ng/mL macrophage colony-stimulating factor and inoculated in 6-well plates according to experimental requirements. On day 7, they were differentiated into mature mouse bone marrow-derived macrophages (M0 type). Then, 100 ng/mL lipopolysaccharide was used to induce polarization to M1 type, and 20 ng/mL interleukin-4 was used to induce polarization to M2 type. Flow cytometry and RT-qPCR were used to detect the expression of corresponding markers in macrophages under different polarization states, and Western blot was used to detect the expression of M1 macrophage marker pathway proteins p-STAT1, STAT1 and M2 macrophage marker pathway proteins p-STAT6, STAT6. RESULTS AND CONCLUSION: (1) After stimulation with 20 ng/mL macrophage colony-stimulating factor for 7 days, flow cytometry showed that the positive rate of macrophage surface marker F4/80 reached 98.1%. (2) After stimulation with 100 ng/mL lipopolysaccharide for 6 h, the positive rates of F4/80 and CD86 were about 35%, and RT-qPCR showed that the mRNA expression of M1 macrophage markers inducible nitric oxide synthase, interleukin-6, macrophage inflammatory protein 1α, and monocyte chemoattractant protein 1 were significantly higher than those in the control group (P < 0.01). (3) After stimulation with 20 ng/mL interleukin-4 for 24 h, the mean fluorescence intensity of CD206 was significantly increased, and RT-qPCR showed that the mRNA expression of M2 macrophage markers Chi3l3 (Ym1), interleukin-10, and arginase 1 were significantly higher than those in the control group (P < 0.01). (4) Western blot results showed that lipopolysaccharide-induced M1 macrophage marker pathway protein p-STAT1 was significantly activated; interleukin-4-induced M2 macrophage marker pathway protein p-STAT6 was significantly activated. These results indicate that lipopolysaccharide and interleukin-4 effectively induced polarization of bone marrow-derived macrophages to M1 and M2 types, respectively.

1. Introduction

Macrophages are key cells in the human immune system and play an important role in disease pathogenesis and inflammatory microenvironment formation [1]. Macrophage polarization refers to the process by which macrophages change their phenotype according to microenvironmental changes, thereby exerting multiple functions, also known as macrophage phenotype switching or reprogramming. Generally, macrophage polarization induced by microenvironmental changes is highly heterogeneous and plastic, and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2) [2-3]. The induction of M1 macrophages mainly depends on the combined action of Th1 cytokines interferon-γ and/or lipopolysaccharide. When pathogens invade the body, macrophages, as the main effector cells for eliminating pathogens, polarize to M1 type, recognize antigens, and release a large number of pro-inflammatory mediators (such as interleukin-1, interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase) and chemokines (such as chemokine 2-4 and chemokine 8-11) [4-5], thereby producing reactive oxygen species and reactive nitrogen species, and enhancing the expression of surface markers such as MHC-I/II, CD80, CD86, exerting pro-inflammatory effects [6-7]. The induction of M2 macrophages depends on the action of Th2 cytokines interleukin-4 and/or interleukin-13. M2 macrophages include three subgroups: M2a, M2b, and M2c, which can respond specifically to different stimuli. M2a macrophages are induced by interleukin-4 and interleukin-13, M2b macrophages are induced by interleukin-1β, and M2c macrophages are induced by glucocorticoids, interleukin-10, and transforming growth factor-β. All three subtypes highly express anti-inflammatory factors and participate in tissue repair and immune regulation.