Background Keloid is a pathological scar caused by abnormal wound healing and characterized by a persistent local inflammatory response and excessive collagen deposition. Macrophage serves as the primary inflammatory cell for tissue remodeling after injury, and several studies have demonstrated that macrophage polarization affects wound healing outcomes.

Objective To explore the biological behavior of macrophages with different polarization states on keloid fibroblasts and the effects on TGF-β/Smad and Hippo pathways.

Methods The excised human keloid tissue was subjected to in vitro culturing to obtain human keloid fibroblasts. Human THP-1 cells were induced to differentiate into M0 macrophages by adding Phorbol 12-myristate 13-acetate (PMA) 100 mg/L for 48 h. Then Based on M0 macrophages, lipopolysaccharide (LPS) 100 mg/L + Interferon-γ (IFN-γ) 20 mg/L were added to induce the differentiation of M1 macrophages for 72 h, and Interleukin-4 (IL-4) 20 mg/L + Interleukin-13 (IL-13) 20 mg/L were used to induce the differentiation of M2 macrophages for 72 h. The macrophage intervention experiments were divided into blank control group, M0 macrophage group, M1 macrophage group and M2 macrophage group. Each group was subsequently co-cultured with keloid fibroblasts in a Transwell chamber for 24 hours, 48 hours and 72 hours, respectively, and the co-cultured keloid fibroblasts and their supernatant were taken. The hydroxyproline content in the supernatant of keloid fibroblasts in each group was detected, and the levels of IL-6, IL-10, and TGF-β1 in the supernatant were determined by ELISA; the proliferation rate of fibroblasts was measured by CCK-8 assay, the fibroblast migration rate was determined by scratch assay, and the transcript levels of TGF-β1, Smad3, col Ⅰ, col Ⅲ, YAP, and TAZ in fibroblasts were detected by RT-qPCR.

Results Compared with the control group, after 24 hours of co-culture, the migration and collagen secretion of keloid fibroblasts in the M2 macrophage group were enhanced (P<0.01), while the transcription levels of COL Ⅰ, COL Ⅲ, TGF-β1, Smad3, YAP, and TAZ were elevated (P<0.01), and keloid fibroblasts had inhibited proliferation at 24 h (P<0.01). There was no significant difference in proliferation ability between the M2 macrophage group and the control group at 48 h and 72 h. However, the proliferation of keloid fibroblasts in the M1 macrophage group was inhibited (P<0.01), and there was no significant difference in cell migration, collagen secretion, TGF-β/Smad and Hippo signaling pathway activity between the M1 macrophage group and control group.

Conclusion M2 macrophages enhance fibroblast invasive ability and fibrosis function, activate TGF-β/Smad and Hippo pathway, and macrophage polarization toward M2 is an important cause of keloid formation. And polarization toward M1 may play an inhibitory role in keloid formation.