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Ag85B-induced M1 Macrophage Polarization via the TLR4/TRAF6/NF-κB Axis Leading to Bronchial Epithelial Cell Damage
Lei Zhou1, Li Luo1, Linzi Luo1
1Endoscopy Center, Hunan Chest Hospital, Hunan Provincial Center for Disease Control and Prevention, Changsha, 410016, China.
Background:
Antigen 85B (Ag85B) is a signature antigen of Mycobacterium tuberculosis (MTB). In this study, we aimed to investigate the impact of macrophages stimulated with Ag85B on bronchial epithelial cells and T cells, as well as the underlying mechanisms involved.
Methods:
We used Ag85B to stimulate macrophage and investigated the impact of Ag85B on macrophage polarization. We assessed the impact of TLR4 on Ag85Bmediated macrophage polarization by silencing TLR4. Additionally, the regulatory role of TLR4 on the TRAF6/NF-κB pathway was evaluated through immunoblotting. Activated macrophages with Ag85B were co-cultured with mouse bronchial epithelial cells (MBECs) and T cells, respectively. Through immunoblotting quantification, biochemical methods, and flow cytometry, we explored the effects and molecular mechanisms of Ag85B-induced macrophage activation on bronchial epithelial cell damage and T-cell transformation.
Results:
In macrophages stimulated with Ag85B, levels of M1 polarization-related genes (CXCL9, CXCL10, and iNOS) and cytokines (IL-6, TNF-α, IL-1β, and IL-12) were increased, and the M1/M2 ratio was elevated. TLR4 silence inhibited the effects of Ag85B on macrophages and decreased TRAF6 and p-NF-κB/NF-κB levels. TRAF6 overexpression reversed the inhibitory effect of TLR4 on macrophage stimulation with Ag85B. After co-culturing with macrophages induced by Ag85B, MBEC cell proliferation was inhibited, apoptosis was promoted, and the TH17/Treg ratio of T cells was increased. Silencing TLR4 reversed the impact of Ag85B-induced macrophage polarization on bronchial epithelial cells and T cells, which was further reversed by TRAF6 overexpression.
Conclusion:
Ag85B promoted M1 polarization in macrophages through the TLR4/TRAF6/NF-κB axis, resulting in bronchial epithelial cell damage and an imbalance in TH17/Treg cells.
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