ALKBH5-Driven m6A Demethylation Boosts Inflammation and Autophagy in LPS-Stimulated Macrophages

Purpose: Acute Respiratory Distress Syndrome (ARDS) remains a critical health threat with limited pharmacological treatments. This study investigates the role of the N⁶-methyladenosine (m⁶A) demethylase AlkB homolog 5 (ALKBH5) in alveolar macrophages and its subsequent impact on inflammation and Autophagy during ARDS pathogenesis.

Methods: Primary mouse alveolar macrophages were stimulated with Lipopolysaccharide (LPS) and transfected with ALKBH5 knockdown or overexpression plasmids. Global m6A levels were assessed via Dot Blot, while m⁶A modification of the target gene ULK1 was analyzed using MeRIP-qPCR. Macrophage polarization, migration, and Autophagy (LC3-II/p62 flux) were evaluated in vitro. The therapeutic potential of ALKBH5 downregulation was validated in an LPS-induced murine ARDS model through lung histology, cytokine analysis (ELISA), and microvascular permeability assessments.

Results: ALKBH5 was found to be a critical regulator of m⁶A methylation in alveolar macrophages. LPS stimulation decreased m⁶A modification of ULK1 mRNA, a process reversed by ALKBH5 downregulation. ALKBH5 knockdown significantly suppressed LPS-induced Autophagy by reducing autophagosome formation and inhibited M1 pro-inflammatory polarization. In vivo, ALKBH5 downregulation significantly mitigated lung tissue damage, reduced pulmonary edema, and lowered levels of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-17.

Conclusions: Our findings demonstrate that ALKBH5-driven m⁶A demethylation of ULK1 exacerbates ARDS by promoting macrophage Autophagy and pro-inflammatory responses. These results suggest that targeting ALKBH5 may disrupt pathogenic m6A demethylation, offering a novel therapeutic strategy for mitigating ARDS progression.