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ALKBH5-Driven m6A Demethylation Boosts Inflammation and Autophagy in LPS-Stimulated Macrophages
Gui Wang1, Ting Zhou1, Shujun Zhou1
1Department of Critical Care Medicine, The First People's Hospital of Changzhou, The Third Affiliated Hospital of Soochow University, Changzhou, China.
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.
Insights
Targeting ALKBH5, an m6A demethylase, may offer a novel therapy for Acute Respiratory Distress Syndrome (ARDS). Downregulating ALKBH5 in macrophages reduces inflammation and autophagy, mitigating ARDS severity.
Area of Science:
- Immunology
- Molecular Biology
- Respiratory Medicine
Background:
- Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with limited treatment options.
- Alveolar macrophages play a key role in ARDS pathogenesis.
- The role of m6A modification and its regulators in ARDS is not fully understood.
Purpose of the Study:
- To investigate the function of ALKBH5 (N6-methyladenosine demethylase) in alveolar macrophages during ARDS.
- To determine the impact of ALKBH5 on inflammation and autophagy in ARDS.
- To explore ALKBH5 as a potential therapeutic target for ARDS.
Main Methods:
- Primary mouse alveolar macrophages were stimulated with LPS and manipulated for ALKBH5 expression (knockdown/overexpression).
- m6A levels, ULK1 mRNA modification, macrophage polarization, and autophagy were assessed in vitro.
- Therapeutic effects of ALKBH5 downregulation were evaluated in an LPS-induced murine ARDS model.
Main Results:
- ALKBH5 regulates m6A methylation in alveolar macrophages, with its downregulation reversing LPS-induced ULK1 mRNA m6A decrease.
- ALKBH5 knockdown suppressed LPS-induced autophagy and M1 pro-inflammatory macrophage polarization.
- In vivo, ALKBH5 downregulation attenuated lung injury, edema, and pro-inflammatory cytokine release in an ARDS model.
Conclusions:
- ALKBH5-mediated m6A demethylation of ULK1 exacerbates ARDS by promoting macrophage autophagy and inflammation.
- Targeting ALKBH5 presents a novel therapeutic strategy to disrupt pathogenic m6A demethylation and mitigate ARDS progression.
