CRISPR screening identifies DTX4 governing alveolar macrophage cholesterol efflux in pulmonary alveolar proteinosis
Zimu Wang1, Jingwei Shi2, Xu Ye3
1Department of Respiratory and Critical Care Medicine, Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
Abstract:
Pulmonary alveolar proteinosis (PAP) is a rare pulmonary syndrome characterized by impaired surfactant clearance, driven by dysfunctional cholesterol efflux in alveolar macrophages (AMs). However, the molecular determinants governing AM cholesterol homeostasis remain incompletely defined. Here, through a genome-wide CRISPR screen in foamy macrophages and bulk RNA sequencing of AMs from PAP patients, we identify DTX4 as a pivotal regulator of cholesterol efflux in AMs. In mice, AAV-mediated silencing of DTX4 led to excessive AM lipid accumulation, exacerbated proteinosis, increased lung opacities, and deteriorated pulmonary function. Similarly, DTX4 depletion in primary AMs impaired cholesterol efflux and promoted intracellular lipid deposition. Conversely, AM-specific overexpression of DTX4 in the Csf2ra-/- PAP model markedly alleviated lipid accumulation, mitigated alveolar proteinosis, restored lung densities, and rescued pulmonary function. Mechanistically, DTX4 stabilizes the GM-CSF receptor via an E3-independent interaction to sustain JAK2/STAT5 signaling, which reciprocally maintains DTX4 transcription. This positive-feedback loop drives PPARγ expression, and its disruption in PAP impairs cholesterol efflux, a defect partially reversible by ectopic PPARγ expression. Collectively, our findings identify DTX4 as a central orchestrator of AM cholesterol efflux and surfactant homeostasis, positioning it as a promising therapeutic target for PAP.
Insights
Researchers identified DTX4 as a key regulator of cholesterol efflux in alveolar macrophages (AMs), crucial for surfactant clearance in pulmonary alveolar proteinosis (PAP). Restoring DTX4 function improved lung function in PAP models.
Area of Science:
- Pulmonary Medicine
- Cell Biology
- Molecular Genetics
Background:
- Pulmonary alveolar proteinosis (PAP) is a rare lung disease caused by impaired surfactant clearance in alveolar macrophages (AMs).
- Cholesterol homeostasis in AMs is critical for surfactant metabolism, but the underlying molecular mechanisms are not fully understood.
- Dysfunctional cholesterol efflux in AMs is a key feature of PAP pathogenesis.
Purpose of the Study:
- To identify novel molecular regulators of cholesterol efflux in AMs relevant to PAP.
- To investigate the role of DTX4 in AM cholesterol homeostasis and its potential as a therapeutic target for PAP.
Main Methods:
- Genome-wide CRISPR screen in foamy macrophages.
- Bulk RNA sequencing of AMs from PAP patients and control subjects.
- In vivo studies using AAV-mediated gene manipulation in mouse models of PAP.
- Analysis of GM-CSF receptor signaling, JAK2/STAT5 pathway, and PPARγ expression.
Main Results:
- DTX4 was identified as a pivotal regulator of cholesterol efflux in AMs.
- DTX4 deficiency in mice led to lipid accumulation in AMs, exacerbated proteinosis, and impaired lung function.
- Overexpression of DTX4 in a mouse model of PAP alleviated lipid accumulation and improved pulmonary function.
- DTX4 stabilizes the GM-CSF receptor, sustaining JAK2/STAT5 signaling and driving PPARγ expression, which is crucial for cholesterol efflux.
Conclusions:
- DTX4 is a central regulator of cholesterol efflux and surfactant homeostasis in AMs.
- The DTX4-GM-CSF receptor-JAK2/STAT5-PPARγ axis is critical for maintaining AM cholesterol balance.
- DTX4 represents a promising therapeutic target for pulmonary alveolar proteinosis.
