CRISPR screening identifies DTX4 governing alveolar macrophage cholesterol efflux in pulmonary alveolar proteinosis
Zimu Wang, Jingwei Shi, Xu Ye, Xinye Xia, Huihui Zhu, Qi Li, Min Chen, Yichao Zhao, Yingwei Zhang, Mengshu Cao, Yonglong Xiao, Xinmei Huang
Zimu Wang, Jingwei Shi, Xu Ye, Xinye Xia, Huihui Zhu, Qi Li, Min Chen, Yichao Zhao, Yingwei Zhang, Mengshu Cao, Yonglong Xiao, Xinmei Huang
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Research Article Metabolism Pulmonology

CRISPR screening identifies DTX4 governing alveolar macrophage cholesterol efflux in pulmonary alveolar proteinosis

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.

Authors

Zimu Wang, Jingwei Shi, Xu Ye, Xinye Xia, Huihui Zhu, Qi Li, Min Chen, Yichao Zhao, Yingwei Zhang, Mengshu Cao, Yonglong Xiao, Xinmei Huang

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Figure 4

AM-specific DTX4 knockdown impairs pulmonary cholesterol clearance and lung function.

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AM-specific DTX4 knockdown impairs pulmonary cholesterol clearance and l...
(A) Schematic showing the design of the AAV vector (left) and the strategy for AM-specific knockdown of DTX4 in vivo (right). (B and C) Quantification of AAV transduction efficiency (B) and targeting specificity (C) in AMs by flow cytometry (n = 3). (D) Western blot analysis of indicated protein in lung tissue; GAPDH was used as loading control (n = 5). (E) Representative images of DTX4 (red) and GFP (green) expression in the lung; nuclei were stained with Hoechst (blue). Scale bars: 50 μm. (F) Gross lung morphology of mice from different groups. (G) Representative images of BALF collected from each group. (H) Quantification of BALF turbidity, total protein concentration, and total cholesterol levels across groups (n = 8). (I) Quantification of GM-CSF, M-CSF, and MCP-1 levels in BALF by ELISA (n = 8). (J) Representative micro-CT images of the lungs from each group, including transverse, dorsal, and 3D reconstructed views. (K and L) Quantification of poorly aerated lung tissue (K) and relative total lung volume (L) based on micro-CT analysis (n = 4). (M) H&E staining of lung sections showing tissue structure (n = 8). Scale bars: 1 mm (top), 50 μm (bottom). (N and O) PAS staining of lung sections with representative images (N) and quantification (O) (n = 8). Scale bars: 1 mm (top), 50 μm (bottom). (P and Q) Oil Red O staining of lung sections with representative images (P) and quantification (Q) (n = 8). Scale bars: 100 μm. Statistical comparisons were performed using a 2-tailed unpaired t test; P values are indicated (*P < 0.05, ****P < 0.0001).