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DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries
Yu-Chang Ku1, Min-Hua Lai1, Chen-Chia Lo1
1Department of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.
Abstract:
Recent studies have suggested that DDX3 functions in antiviral innate immunity, but the underlying mechanism remains elusive. We previously identified target mRNAs whose translation is controlled by DDX3. Pathway enrichment analysis of these targets indicated that DDX3 is involved in various infections and inflammation. Using immunoblotting, we confirmed that PACT, STAT1, GNB2, Rac1, TAK1, and p38 mitogen-activated protein kinase (MAPK) proteins are downregulated by DDX3 knockdown in human monocytic THP-1 cells and epithelial HeLa cells. Polysome profiling revealed that DDX3 knockdown reduces the translational efficiency of target mRNAs. We further demonstrated DDX3-mediated translational control of target mRNAs by luciferase reporter assays. To examine the effects of DDX3 knockdown on macrophage migration and phagocytosis, we performed in vitro cell migration assay and flow cytometry analysis of the uptake of green fluorescent protein-expressing Escherichia coli in THP-1 cells. The DDX3 knockdown cells exhibited impaired macrophage migration and phagocytosis. Moreover, we used a human cytokine antibody array to identify the cytokines affected by DDX3 knockdown. Several chemokines were decreased considerably in DDX3 knockdown THP-1 cells after lipopolysaccharide or poly(I·C) stimulation. Lastly, we demonstrated that DDX3 is crucial for the recruitment of phagocytes to the site of inflammation in transgenic zebrafish.
Insights
The DEAD-box helicase 3 (DDX3) protein regulates gene translation and is vital for macrophage functions, including migration and phagocytosis, impacting innate immunity during infections.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- The role of DEAD-box helicase 3 (DDX3) in antiviral innate immunity is suggested but not fully understood.
- Previous research identified DDX3 as a regulator of target mRNA translation.
- Pathway analysis of DDX3 targets indicates involvement in infection and inflammation pathways.
Purpose of the Study:
- To elucidate the mechanism of DDX3's function in antiviral innate immunity.
- To investigate the impact of DDX3 on macrophage functions like migration and phagocytosis.
- To identify specific molecular pathways and cytokines regulated by DDX3.
Main Methods:
- DDX3 knockdown in human monocytic (THP-1) and epithelial (HeLa) cells.
- Immunoblotting to assess protein levels (PACT, STAT1, GNB2, Rac1, TAK1, p38 MAPK).
- Polysome profiling and luciferase reporter assays for translational efficiency.
- In vitro cell migration assays and flow cytometry for phagocytosis.
- Human cytokine antibody array and zebrafish inflammation models.
Main Results:
- DDX3 knockdown led to downregulation of key immune proteins and reduced translational efficiency of target mRNAs.
- DDX3-depleted cells showed impaired macrophage migration and phagocytosis of Escherichia coli.
- Cytokine antibody arrays revealed decreased chemokine levels in DDX3 knockdown cells post-stimulation.
- DDX3 was essential for phagocyte recruitment to inflammatory sites in zebrafish.
Conclusions:
- DDX3 plays a critical role in regulating mRNA translation, impacting innate immune responses.
- DDX3 is essential for macrophage migration, phagocytosis, and chemokine production.
- DDX3 is crucial for effective inflammatory responses and phagocyte recruitment in vivo.
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