The homeobox transcription factor VentX controls human macrophage terminal differentiation and proinflammatory activation
Xiaoming Wu, … , Roger W. Giese, Zhenglun Zhu
Xiaoming Wu, … , Roger W. Giese, Zhenglun Zhu
Published June 13, 2011
Citation Information: J Clin Invest. 2011;121(7):2599-2613. https://doi.org/10.1172/JCI45556.
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Research Article Immunology

The homeobox transcription factor VentX controls human macrophage terminal differentiation and proinflammatory activation

Abstract

Macrophages are critical players in both innate and adaptive immunity. While the exogenous signaling events leading to the terminal differentiation of macrophages from monocytes have been studied extensively, the underlying intracellular transcriptional mechanisms remain poorly understood. Here we report that the homeobox transcription factor VentX plays a pivotal role in human macrophage terminal differentiation and proinflammatory function. Our study showed that VentX expression was upregulated upon human primary monocyte-to-macrophage differentiation induced by cytokines such as M-CSF, GM-CSF, and IL-3. Moreover, ablation of VentX expression in primary monocytes profoundly impaired their differentiation to macrophages, and ectopic expression of VentX in a myeloid progenitor cell line triggered its differentiation with prominent macrophage features. Further analysis revealed that VentX was pivotal for the proinflammatory response of terminally differentiated macrophages. Mechanistically, VentX was found to control expression of proteins key to macrophage differentiation and activation, including M-CSF receptor. Importantly, preliminary analysis of gene expression in leukocytes from patients with autoimmune diseases revealed a strong correlation between levels of VentX and those of proinflammatory cytokines. Our results provide mechanistic insight into the crucial roles of VentX in macrophage differentiation and proinflammatory activation and suggest that dysregulation of VentX may play a role in the pathogenesis of autoimmune diseases.

Authors

Xiaoming Wu, Hong Gao, Weixiong Ke, Roger W. Giese, Zhenglun Zhu

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Figure 8

VentX expression is required for classical macrophage activation.

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VentX expression is required for classical macrophage activation. Macrop...
Macrophages were generated through incubation of freshly isolated monocytes with 100 ng/ml M-CSF for 4 days. These macrophages were then transfected with siGFP or siVentX and further cultured in RPMI 1640 medium for an additional 3 days. Thereafter, cells were exposed to 1 μg/ml LPS plus 20 ng/ml IFN-γ for 6 hours. (A) Surface staining of CD40, CD80, CD86, and HLA-DR and intracellular staining of TNF-α and IL-1β were analyzed by flow cytometry. (B) Levels of secreted TNF-α, IL-1β, and IL-12p70 from culture supernatants were determined with ELISA kits. (C) ROS from siGFP- or siVentX-transfected macrophages was analyzed with fluorescence microscopy (left; original magnification, ×200) and flow cytometry (middle). Right panel: Mean + SD of 3 different flow cytometry experiments. (D) Nitrate level from siGFP- and siVentX-transfected macrophages. Data represent mean + SD of 3 different experiments. *P < 0.05 (B–D). (E) Phagocytosis of siGFP- and siVentX-transfected macrophages. Red histogram represents transfection with siGFP; green histogram represents transfection with siVentX. Left panel: Cells were incubated on ice. Right panel: Cells were incubated at 37°C. (F) Effects of VENTX knockdown on mixed lymphocyte reaction. Irradiated macrophages that had been transfected with siGFP or siVentX were utilized to stimulate allogenic naive CD4+ T cell proliferation. The proliferation rates are presented as cpm. Results show mean + SD of triplicate wells of 1 representative experiment. Statistically significant difference (*P < 0.05) was observed when 4 × 102 or 2 × 103 cells were added.