The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and limits antimicrobial immunity
Xiang Chen, … , Seon Hee Chang, Chen Dong
Xiang Chen, … , Seon Hee Chang, Chen Dong
Published September 15, 2022
Citation Information: J Clin Invest. 2022;132(18):e147566. https://doi.org/10.1172/JCI147566.
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Research Article Immunology

The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and limits antimicrobial immunity

Abstract

Forkhead box O transcriptional factors, especially FoxO1 and FoxO3a, play critical roles in physiologic and pathologic immune responses. However, the function of FoxO4, another main member of the FoxO family, in lymphoid cells is still poorly understood. Here, we showed that loss of FoxO4 in T cells augmented IFN-γ production of Th1 cells in vitro. Correspondingly, conditional deletion of FoxO4 in CD4+ T cells enhanced T cell–specific responses to Listeria monocytogenes infection in vivo. Genome-wide occupancy and transcriptomic analyses identified Dkk3 (encoding the Dickkopf-3 protein) as a direct transcriptional target of FoxO4. Consistent with the FoxO4-DKK3 relationship, recombinant DKK3 protein restored normal levels of IFN-γ production in FoxO4-deficient Th1 cells through the downregulation of lymphoid enhancer–binding factor 1 (Lef1) expression. Together, our data suggest a potential FoxO4/DKK3 axis in Th1 cell differentiation, providing what we believe to be an important insight and supplement for FoxO family proteins in T lymphocyte biology and revealing a promising target for the treatment of immune-related diseases.

Authors

Xiang Chen, Jia Hu, Yunfei Wang, Younghee Lee, Xiaohong Zhao, Huiping Lu, Gengzhen Zhu, Hui Wang, Yu Jiang, Fan Liu, Yongzhen Chen, Byung-Seok Kim, Qinghua Zhou, Xindong Liu, Xiaohu Wang, Seon Hee Chang, Chen Dong

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Figure 4

Identification of Dkk3 as a direct target gene of FoxO4.

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Identification of Dkk3 as a direct target gene of FoxO4. (A) Flow cytome...
(A) Flow cytometry of CD4+ T cells cultured under Th1-polarizing conditions and transduced with an empty vector (RV) or a vector encoding FoxO4 (RV-FoxO4), followed by restimulation of the cells and intracellular staining for IFN-γ. Data were analyzed in GFP– and GFPhi cell populations (n = 4). (B) Frequency of IFN-γ–expressing cells as in A (n = 4). (C) A gene expression microarray experiment was performed using sorted GFP+ cells transduced with RV or RV-FoxO4 as in A. Heatmap shows the gene expression intensities of some of the most significantly regulated genes (FDR <0.05 and fold change >1.5), including cytokine and chemokine genes (left) and transcriptional factor genes (right). (D) Real-time qPCR analysis was performed to validate gene expression changes [log2(KO/WT)], as in C, in polarized Th1 cells from WT and FoxO4-cKO mice after 5 hours of stimulation with plate-bound anti-CD3. (E) Distribution of genome-wide FoxO4-binding sites in polarized Th1 cells in vitro relative to annotated features of known genes. (F) Venn diagram of FoxO4-bound genes and FoxO4-bound genes containing the FoxO-binding motif. (G) Venn diagram of genes regulated by FoxO4 and FoxO4-bound genes containing the FoxO-binding motif. (H) The base sequence represents the consensus FoxO-binding motif (JASPAR), which was found at the Dkk3 locus with a score of 11.3. (I) FoxO4-binding peaks located at the Dkk3 gene locus. MACS software was applied to determine peak significance within ChIP-Seq data, and a threshold of P < 1 × 10–5 was used for peak calling. ***P < 0.001, by unpaired, 2-tailed Student’s t test (B). Data are representative of 2 independent experiments (mean ±SD in B).