Zfp335 establishes eTreg lineage and neonatal immune tolerance by targeting Hadha-mediated fatty acid oxidation
Xin Wang, Lina Sun, Biao Yang, Wenhua Li, Cangang Zhang, Xiaofeng Yang, Yae Sun, Xiaonan Shen, Yang Gao, Bomiao Ju, Yafeng Gao, Dan Liu, Jiapeng Song, Xiaoxuan Jia, Yanhong Su, Anjun Jiao, Haiyan Liu, Lianjun Zhang, Lan He, Lei Lei, WanJun Chen, Baojun Zhang
Xin Wang, Lina Sun, Biao Yang, Wenhua Li, Cangang Zhang, Xiaofeng Yang, Yae Sun, Xiaonan Shen, Yang Gao, Bomiao Ju, Yafeng Gao, Dan Liu, Jiapeng Song, Xiaoxuan Jia, Yanhong Su, Anjun Jiao, Haiyan Liu, Lianjun Zhang, Lan He, Lei Lei, WanJun Chen, Baojun Zhang
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Research Article Autoimmunity Immunology

Zfp335 establishes eTreg lineage and neonatal immune tolerance by targeting Hadha-mediated fatty acid oxidation

Abstract

Regulatory T cells (Tregs) are instrumental in maintaining immune tolerance and preventing destructive autoimmunity, but how heterogeneous Treg populations are established remains largely unknown. Here, we show that Zfp335 deletion in Tregs failed to differentiate into effector Tregs (eTregs) and lose Treg-suppressive function and that KO mice exhibited early-onset lethal autoimmune inflammation with unrestricted activation of conventional T cells. Single-cell RNA-Seq analyses revealed that Zfp335-deficient Tregs lacked a eTreg population and showed dramatic accumulation of a dysfunctional Treg subset. Mechanistically, Zfp335-deficient Tregs displayed reduced oxidative phosphorylation and dysfunctional mitochondrial activity. Further studies revealed that Zfp335 controlled eTreg differentiation by regulating fatty acid oxidation (FAO) through direct targeting of the FAO enzyme Hadha. Importantly, we demonstrate a positive correlation between ZNF335 and HADHA expression in human eTregs. Our findings reveal that Zfp335 controls FAO-driven eTreg differentiation to establish immune tolerance.

Authors

Xin Wang, Lina Sun, Biao Yang, Wenhua Li, Cangang Zhang, Xiaofeng Yang, Yae Sun, Xiaonan Shen, Yang Gao, Bomiao Ju, Yafeng Gao, Dan Liu, Jiapeng Song, Xiaoxuan Jia, Yanhong Su, Anjun Jiao, Haiyan Liu, Lianjun Zhang, Lan He, Lei Lei, WanJun Chen, Baojun Zhang

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Figure 6

Zfp335 facilitates eTreg differentiation via direct targeting of Hadha.

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Zfp335 facilitates eTreg differentiation via direct targeting of Hadha. ...
(A) Venn diagram shows the genes shared among the upregulated genes in eTregs and rTregs (WT vs. KO) and genes associated with metabolic pathways. (B) Heatmaps of the indicated genes between ERCre and ERCre Zfp335fl/fl Tregs. (C) ChIP-Seq peaks of Zfp335-bound regions in the indicated genes in WT Tregs compared with input. (D and E) CD4+YFP+CD44–ICOS– Tregs were isolated from 8-day-old WT and KO mice. WT Tregs were transfected with mock, while KO Tregs were transfected with mock, Ndufa4, Hadha, and Actr2 for 4 days. Representative FACS plots (D) and frequencies (E) of ICOS+ Tregs in different groups (n = 3). (F and G) Representative FACS plots (F) and MFI of MitoTracker Deep Red (G) in the indicated groups (n = 3). (H) Frequencies of each cell division of Tcons alone or in the presence of WT eTregs transfected with mock and KO eTregs transfected with mock or Hadha. (I–L) CD4+CD25+CD44–ICOS– rTregs sorted from ERCre and ERCre Zfp335fl/fl mice were activated by anti-CD3/anti-CD28 Abs and IL-2 in the presence or absence of 30 mM malate. Two days later, the cells were collected and subjected to FACS analysis. (I) Representative FACS plots of ICOS+ Tregs in different groups. (J) Frequencies of ICOS+ Tregs (n = 3). (K and L) Representative FACS plots (K) and MFI of MitoTracker Deep Red (L) (n = 3). Data are representative of 2 or 3 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test.