TLR4 maintains Treg-mediated protection against adverse outcomes in a model of hepatic surgical stress
Hongji Zhang, Yunwei Zhang, Tianxing Ren, Carolyn Tsung, Peng Song, Peng Xu, Guoliang Wang, Chunyan Cao, Changyan Wang, Ping Sun, Qi Zhang, Yanhong Zhu, Xin Zhong, Yong Guan, Xiaofei Zhang, Han Wang, Jinxiang Zhang, Hui Wang
Hongji Zhang, Yunwei Zhang, Tianxing Ren, Carolyn Tsung, Peng Song, Peng Xu, Guoliang Wang, Chunyan Cao, Changyan Wang, Ping Sun, Qi Zhang, Yanhong Zhu, Xin Zhong, Yong Guan, Xiaofei Zhang, Han Wang, Jinxiang Zhang, Hui Wang
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Research Article Hepatology Immunology Inflammation

TLR4 maintains Treg-mediated protection against adverse outcomes in a model of hepatic surgical stress

Abstract

Surgical stress, such as hepatic ischemia-reperfusion (I/R) injury, induces excessive inflammation and adversely affects liver surgery outcomes. Regulatory T cells (Tregs) are crucial for immune homeostasis, yet their protective mechanisms against liver I/R injury remain unclear. In this study, we demonstrated that decreased hepatic Treg abundance correlates with increased liver injury in patients undergoing hepatic hemangioma resections. In murine models, Treg depletion worsened liver I/R injury. Bulk RNA-seq of hepatic Tregs showed enrichment of Toll-like receptor (TLR) signaling pathways, with flow cytometry identifying TLR4 as the most increased TLR after I/R. Treg-specific Tlr4 knockout mice (Treg-Tlr4–/– mice) exhibited exacerbated liver injury following I/R. Adoptive transfer of WT Tregs, but not Tlr4-deficient Tregs, alleviated liver injury in both Treg-depleted and Treg-Tlr4–/– mice. Transcriptomic analysis revealed that IL-10 production was impaired in Tlr4-deficient Tregs. Mechanistically, Tlr4-deficient Tregs showed reduced activation of the MyD88/ERK/CREB pathway, resulting in diminished IL-10 production. Myd88–/– and IL-10–/– Tregs failed to confer protection against liver I/R injury, whereas exogenous IL-10 administration rescued the hepatic dysfunction in Treg-Tlr4–/– mice. Our findings implicate the vital role of TLR4 in Tregs to mitigate liver I/R injury and offer a potential therapeutic option to reduce postoperative complications following liver surgery.

Authors

Hongji Zhang, Yunwei Zhang, Tianxing Ren, Carolyn Tsung, Peng Song, Peng Xu, Guoliang Wang, Chunyan Cao, Changyan Wang, Ping Sun, Qi Zhang, Yanhong Zhu, Xin Zhong, Yong Guan, Xiaofei Zhang, Han Wang, Jinxiang Zhang, Hui Wang

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Figure 3

Specific deletion of Tlr4 in Tregs exacerbates liver I/R injury.

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Specific deletion of Tlr4 in Tregs exacerbates liver I/R injury. (A) Flo...
(A) Flow cytometry quantification of hepatic CD4+FOXP3+ Tregs in Tlr4fl/fl and Treg-Tlr4–/– mice before and after liver I/R (n = 5 per group). (B) Serum ALT and AST levels in Tlr4fl/fl and Treg-Tlr4–/– mice before and after liver I/R (n = 5 per group). (C) Representative H&E staining images of ischemia liver lobe from Tlr4fl/fl and Treg-Tlr4–/– mice before and after liver I/R (n = 5 per group). Scale bars: 200 μm. (D) Quantification of hepatic infiltrating CD11b+Ly6G+ neutrophils and CD11b+ Ly6C+ monocytes by flow cytometry in Tlr4fl/fl and Treg-Tlr4–/– mice before and after liver I/R (n = 5 per group). (E) Quantitative PCR analysis of Il1b, Tnf, and Il10 mRNA expression in liver from sham and I/R Tlr4fl/fl and Treg-Tlr4–/– mice (n = 5 per group). (F) Serum IL-1β, TNF-α, and IL-10 levels from Tlr4fl/fl and Treg-Tlr4–/– mice before and after liver I/R (n = 5 per group). Statistical analyses were performed using unpaired, 2-tailed t tests (A) and 2-way ANOVA with Šidák’s post test (B–F). *P < 0.05, **P < 0.01, ***P < 0.001. ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; I/R, ischemia/reperfusion.