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 7

TLR4/MyD88/ERK/CREB-mediated IL-10 production in Tregs alleviates liver I/R injury.

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TLR4/MyD88/ERK/CREB-mediated IL-10 production in Tregs alleviates liver ...
(A) Quantification of IL-10 expression in Tregs by flow cytometry from Tlr4fl/fl mice and Treg-Tlr4–/– mice before and after I/R (n = 6 per group). (B) Quantitative PCR analysis of Myd88, Mapk1, Mapk14, and Creb mRNA expression in Tregs from Tlr4fl/fl mice and Treg-Tlr4–/– mice before and after I/R (n = 5 per group). (C) Western blot analysis of MyD88, p-ERK, p-p38, and p-CREB protein expression levels in Tregs from Tlr4fl/fl mice and Treg-Tlr4–/– mice before and after I/R (n = 3 per group). (D) Experimental design for adoptive transfer of PBS, WT Tregs, or Myd88–/– Tregs into Treg-Tlr4–/– mice via tail vein injection 12 hours before I/R. (E) Serum ALT and AST levels in Treg-Tlr4–/– mice receiving PBS, WT Tregs, or Myd88–/– Tregs after liver I/R (n = 5 per group). (F) Serum IL-10 levels from Treg-Tlr4–/– mice adoptively transferred with PBS, Myd88–/– Tregs or WT Tregs after liver I/R (n = 5 per group). (G) Experimental design for adoptive transfer of PBS, WT Tregs, or IL-10–/– Tregs into Treg-Tlr4–/– mice via tail vein injection 12 hours before I/R. (H) Serum ALT and AST levels in Treg-Tlr4–/– mice receiving PBS, WT Tregs, or IL-10–/– Tregs after liver I/R (n = 5 per group). (I) Serum IL-10 levels from Treg-Tlr4–/– mice adoptively transferred with PBS, IL-10–/– Tregs or WT Tregs after liver I/R (n = 5 per group). (J) Experimental design for administration of recombinant IL-10 and its control into Treg-Tlr4–/– mice. (K) Serum ALT and AST levels in Tlr4fl/fl mice and Treg-Tlr4–/– mice receiving recombinant IL-10 or its control after I/R (n = 5 per group). (L) Serum IL-10 levels from Treg-Tlr4–/– mice receiving recombinant IL-10 or its control after I/R (n = 5 per group). Statistical analyses were performed using unpaired, 2-tailed t tests (A), 2-way ANOVA with Šidák’s post test (B, C, K, and L) and 1-way ANOVA with Tukey’s post test (E, F, and H–I). *P < 0.05, **P < 0.01, ***P < 0.001. ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; I/R, ischemia/reperfusion; rIL-10, recombinant IL-10.