Prof.  Yi  Wang
University of Electronic Science and Technology of China,  China

Title: Nuclear Translocation of PFKFB3 in Kupffer Cells Regulates PD-1 Expression During Sepsis-Induced Acute Liver Injury

Abstract:

Objective: Sepsis is one of the main causes of clinical mortality. The liver is an important organ for maintaining homeostasis and regulating host defense. During sepsis, the liver transitions from a tolerable state to an active immune state, forming the first line of defense against microbes and endotoxins. However, excessive inflammatory activation can lead to hepatic injury and immunosuppression. In the current study, we investigated the specific molecular mechanism by which PFKFB3, a key protein in glycolysis, regulates the function of KCs in sepsis.

Methods: We established three types of sepsis mouse models, including cecal ligation (CLP), LPS injection, and Escherichia coli (E. coli) injection. Immunohistochemical analysis was performed to assess PFKFB3 expression and subcellular localization in KCs across these models. We also isolated KC cells, a kind of resident macrophage, from three model animals, extracted nuclear proteins and cytoplasmic proteins, and performed immunoblotting on the expression of PFKFB3 protein in the nucleus. These experiments jointly verify the nuclear translocation of chromatin immunoprecipitation (ChIP)-PCR assay was conducted to screen downstream regulatory targets of PFKFB3. Additionally, we evaluated expression changes of associated proteins in animal models treated with the PFKFB3 inhibitor 3PO.

Results: Immunohistochemical staining and immunoblotting showed that the expression of PFKFB3 was significantly up-regulated and transferred from cytoplasm to nucleus in KCs in three animal models of sepsis. But its phosphorylation level remained unchanged, indicating that the total protein of PFKFB3 entered the nucleus to play a messenger role. The anti-PFKFB3 antibody was co-immunoprecipitated with the chromosomes of LPS-stimulated KCs, and then the result of PCR amplification showed that PFKFB3 binds to the CR-C region of the promoter of the Pdcd1 gene. The transcription level of the Pdcd1 gene was significantly increased in liver KCs of three animal models of sepsis. But the expression of the Pdcd1 gene in KCs isolated from animal liver was significantly reduced after administration of the PFKFB3 protein inhibitor 3PO. This result suggests to us that PFKFB3 may promote immunosuppression-induced liver injury by upregulating PD-1 expression through transfer into the KC nucleus.

Conclusion: In sepsis, PFKFB3 transfers into the KC nucleus and binds to the promoter region of the Pdcd1 gene to promote its transcription to induce immune tolerance and exacerbate the inflammatory situation of the patients.

Biography: