Effect Of Anti-PD-L1Antibody On Liver Function In Mice With Experimental Sepsis

ObjectiveThis study examined the PD-L1expression in sepsis mice liver, and investigated howanti-PD-L1antibody took part in the process of sepsis. Otherwise, some cytokines in liverwere also examined to elucidate how PD-1/PD-L1pathway participated the liver injury insepsis.Methods1. Experimental sepsis was induced by cecal ligation and puncture (CLP).16C57BL/6mice were randomly divided into sham group (n=8) and CLP group (n=8).4μmfrozen sections of mice livers were made24hour after operation. Immunohistochemisty(IHC) was performed by anti-PD-L1antibody as first antibody, and second antibody wasconjugated with horseradish peroxidase (HRP). Sections were observed under microscopeto analyze the expression of PD-L1. On the other side, mice livers were homogenated toextract RNA, and Q-RT-PCR was performed to examine the mRNA level of PD-L1inliver.2. Experimental sepsis was induced by CLP.32C57BL/6mice were randomlydivided into sham group (n=8), CLP group (n=8), isotype group (n=8) and anti-PD-L1group (n=8). Sepsis was induced in all mice except those in sham group. Isotype antibodyand anti-PD-L1antibody (50μg/200μl) were inject to mice in isotype group andanti-PD-L1group respectively. Blood was collected24hour after operation to detect thealanine aminotransferase (ALT) and aspartate aminotransferase (AST) level in serum andliver HE staining sections were made to evaluate the liver injury degree by pathologist.Otherwise, RNA was also extracted and Q-RT-PCR was performed to compare the mRNAlevel of TNF-α、IFN-γ、IL-6and IL-10in mice livers of each group.Results1、Positive expression was observed on parenchyma cells in frozen liver sections ofCLP mice under microscope after IHC, while negative result was obtained in sham group.Q-RT-PCR results showed higher expression of PD-L1mRNA in mice liver of CLP groupwhen compared with sham group (p <0.01).2、Serum ALT and AST levels of CLP group and isotype group were significantly higher than those of sham group (p <0.01) and lower than those of anti-PD-L1group (p <0.01). Obvious pathological change was found in liver HE staining sections of CLP andisotype groups, and the pathological display was quite relieved in anti-PD-L1group.Q-RT-PCR of TNF-α、 IFN-γ、 IL-6and IL-10showed that these cytokines weresignificantly up-regulated in mice livers of CLP or isotype groups when compared withsham group (p <0.01), and mRNA levels of TNF-α、IL-6and IL-10of anti-PD-L1groupwere lower than that of CLP or isotype groups (p <0.01), while mRNA level of IFN-γ washigher (p <0.01).ConclusionThe expression of PD-L1in mice liver were significantly up-regulated after CLPoperation. Denaturation and inflammatory infiltration occurred in CLP mice liver, togetherwith high level of serum ALT and AST. Injection of anti-PD-L1antibody could obviouslyprotect liver in sepsis, which was represented as lower serum ALT and AST level andreliefed pathological change. The Q-RT-PCR result of cytokine mRNA showed anti-PD-L1antibody could down-regulate the mRNA ofIL-6, IL-10and TNF-α and up-regulate themRNA of IFN-γ in liver of sepsis mice, which may help to suppress the excessiveinflammatory response and to avoid the severe liver injury induced by “cytokine storm”.