Anti-TLR4 IgG2 Prevents Acetaminophen-induced Acute Liver Injury through the Toll-like Receptor 4/MAPKs Signaling Pathway in Mice.

BACKGROUND AND OBJECTIVE: Acetaminophen (APAP) is a widely used antipyretic and analgesic. If taken in excess, it can cause severe drug-induced acute liver injury. The purpose of this study was to investigate the effects of anti-TLR4 IgG2 on APAP-induced liver injury and its underlying mechanisms. METHODS: We injected APAP into the abdominal cavity of mice to establish a liver injury model. Mice were divided into the control group, APAP group, and APAP + anti-TLR4 IgG2 group. In order to verify the implication of the toll-like receptor4 and mitogen-activated protein kinases activation (TLR4/MAPKs) signaling pathway, mice were intraperitoneally injected with a TLR4 / MAPKs inhibitor anti-TLR4 IgG2. We evaluated the effects of TLR4 IgG2 on the antioxidant, anti-apoptotic, anti-inflammatory, and liver histopathology of APAP mice. In addition, the expression of the TLR4 / MAPKs signaling pathway was detected by Western blot. RESULTS: Our study showed that APAP mouse models were successfully established; however, pretreatment with anti-TLR4 IgG2 alleviated APAP-induced hepatic injury, as evidenced by the 24-h survival rate. Meanwhile, anti-TLR4 IgG2 prevented the elevation of serum biochemical parameters and lipid profile. Furthermore, compared with the APAP group, hepatic antioxidants, including 3- Nitrotyrosine, high mobility group protein B1, superoxide dismutase, catalase, and glutathione, were increased in APAP + anti-TLR4 IgG2 group. In contrast, a significant decrease was observed in the levels of the malondialdehyde, which is a lipid peroxidation product. Moreover, the western blotting analysis showed that anti-TLR4 IgG2 treatment inhibited the activation of the apoptotic pathway by increasing Bcl-2 and decreasing Bax, P53, and cleaving caspase-3 / caspase-3 protein expression. These results were further validated by TUNEL staining and immunohistochemical. Histopathological observation also revealed that pretreat-ment with anti-TLR4 IgG2 could significantly reverse hepatocyte inflammatory infiltration, congestion, and necrosis in liver tissues by APAP. Importantly, anti-TLR4 IgG2 effectively alleviated APAP-induced liver injury by inhibiting tolllike receptor4 and mitogen-activated protein kinases activation signaling pathways (TLR4/MAPKs). CONCLUSION: The results clearly suggest that the underlying molecular mechanisms in the hepatoprotection of anti-TLR4 IgG2 in APAP-induced hepatotoxicity may be due to its antioxidation, anti-apoptosis, and anti-inflammation effects through inhibition of the TLR4/MAPKs signaling axis.

Human monoclonal anti­TLR4 antibody negatively regulates lipopolysaccharide­induced inflammatory responses in mouse macrophages.

Previous studies have revealed that activation of the Toll­like receptor 4 (TLR4)­mediated proinflammatory signaling pathway plays an important role in acute inflammation, sepsis and chronic inflammatory disorders. Moreover, TLR4 significantly contributes to lipopolysaccharide (LPS)­induced immune response. Thus, modulation of the TLR4 pathway is an important strategy to specifically target these pathologies. The aim of the present study was to develop a complete human anti­TLR4 IgG2 antibody by screening human TLR4 Fab from a phage­display library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescence­activated cell sorting were used to assess its affinity. Furthermore, mouse­derived peritoneal macrophages were treated with human anti­TLR4 IgG2 and induced with LPS in vitro. Reverse transcription­quantitative PCR and western blotting were used to determine mRNA expression levels of cytokines and phosphorylation levels of signaling pathways, respectively. It was found that human anti­TLR4 IgG2 bound to TLR4 with a high affinity of 8.713x10­10 M, and that preincubation with anti­TLR4 IgG2 inhibited the LPS­induced production of tumor necrosis factor­α, interferon­ß and interleukin­6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human anti­TLR4 IgG2 inhibited LPS­induced TLR4 signaling by reducing the phosphorylation of the NF­κB, mitogen­activated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human anti­TLR4 IgG2 protected mice from LPS challenge with a survival rate of 40% and also significantly increased the survival time in the cecal ligation and puncture model. Therefore, it was speculated that human anti­TLR4 IgG2 plays a protective role against sepsis­associated injury and is potentially applicable for the treatment of infection­associated immune dysfunction.