Human monoclonal antiTLR4 antibody negatively regulates lipopolysaccharideinduced inflammatory responses in mouse macrophages.
2020
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, interferons 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.
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