Human monoclonal anti-TLR4 antibody negatively regulates lipopolysaccharide-induced inflammatory responses in mouse macrophages

Previous studies have revealed that activation of the Toll-like 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 anti-TLR4 IgG2 antibody by screening human TLR4 Fab from a phage-display library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescence-activated cell sorting were used to assess its affinity. Furthermore, mouse-derived peritoneal macrophages were treated with human anti-TLR4 IgG2 and induced with LPS in vitro. Reverse transcription-quantitative 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 anti-TLR4 IgG2 bound to TLR4 with a high affinity of 8.713×10(−10) M, and that preincubation with anti-TLR4 IgG2 inhibited the LPS-induced production of tumor necrosis factor-α, interferon-β and interleukin-6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human anti-TLR4 IgG2 inhibited LPS-induced TLR4 signaling by reducing the phosphorylation of the NF-κB, mitogen-activated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human anti-TLR4 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 anti-TLR4 IgG2 plays a protective role against sepsis-associated injury and is potentially applicable for the treatment of infection-associated immune dysfunction.