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

Wang,Yiwen; Gong,Dandan; Yao,Chuanxia; Zheng,Feng; Zhou,Tingting; Cao,Qingxin; Zhu,Xuhui; Wang,Maorong; Zhu,Jin

doi:10.3892/mmr.2020.11500

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

Authors:
- Yiwen Wang
- Dandan Gong
- Chuanxia Yao
- Feng Zheng
- Tingting Zhou
- Qingxin Cao
- Xuhui Zhu
- Maorong Wang
- Jin Zhu
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Affiliations: Department of Epidemiology and Microbiology, Huadong Medical Institute of Biotechniques, Nanjing, Jiangsu 210002, P.R. China, Department of Obstetrics and Gynecology, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210004, P.R. China, Department of Infectious Disease, Anhui Medical University Affiliated with Bayi Clinical College, Hefei, Anhui 230000, P.R. China, Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing, Jiangsu 210002, P.R. China
Published online on: September 9, 2020 https://doi.org/10.3892/mmr.2020.11500
Pages: 4125-4134
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

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 8.713x10‑10 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.

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