The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage.

Binggang Cai,Jing Xu,Zhenqing Feng,Maorong Wang,Xuhui Zhu,Feng Zheng,Jin Zhu,Yiqing Zhang,Wenkai Zheng

doi:10.3390/ijms161025502

Abstract

Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

Highlights

Toll-like receptor 4 (TLR4), the human Toll homolog originally known as hToll [1], is one of the first members of the TLR family and has been well characterized as a pattern-recognition receptor (PPR) [2].TLRs can recognize pathogen-associated molecular patterns (PAMPs) or endogenously sourced damage-associated molecular pattern molecules (DAMPs) produced by the pathogen, resulting in the activation of innate immune responses
To confirm that the release of LPS-induced inflammatory cytokines could be inhibited by the humanized anti-Toll receptor 4 (TLR4) antibody in mouse macrophages, we examined the production of mRNAs encoding interleukin (IL)-6, TNF-α, IL-1, and IFN-β by quantitative polymerase chain reaction
The results showed that the IL-6, TNF-α, IL-1, and IFN-β expression levels were significantly increased in LPS-induced mouse macrophages as compared with those in the untreated control

Summary

Introduction

Toll-like receptor 4 (TLR4), the human Toll homolog originally known as hToll [1], is one of the first members of the TLR family and has been well characterized as a pattern-recognition receptor (PPR) [2]. TLRs can recognize pathogen-associated molecular patterns (PAMPs) or endogenously sourced damage-associated molecular pattern molecules (DAMPs) produced by the pathogen, resulting in the activation of innate immune responses. The secretion of pro-inflammatory cytokines and type I interferons (IFNs) could be induced after the activation of innate immune responses, eventually removing the invading pathogenic microorganisms [3,4]. TLR4 is mainly expressed in antigen-presenting cells (APCs), including macrophages, monocytes, and dendritic cells (DCs) [5,6]. TLR4 is different from other TLRs in many ways. TLR4 can activate both myeloid differentiation factor 88

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