A humanized anti-Toll like receptor 4 antibody Fab fragment inhibits pro-inflammatory responses induced by lipopolysaccharide through TLR4 in vitro and in vivo

Abstract

Introduction: Toll like receptor 4 (TLR4) and its co-receptor MD-2 recognize bacterial lipopolysaccharide (LPS), initiating responses to infections caused by Gram-negative bacteria. TLR4 also plays a role in various pathological processes, including viral infections and sterile inflammation. However, effective methods to inhibit LPS/TLR4-mediated inflammation remain elusive. This study aimed to evaluate the inhibitory effects of a constructed hTLR4-Fab on LPS-induced inflammation in both in vitro and in vivo settings.

Methodology: In vitro, mouse dendritic cells (DCs), human macrophages, and human DCs were incubated with hTLR4-Fab and then stimulated with LPS. In vivo, mice were pre-treated with a humanized anti-TLR4 antibody Fab prior to LPS injection. We examined the activation of various signaling pathways to elucidate the molecular mechanism underlying the inhibition of LPS-induced inflammation by hTLR4-Fab.

Results: We observed that the binding affinity of hTLR4-Fab to TLR4 on mouse bone marrow-derived dendritic cells (DCs) was approximately 81.8%, while the binding affinity to human blood monocyte-derived macrophages and DCs exceeded 90%. Pretreatment with hTLR4-Fab significantly reduced both mRNA and protein levels of LPS-induced proinflammatory cytokines. In vivo, a significant suppression of serum cytokine expression was driven by hTLR4-Fab treatment.

Conclusions: The results demonstrated that the antibody Fab could impede the phosphorylation of downstream components, including the nuclear factor κB (NF-κB) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and IFN regulatory factor 3 (IRF-3), all of which are activated by TLR4. Consequently, our study demonstrates that our hTLR4-Fab is effective in mitigating LPS-induced inflammation, both in vitro and in vivo.

Keywords: TLR4 signaling; anti-inflammatory therapy lipopolysaccharide; inflammation; sepsis; toll like receptor 4 signaling.