A Human Anti-Toll Like Receptor 4 Antibody Fab Fragment Inhibits Lipopolysaccharide-Induced Inflammation In Vitro And In Vivo

Toll-like receptor 4(TLR4) is one of the firstmembers of the TLR family and has been well characterized as a pattern-recognition receptor(PPR). TLR4 plays an important role in innate immune. TLR4 recognizes bacterial lipopolysaccharude(LPS) and induces acute inflammation, sepsis, or chronic inflammatory disorders.TLR4 is the PRRs for LPS pathway activation, which plays a key role in LPS signaltransduction. Blocking TLR4 could be most effective in control of endotoxinbiological results. Therefore, the research and development of therapeutic TLR4 antibody should be helpful in the treatment of G- bacteria-related infectiousdiseases and septic shock. In our previous study, we prepared a chimeric Fab antibody using variable regions of anti-TLR4 m Ab combined with human constant regions by splicing by overlap extension. Then the Fab antibody was expressed and named h TLR4-Fab04. In this study, we aimed to investigate the inhibition of h TLR4-Fab04 which we built on LPS-induced inflammation in vitro and in vivo.Methods1.To detect whether the h TLR4-Fab04 can specific bind to TLR4 on mousemacrophage surfaces.Then to examine the inhibitory of h TLR4-Fab04 on LPS-induced the expression of m RNA and protein levels of pro-inflammatory cytokines. The m RNA levels were detected by Q-PCR and protein levels were detected by ELISA. To understand the molecular mechanism by which h TLR4-Fab04 inhibited the inflammatory responses induced by LPS. We examined the effect of h TLR4-Fab04 on the phosphorylation levels of MAPKs、NF-κB and IRF-3 pathway.2. To detect whether the hTLR4-Fab04 can specific bind to TLR4 onmurine bone marrow derived DCs surfaces. Then to examine the inhibitory of h TLR4-Fab04 on LPS-induced the expression of m RNA and protein levels of pro-inflammatory cytokines.The m RNA levels were detected by Q-PCR and protein levels were detected by ELISA. To understand the molecular mechanism by which h TLR4-Fab04 inhibited the inflammatory responses induced by LPS. We examined the effect of h TLR4-Fab04 on the phosphorylation levels of MAPKs、NF-κB and IRF-3 pathway.3.To detect whether the h TLR4-Fab04 can specific bind to TLR4 on human blood monocytes derived macrophages surfaces. Then to examine the inhibitory of h TLR4-Fab04 on LPS-induced the expression of m RNA and protein levels of pro-inflammatory cytokines. The m RNA levels were detected by Q-PCR and protein levels were detected by ELISA. To understand the molecular mechanism by which h TLR4-Fab04 inhibited the inflammatory responses induced by LPS. We examined the effect of h TLR4-Fab04 on the phosphorylation levels of MAPKs、NF-κB and IRF-3 pathway.4. To detect whether the h TLR4-Fab04 can specific bind to TLR4 on human blood monocytes derived DCssurfaces. Then to examine the inhibitory of h TLR4-Fab04 on LPS-induced the expression of m RNA and protein levels of pro-inflammatory cytokines. The m RNA levels were detected by Q-PCR and protein levels were detected by ELISA. To understand the molecular mechanism by which h TLR4-Fab04 inhibited the inflammatory responses induced by LPS. We examined the effect of h TLR4-Fab04 on the phosphorylation levels of MAPKs、NF-κB and IRF-3 pathway.5. To test the in vivo inhibitory effects of h TLR4-Fab04 on LPS-induced the production of pro-inflammatory cytokines in serum.We established a mouse model of endotoxemia.Results1.The binding rate of h TLR4-Fab04 with TLR4 was about 60 % on murinebone marrow derived DCs and mouse macrophages, and the binding rate could reached more than 90 % on human blood monocytes derived macrophages and DCs.2. The h TLR4-Fab04 could inhibit the expression of m RNA and proteinlevels of pro-inflammatory cytokines stimulated by LPS on mouse macrophages;murine bone marrow derived DCs; human blood monocytes derived macrophages and DCs. And all of the inhibitory effect of h TLR4-Fab04 could reach 50 %.3. The h TLR4-Fab04 could inhibiton the phosphorylation levels of MAPKs、NF-κB and IRF-3 pathway stimulated of LPS on mouse macrophages;murine bone marrow derived DCs; human blood monocytes derived macrophages and DCs.4. Different concentrations of h TLR4-Fab04 couldsignificantly down-regulated the production of pro-inflammatory cytokines in serum induced by LPS in vivo, and the inhibitory rates were strongest when the concentrations of h TLR4-Fab04 is 2 mg/kg. All inhibitory rates could reach50 %.ConclusionsIn this study, we confirmed that the h TLR4-Fab04 could specific bind to TLR4 on differentspecies cells surfaces, and the h TLR4-Fab04 could inhibit the inflammatory responses stimulated by LPS in vivo and in vitro. This study was prepared for the clinical research of h TLR4-Fab04, and also provided a new way to treat LPS-mediated inflammation, such as SIRS.