title: HBD-3 regulation of the immune response and the LPS/TLR4-mediated signaling pathway.
abstract: The aim of the study was to investigate the mechanisms of human β-defensin 3 (HBD-3) regulation of the immune response and the lipopolysaccharide/Toll-like receptor-4 (LPS/TLR4)-mediated signaling pathway. A TLR4 extracellular gene fragment was cloned into the pET32a plasmid to determine its expression inEscherichia coli (E. coli)and purification. A dialysis labeling method was used to stain HBD-3 with fluorescein isothiocyanate (FITC). FITC-HBD-3 was used to induce the differentiation of human peripheral blood mononuclear cells (MNC) into immature dendritic cells (imDC)in vitro. Binding reactions were established using FITC-HBD-3 and sTLR4 into cell suspensions. Flow cytometry (FCM) was used to analyze the results. Western blot analysis confirmed the identity of nuclear factor-κB (NF-κB) and was used to quantify its nuclear translocation. The results showed that, HBD-3 bound to imDC in a Ca2+-dependent manner, and sTLR4 and LPS competitively inhibited the binding. HBD-3 competitively blocked the binding of LPS and imDC by binding to imDC. HBD-3 significantly decreased the translocation of LPS-induced NF-κB into the nucleus. In conclusion, HBD-3 can competitively inhibit the binding of LPS and imDC through its binding to TLR4 molecules, which are expressed in imDC, thereby preventing LPS from inducing the maturity of the imDCs.
authors.affiliation: Department of Orthopaedic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Department of Orthopaedic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Ni-Rong
authors.initials: NR
authors.lastname: Bao
authors.affiliation: Department of Orthopaedic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Jian-Ning
authors.initials: JN
authors.lastname: Zhao
keywords: Toll-like receptor-4; human β-defensin 3; immature dendritic cells; nuclear factor-κB