Studies On The Regulation Of Rat β-Defensin-2 Gene Expression And Gene Transfer In Respiratory Tract

Endogeneous antimicrobial peptides are important mediators in innate immunity. The p-defensins are a relatively recently described family of antimicrobial peptides that are widely expressed at mucosal surface, including airway and submucosal gland epithelia. These small cationic peptides exhibit broad-spectrum activity against bacteria, fungi, and some enveloped virus. Their expression in airway epithelia may be constitutive or inducible by bacteria and pro-inflammatory cytokines. hBD-1, b.BD-2, and hBD-3 are all expressed in airway epithelia and play an important role in the lung defense against infections. Bacteria and pro-inflammatory cytokines can induce hBD-2 and hBD-3 expression in airway mucosa. (3-defensins also act as chemokines and provide a link between innate and adaptive immunity. Enhancing the local concentration of antimicrobial peptides is an ideal remedy for treatment of the antibiotic-resistant bacterial infection, especially for CF patients. The antimicrobial peptides produced by the innate immune system are not only widely effective but also may provide a new source of antibiotics.The purpose of this study is to investigate the regulation of rat P-defensin-2 (rBD-2) gene expression in respiratory tract and evaluatewhether mucosal rBD-2 gene transfer or BCG cell wall protein would enhance airway defense against infections.Total RNAs isolated from the pulmonary tissues of the fetal, neonatal, and adult rat were used as templates, and RT-PCR was performed to amplify rBD-2 cDNA fragment. Rat P-defensin-2 transcripts were detected in all the pulmonary tissues of the rats during different developmental stages, suggesting that rBD-2 gene is constitutively expressed in the lung tissues. We also found that the supernatant of E. Coli down-regulated the rBD-2 gene expression in the lung.Rat p-defensin-2 recombinant pBK-CMV-rBD2 and pcDNA/3.1-Myc-His(+)-rBD2 were constructed. By using cationic liposome as delivery carrier, the recombinants were transferred into COS-7 cell and the rat. Rat P-defensin-2 mRNA and protein expression were detected in the transfected cells by RT-PCR and Western Blotting. Agar radial diffusion assay showed that there was an antibacterial activity against E.Coli ML-35p to be detected in the supernatant of the transfected cells. The rBD-2 mRNA expression was detected in the trachea of rat transfected with pcDNA/3.1-Myc-His(+)-rBD2 by RT-PCR and its protein expression was determined in the lungs by the tag 6 X His immnunostaining. When P. aeruginosa ATCC27853 were inoculated via trachea, the CPU markedly reduced in the lung of rat transfected with pBK-CMV-rBD2, compared with the control (n=8, P=0.003). The data presented in this study provide evidence that antimicrobial peptides protect the consequences of bacterial infection in vivo, highlighting the role of these substances as part of the innate host defense system.The components of BCG cell wall protein fractionated by Sephadex G-150 chromatography, molecular weight between 18kDa and 29kDa, were injected intraabdominally. After 48 hours, 5×106 CPU of P. aeruginosa ATCC27853 were inoculated via trachea. The data suggested that the components of BCG cell wall protein could significantly enhance the bacterial clearance of rat lung (n=8, P=0.004). The results bring us a new ideal to develop innovative anti-infection adjuvant enhancing the lung defense function.