Generation And Disease-resistance Evaluation Of Porcine Beta Defensin 2 Transgenic Mice And Pigs

Antibiotic-resistant bacteria are a growing public health concern.To reduce the development of antimicrobial resistance,alternative approaches to decrease antibiotic use are needed.Porcine beta-defensin 2(PBD-2)is highly expressed in epithelial cells and thought to be an important defensin in pig.PBD-2 demonstrates excellent antimicrobial activity with broad spectrum but with low hemolytic activity under physiological conditions.We hypothesized that expression PBD-2 in PK-15 cells,mice and pigs should enhance the resistance to bacterial infection.This study lays the foundations for development of infection-resistant animals.1.Overexpression of PBD-2 in porcine kidney cells confers antimicrobial activity to cells.To study the functional consequences of a supplemental defensin in cells,the recombinant plasmid pcCAG-PBD2 was generated and transfected into porcine kidney(PK-15)cells with lipofectamine.The stably transfected cells were selected with G418 and then verified by Indirect immunofluorescence.Culture supernatant of stably transfected cells showed obvious antibacterial effects on disk zone inhibition assay,but the cell lysates did not.These findings confirmed the requirement for post-translational processing of defensin to activate antimicrobial activity.2.Expression of porcine beta-defensin 2 enhances resistance to Salmonella typhimurium infection in mice.Although in vitro antibacterial activity of PBD-2 has been declared,there are few biology functional researches of PBD-2 for the lack of in vivo model.We developed a transgenic mouse to express PBD-2 propeptides and compare their susceptibilities to Salmonella typhimurium infection with those of wild type mice.Here we show that mortality was 100%in wild type mice after a lethal dose infection of S.typhimurium,whereas 43%of the transgenic mice survived after that.The significantly lower bacterial load was found in the distal small intestine of PBD-2 transgenic mice compared with wild type mice.These findings suggest that expression of PBD-2 in transgenic mice enhanced the resistance to bacterial infection and provides support for a critical in vivo role of defensins in host defense.3.Enhanced resistance to Actinobacillus pleuropneumoniae in transgenic porcine overexpressing PBD-2.To address whether overexpression of a defensin will increase resistance in pigs,we generated transgenic pigs constitutively overexpressing PBD-2 by using somatic cell cloning method.Resistance to bacterial infection was evaluated by infection with A.pleuropneumoniae.PBD-2 transgenic pigs had significantly lower bacterial loads,reduced clinical scores and lung lesions scores,in the lungs after direct or cohabitation infection.Thus,overexpression of PBD-2 in pigs enhanced the resistance against bacterial infection.4.PBD-2 attenuates the lipopolysaccharide-mediated inflammatory responses in vitro and in vivo.We presented evidence here that PBD-2 attenuated lipopolysaccharide(LPS)-induced production of proinflammatory cytokines(TNF-a or IL-6)in mouse and RAW264.7 cells.PBD-2 also showed similar effects on the LPS-induced activation of inflammatory transcription factor nuclear factor-kappaB(NF-kB)and phosphorylation of p65.Without LPS,PBD-2 alone displayed no detectable effect on the TNF-a and IL-6 production,NF-kB activation as well as phosphorylation of p65.Further analysis showed that the PBD-2 peptide could bind with LPS,but this binding did not affect the endotoxin activity of LPS.Our data disclosed that PBD-2 could down-regulate the LPS-mediated cytokine production.