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Porcine beta-defensin 2 (PBD-2) which is a member of the family of antimicrobial peptides, is widely expressed in pig organs with a broad spectrum of bactericidal activities confirmed  in vitro . We previously demonstrated that transgenic (TG) pigs overexpressing PBD-2 could resist the infection by the porcine pathogen  Actinobacillus pleuropneumoniae . In this study, the roles of PBD-2 in protecting against bacterial infection were further investigated. The biochemical indexes of the blood sample, body weights, histological morphologies, and weights of the organs of TG mice expressing PBD-2 were measured. Results confirmed that these mice showed normal physiological features. An assay of  Salmonella  Typhimurium infection was conducted on wild-type (WT) and TG mice. The TG mice possessed higher survival rate, less body weight loss, and pathological changes and smaller recovery rates of bacteria after infection with  S . Typhimurium. The  in vitro  synthetic PBD-2 and the serum and tissue homogenates from the TG mice displayed a direct bactericidal activity. Moreover, PBD-2 could inhibit the release of the proinflammatory cytokines, including IL-6, TNF-α, IL-1β, and IL-12, in the TG mice infected with  S . Typhimurium or treated with lipopolysaccharide (LPS). The WT mice treated with PBD-2 and  S . Typhimurium or LPS showed reduced levels of proinflammatory cytokines. The mouse macrophage cell line RAW 264.7 which expressed PBD-2 was constructed to detect the signal pathways affected by PBD-2. The suppressing effect of PBD-2 on the release of the proinflammatory cytokines was confirmed using RAW 264.7 either expressing PBD-2 or supplemented with PBD-2. The promoter activity and mRNA level of NF-κB were detected, and PBD-2 was shown to significantly inhibit the activation of the NF-κB pathway induced by LPS. The direct interaction of PBD-2 with TLR4 was revealed by isothermal titration calorimetry and far-Western blot  in vitro  and the coimmunoprecipitation of PBD-2 with TLR4 on RAW 264.7 cells. This interaction indicates one reason for the interference of NF-κB activation. Overall, this study showed that PBD-2 protected against bacterial infection through a direct bactericidal activity and alleviated inflammation by interfering with the TLR4/NF-κB pathway.

frontiers in immunology

Porcine Beta-Defensin 2 Provides Protection Against Bacterial Infection by a Direct Bactericidal Activity and Alleviates Inflammation via Interference With the TLR4/NF-κB Pathway