Abstract
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.
Highlights
Bacterial drug resistance has become a global concern
The results presented that Porcine beta-defensin 2 (PBD-2) TG mice showed an increased resistance to bacterial infection compared with wild-type (WT) mice
PCR was performed using the genomic DNA from the offspring of the F0 TG mice, and the results showed the successful expression of pbd-2 (Figure 1B)
Summary
Bacterial drug resistance has become a global concern. Endeavors in search for antibiotic substitutes are desperately needed. Antimicrobial peptides (AMPs) are natural anti-infective agents identified in various species and implicated in innate immunity [1]. These agents have been suggested to be a prominent alternative to antibiotics in fighting against infectious diseases because of their significant antibacterial activity [2]. AMPs can modulate both innate, and adaptive immunity [2]. Many host peptides can serve as chemokines to recruit and activate immune cells, modulate the release of cytokines, suppress the apoptosis of neutrophils, and act as a link between innate and adaptive immune systems [5]. The process of the immune regulatory and anti-inflammatory effects of AMPs is complicated and yet to be fully explored
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