NF-κB/AP-1-mediated expression of the porcine cathelicidin proPMAP-37 suppresses inflammation by neutralizing LPS and inhibiting M1 macrophage polarization

Cathelicidins are short cationic peptides secreted in pro-peptide, characterized by a conserved N-terminal cathelin domain and a diverse C-terminal mature peptide. While the antimicrobial and immunomodulatory properties of mature cathelicidins are well-characterized, whether their precursor forms (pro-forms) possess biological activity remains a subject of debate. Amid the global challenge of antimicrobial resistance (AMR) and the mandate to reduce antibiotic use in livestock, cathelicidins represent promising alternatives for managing bacterial infection in agriculture. However, high production costs hamper their large-scale application. Therefore, elucidating the regulation of expression of porcine myeloid antimicrobial peptide 37 (PMAP-37), investigating the function of its pro-form (proPMAP-37), and developing a cost-effective production strategy are of significant importance for both basic immunology in porcine systems and agricultural applications. This study demonstrated that porcine myeloid antimicrobial peptide 37 ( PMAP-37 ) exhibited widespread tissue distribution in disease-susceptible Duroc × Landrace × Yorkshire (DLY) pigs, whereas its constitutive expression level was significantly higher in disease-resistant Tibetan pigs (TB). Furthermore, Escherichia coli K88 ( E. coli K88) upregulated PMAP-37 expression in the intestine of DLY pigs. Mechanistic investigations revealed that the induction of PMAP-37 mediated by E. coli K88 depends on NF-κB and AP-1 transcription factors. Importantly, proPMAP-37 (the precursor of PMAP-37) was successfully expressed in a heterologous system and purified to homogeneity. Functional characterization demonstrated negligible hemolytic activity and cytotoxicity toward mammalian cells. Although no direct bactericidal effect was observed in vitro, proPMAP-37 functioned as an immunomodulator. It exhibited high affinity with Lipopolysaccharide (LPS) and inhibited pro-inflammatory M1 macrophage polarization, thereby reducing the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). This comprehensive study provides novel insights into the immunological functions of porcine cathelicidins and promotes the industrial development of proPMAP-37 as a novel anti-inflammatory agent.