Inter-relation analysis of signaling adaptors of porcine innate immune pathways

To study the interrelationship between the signaling adaptors of innate pattern recognition receptor (PRR) pathways including toll-like receptor (TLR), retinoic acid-inducible gene-1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and cytoplasmic DNA recognition receptors (CDR) pathways. The coding genes of porcine TRIF, MAVS, STING, MyD88, RIPK2, and ASC were isolated from PK15 cells. Phylogenetic analysis of the six adaptor proteins in pig, cattle, goat, horse, human, mouse, chicken, and duck performed by MEGA 5.05 showed that these adaptors have slightly different similarity across species. The expression of these proteins in transfected cells were detected by both Western blotting and confocal microscopy. All six adaptors were visualized in cytoplasm but with different distribution patterns. The activities of the six adaptors triggering NF-κB and ISRE signaling and downstream gene productions were examined by dual-luciferase reporter assay and real-time RT-PCR, respectively. The results showed that STING has an ability to activate ISRE signaling, MyD88, RIPK2 and ASC possess NF-κB signal activity, while TRIF and MAVS can activate both. Furthermore, the mutual signaling effects were assessed by NF-κB and ISRE dual-luciferase reporter assay in the co-expression experiments. STING was shown to enhance MAVS activated NF-κB signaling and MyD88 could heighten STING activated ISRE signaling. However, all other adaptors inhibited each other to varying degrees. The work provides a global insight of porcine innate immune signaling pathways and their interaction network.