Immune Pathway Activation in PCLS

Polyriboinosinic:polyribocytidylic acid (poly[I·C]) (LMW)/LyoVec and Pam3Cys-Ser-(Lys)₄ (Pam3CSK4) (Invivogen) were selected as agonists to activate specific immune pathways on PCLSs. Poly(I·C) (LMW)/LyoVec is a synthetic double-stranded RNA (dsRNA) polymer that is complexed with a transfection reagent, and it is sensed by cytosolic helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 (RIG-I/MDA-5) in a specific manner (68 (link)) and forms known essential signaling pathways upon influenza virus infection (69 (link)). Experimental infection in calves with IDV also suggests the activation of this pathway (29 (link)). Pam3CSK4 is a synthetic triacylated lipopeptide that is a potent activator of NF-κB pathway upon binding of TLR2/TLR1 receptor and a MyD88-dependent activation, as described during Mycoplasma spp. infection in different species (41 (link)– (link)43 (link)). To inhibit NF-κB pathway, BAY 11-7082 (Invivogen) was used. BAY 11-7082 was described to inhibit the phosphorylation of IκB-α (which is essential for the release of NF-κB from the cytosolic IκB-α/NF-κB complex) (70 (link)), but it was also suggested to inhibit the inflammasome responses indirectly by preventing the nuclear translocation of NF-κB at the priming step but also to directly inhibitory functions on the NLRP3 inflammasome by blocking the sensor’s ATPase activity (71 (link)). For stimulations, the PCLSs were treated at 0 h with poly(I·C) at a final concentration of 500 ng/mL, followed by stimulation with Pam3CSK4 at a final concentration of 100 ng/mL 24 h later. To inhibit the NF-κB pathway, the PCLSs were pretreated with BAY 11-7082 at a final concentration of 100 ng/mL, followed by M. bovis infection 6 h later.