The Porcine and Chicken Innate DNA Sensing cGAS-STING-IRF Signaling Axes Exhibit Differential Species Specificity.

The innate immune DNA sensing cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) signaling pathway plays a key role in host antiviral function. Although the cGAS-STING pathway has been extensively studied, the cGAS-STING signaling in livestock and poultry is not well understood, and whether the species specificity exists is still unknown. In this study, we found that porcine and chicken STING, but not cGAS, exhibit species differences in regulation of IFN; that is, porcine (p)STING mediates good induction of IFN in mammalian cells and low IFN induction in chicken DF-1 cells; on the contrary, chicken (ch)STING mediates IFN induction only in chicken cells but not in mammalian cells. Furthermore, it was found that the motifs pLxIS of pSTING and pLxVS of chSTING are responsible for the species disparity, with the IFN activity of pSTING and chSTING exchanged by swapping the two pLxI/VS motifs. The pLxI/VS motifs mediated the interactions of various STING with downstream IFN regulatory factors (IRFs), reflecting the species-specific pIRF3 and chIRF7. Next, the STING, IRFs, and STING-IRFs were reconstituted in porcine and chicken macrophages that were genetically knocked out for STING and/or IRFs by the CRISPR-Cas9 approach. The results showed that pSTING plus pIRF3 or chIRF7 are able to induce IFN; however, chSTING plus chIRF7 but not pIRF3 are able to induce IFN, suggesting that pIRF3 is specific and stringent, which underlies the inability of chSTING to induce IFN in mammalian cells. In summary, our findings reveal the differential species specificity in the cGAS-STING pathway and the underlying mechanisms, thus providing valuable insights on the cGAS-STING-IRF signaling axis for comparative immunology.