WB and numerical raw data for Porcine cGAS-STING signaling induced apoptosis negatively regulates STING downstream IFN response and autophagy via different mechanisms

The innate immune cGAS-STING signaling pathway recognizes double-stranded DNA and induces the interferon (IFN) response, autophagy and apoptosis, exerting a broad antiviral effect. However, the mechanisms and interrelationship between STING induced downstream IFN, autophagy, and apoptosis in livestock have not been fully elucidated. Our previous study defined porcine STING (pSTING) induced IFN, autophagy and apoptosis, and showed that IFN does not affect autophagy and apoptosis, whereas autophagy inhibits both IFN and apoptosis, likely by promoting pSTING degradation. In this study, we further explored the underlying mechanism of pSTING induced apoptosis and the regulation of IFN and autophagy by apoptosis. First, pSTING induces endoplasmic reticulum (ER) stress and mitochondrial damage to activate caspases 9, 3, and 7, which drive intrinsic apoptosis. Second, pSTING triggered apoptosis inhibits the IFN response by activating caspase 7, which cleaves pIRF3 at the species specific D197/D198 site. Third, pSTING activated apoptotic caspases 9, 3, and 7 reduce the expression of ATG proteins, and cleave the ATG5-ATG12L1 complex, effectively inhibiting autophagy. Fourth, knockout of pSTING activated apoptosis heightens the IFN response and autophagy, while suppressing the replication of HSV1, VSV and PRV. This study sheds light on the molecular mechanisms of innate immunity in pigs.

固有免疫cGAS-STING信号通路可识别双链DNA，诱导干扰素(IFN)应答、细胞自噬与细胞凋亡，进而发挥广谱抗病毒效应。然而，家畜中STING诱导的下游IFN、自噬与凋亡的具体机制及相互关联尚未完全阐明。我们前期的研究明确了猪STING(pSTING)介导的IFN、自噬与凋亡通路，并证实IFN并不会影响自噬与凋亡，而自噬可通过促进pSTING降解，同时抑制IFN应答与细胞凋亡。本研究进一步探究了pSTING诱导细胞凋亡的潜在机制，以及凋亡对IFN与自噬的调控作用：其一，pSTING可诱导内质网(ER)应激与线粒体损伤，激活半胱天冬酶caspase-9、caspase-3与caspase-7，从而启动内源性细胞凋亡；其二，pSTING触发的细胞凋亡可通过激活caspase-7，在物种特异性D197/D198位点切割猪干扰素调节因子3(pIRF3)，进而抑制IFN应答；其三，pSTING激活的凋亡型caspase-9、3、7可降低自噬相关蛋白(ATG蛋白)的表达水平，并切割ATG5-ATG12L1复合物，有效抑制细胞自噬；其四，敲除pSTING可激活细胞凋亡，进而增强IFN应答与自噬水平，同时抑制单纯疱疹病毒1型(HSV1)、水疱性口炎病毒(VSV)与伪狂犬病病毒(PRV)的复制。本研究阐明了猪固有免疫的分子调控机制。