XIAP Knockdown in Alcohol-Associated Liver Disease Models Exhibits Divergent in vitro and in vivo Phenotypes Owing to a Potential Zonal Inhibitory Role of SMAC

Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from Xiap^Alb.Cre and Xiap^loxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither Xiap^Alb.^Cre nor Xiap^AAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.