Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide. We generated single-cell and spatial transcriptomic and metabolomic maps from 61 human livers, including controls (n = 10), metabolic dysfunction-associated steatotic liver (MASL) (n = 17) and metabolic dysfunction-associated steatohepatitis (MASH) (n = 34). We identified microphthalmia-associated transcription factor (MITF) as a key regulator of the lipid-handling capacity of lipid-associated macrophages (LAMs), and further revealed a hepato-protective role of LAMs mediated through hepatocyte growth factor secretion. Unbiased deconvolution of spatial transcriptomics delineated a fibrosis-associated gene program enriched in advanced MASH, suggesting profibrotic crosstalk between central vein endothelial and hepatic stellate cells within fibrotic regions. Mass spectrometry imaging-based spatial metabolomics demonstrated MASLD-specific accumulation of phospholipids, potentially linked to lipoprotein-associated phospholipase A₂-mediated phospholipid metabolism in LAMs. This spatially resolved multi-omics atlas of human MASLD, which can be queried at the Human Masld Spatial Multiomics Atlas, provides a valuable resource for mechanistic and therapeutic studies.

Cholangiocytes play a crucial role in bile formation. Cholangiocyte injury causes cholestasis, including primary biliary cholangitis (PBC). However, the etiology of PBC remains unclear despite being characterized as an autoimmune disease. Using single-cell RNA sequencing (scRNA-seq), fluorescence-activated-cell-sorting, multiplex immunofluorescence (IF) and RNAscope analyses, we identified unique DUOX2⁺ACE2⁺ small cholangiocytes in human and mouse livers. Their selective decrease in PBC patients was associated with the severity of disease. Moreover, proteomics, scRNA-seq, and qPCR analyses indicated that polymeric immunoglobulin receptor (pIgR) was highly expressed in DUOX2⁺ACE2⁺ cholangiocytes. Serum anti-pIgR autoantibody levels were significantly increased in PBC patients, regardless of positive and negative AMA-M2. Spatial transcriptomics and multiplex IF revealed that CD27⁺ memory B and plasma cells accumulated in the hepatic portal tracts of PBC patients. Collectively, DUOX2⁺ACE2⁺ small cholangiocytes are pathogenic targets in PBC, and preservation of DUOX2⁺ACE2⁺ cholangiocytes and targeting anti-pIgR autoantibodies may be valuable strategies for therapeutic interventions in PBC.

Nonalcoholic fatty liver disease (NAFLD) has become a common health care burden worldwide. The high heterogeneity of NAFLD remains elusive and impairs outcomes of clinical diagnosis and pharmacotherapy. Several NAFLD classifications have been proposed on the basis of clinical, genetic, alcoholic, or serum metabolic analyses. Yet, accurately predicting the progression of NAFLD to cirrhosis or hepatocellular carcinoma (HCC) in patients remains a challenge. Here, on the basis of a Chinese cohort of patients, we classified NAFLD into three distinct molecular subtypes (NAFLD-mSI, NAFLD-mSII, and NAFLD-mSIII) using integrative multiomics including whole-genome sequencing (WGS), proteomics, phosphoproteomics, lipidomics, and metabolomics across a broad range of liver, blood, and urine specimens. We found that NAFLD-mSI had higher expression of CYP1A2 and CYP3A4, which alleviate hepatic steatosis through mediating free fatty acid/bile acid–mTOR–FXR/PPARα signaling. NAFLD-mSII displayed an elevated risk of liver cirrhosis along with increased hepatic infiltration of M1 and M2 macrophages because of lipid-triggered hepatic CCL2 and CRP production. NAFLD-mSIII exhibited a potential risk for HCC development by increased transcription of CEBPB- and ERCC3-regulated oncogenes because of activation of the EGF-EGFR/CHKA/PI3K-PDK1-AKT cascade. Next, we validated the existence of these three NAFLD molecular subtypes in an external cohort comprising 92 patients with NAFLD across three different Chinese hospitals. These findings may aid in understanding the molecular features underlying NAFLD heterogeneity, thereby facilitating clinical diagnosis and treatment strategies with the aim of preventing the development of liver cirrhosis and HCC.

Background and Aims: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. Approach and Results: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5-ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1-P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2, and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid–induced CCL2 and CXCL2 in hepatocytes. Conclusions: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

Semaphorin 7A (SEMA7A) is a membrane‐bound protein that involves axon growth and other biological processes. SEMA7A mutations are associated with vertebral fracture and Kallmann syndrome. Here, we report a case with a mutation in SEMA7A that displays familial cholestasis. WGS reveals a SEMA7A ^R148W homozygous mutation in a female child with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. This patient also carried a SLC10A1 ^S267F allele, but Slc10a1 ^S267F homozygous mice exhibited normal liver function. Similar to the child, Sema7a ^R145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA. Remarkably, liver histology and LC‐MS/MS analyses exhibited hepatocyte hydropic degeneration and increased liver bile acid (BA) levels in Sema7a ^R145W homozygous mice. Further mechanistic studies demonstrated that Sema7a ^R145W mutation reduced the expression of canalicular membrane BA transporters, bile salt export pump (Bsep), and multidrug resistance‐associated protein‐2 (Mrp2), causing intrahepatic cholestasis in mice. Administration with ursodeoxycholic acid and a dietary supplement glutathione improved liver function in the child. Therefore, Sema7a ^R145W homozygous mutation causes intrahepatic cholestasis by reducing hepatic Bsep and Mrp2 expression.

Genetic polymorphisms are associated with the development of nonalcoholic fatty liver disease (NAFLD). Semaphorin7a ( Sema7a ) deficiency in mouse peritoneal macrophages reduces fatty acid (FA) oxidation. Here, we identified 17 individuals with SEMA7A heterozygous mutations in 470 patients with biopsy-proven NAFLD. SEMA7A heterozygous mutations increased susceptibility to NAFLD, steatosis severity, and NAFLD activity scores in humans and mice. The Sema7a ^R145W mutation (equivalent to human SEMA7A ^R148W ) significantly induced small lipid droplet accumulation in mouse livers compared with WT mouse livers. Mechanistically, the Sema7a ^R145W mutation increased N-glycosylated Sema7a and its receptor integrin β1 proteins in the cell membranes of hepatocytes. Furthermore, Sema7a ^R145W mutation enhanced its protein interaction with integrin β1 and PKC-α and increased PKC-α phosphorylation, which were both abrogated by integrin β1 silencing. Induction of PKC α _WT, but not PKC α _dominant negative, overexpression induced transcriptional factors Srebp1, Chrebp, and Lxr expression and their downstream Acc1, Fasn, and Cd36 expression in primary mouse hepatocytes. Collectively, our findings demonstrate that the SEMA7A ^R148W mutation is a potentially new strong genetic determinant of NAFLD and promotes intrahepatic lipid accumulation and NAFLD in mice by enhancing PKC-α–stimulated FA and triglyceride synthesis and FA uptake. The inhibition of hepatic PKC-α signaling may lead to novel NAFLD therapies.