Introduction:Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the leading cause of chronic liver disease and hepatocellular carcinoma. The immune response plays a central role in disease onset and progression and is the focus of many experimental studies. However, traditional models typically rely on terminal sampling procedures that require large tissue quantities, a substantial number of animals per experimental condition, and cross-sectional study designs. Methods:Here, we propose integrating two powerful techniques to longitudinally study a MASLD animal model: image-guided fine-needle aspiration (FNA) and single-cell RNA sequencing (scRNA-seq). Results:This framework enables safe, high-precision longitudinal sampling of a limited number of animals, allowing simultaneous profiling of innate and adaptive immune cells during metabolic dysfunction-associated steatohepatitis (MASH) progression induced by a high-fat, high-cholesterol, and high-fructose diet. We further identified dynamic shifts in the hepatic immune landscape following dietary intervention. Conclusion:Our findings support the feasibility of the FNA-scRNA-seq framework as a novel tool for longitudinal immune profiling in preclinical MASLD and MASH studies and highlight its potential to reduce animal use in chronic liver disease research.

Immune checkpoints (CTLA4 & PD-1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune checkpoint inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune-mediated liver injury from ICIs (ILICI). Learning how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance. We developed a murine model to recapitulate human ILICI using CTLA4+/- mice treated with either combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2. We tested two forms of antisense oligonucleotides to knockdown caspase-3 in a total liver (parenchymal and non-parenchymal cells) or in a hepatocyte-specific manner. We also employed imaging mass cytometry (IMC), a powerful multiplex modality for immunophenotyping and cell interaction analysis in our model. ICI-treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as Nod-like receptor protein 3 (NLRP3) inflammasome activation, but no necroptosis. Total liver knockdown of caspase-3 worsened liver injury, and induced further inflammasome activation, and Gasdermin-D-mediated pyroptosis. Hepatocyte-specific knockdown of caspase-3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed that cC3+ hepatocytes had significantly closer interactions with macrophages, Kupffer cells, and NLRP3hi myeloid cells than other cell types. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8 + T-cells, and macrophages. Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Furthermore, we report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.

Background and Aims The hepatic mitogen‐activated protein kinase (MAPK) cascade leading to c‐Jun N‐terminal kinase (JNK) activation has been implicated in the pathogenesis of nonalcoholic fatty liver (NAFL)/NASH. In acute hepatotoxicity, we previously identified a pivotal role for mitochondrial SH3BP5 (SAB; SH3 homology associated BTK binding protein) as a target of JNK, which sustains its activation through promotion of reactive oxygen species production. Therefore, we assessed the role of hepatic SAB in experimental NASH and metabolic syndrome. Approach and Results In mice fed high‐fat, high‐calorie, high‐fructose (HFHC) diet, SAB expression progressively increased through a sustained JNK/activating transcription factor 2 (ATF2) activation loop. Inducible deletion of hepatic SAB markedly decreased sustained JNK activation and improved systemic energy expenditure at 8 weeks followed by decreased body fat at 16 weeks of HFHC diet. After 30 weeks, mice treated with control–antisense oligonucleotide (control‐ASO) developed steatohepatitis and fibrosis, which was prevented by Sab‐ASO treatment. Phosphorylated JNK (p‐JNK) and phosphorylated ATF2 (p‐ATF2) were markedly attenuated by Sab‐ASO treatment. After 52 weeks of HFHC feeding, control N‐acetylgalactosamine antisense oligonucleotide (GalNAc‐Ctl‐ASO) treated mice fed the HFHC diet exhibited progression of steatohepatitis and fibrosis, but GalNAc‐Sab‐ASO treatment from weeks 40 to 52 reversed these findings while decreasing hepatic SAB, p‐ATF2, and p‐JNK to chow‐fed levels. Conclusions Hepatic SAB expression increases in HFHC diet–fed mice. Deletion or knockdown of SAB inhibited sustained JNK activation and steatohepatitis, fibrosis, and systemic metabolic effects, suggesting that induction of hepatocyte Sab is an important driver of the interplay between the liver and the systemic metabolic consequences of overfeeding. In established NASH, hepatocyte‐targeted GalNAc‐Sab‐ASO treatment reversed steatohepatitis and fibrosis.

INTRODUCTION: Vanishing bile duct syndrome (VBDS) is an uncommon condition of progressive bile duct loss, which often presents with immuno-allergic signs and can be fatal or necessitate liver transplantation. Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a rare spectrum of immune complex-mediated mucocutaneous reactions which share common immuno-allergic features and triggers with VBDS. Multiple previous case reports have described the co-occurrence of SJS/TEN and VBDS that is more common in children. Described here is a case of a young adult male who developed TEN with VBDS and subsequent hemophagocytic lymphohistiocytosis (HLH) after taking ibuprofen for influenza B. CASE DESCRIPTION/METHODS: A 26-year-old previously healthy male presented to an outside hospital with a 5-day history of fevers, chills, dry cough, and sore throat and a subsequent 7-day history of worsening rash over his back, chest, and extremities. He reported significant ibuprofen use for 3 days before the onset of his rash. Upon presentation, the patient was found to be influenza B positive and was started on oseltamivir. Exanthematous eruptions covered approximately 20% body surface area and quickly progressed to 80%. Skin biopsy findings were consistent with TEN. He was transferred to our facility for specialized burn unit care and was treated for TEN with intravenous immunoglobulin and etanercept with gradual improvement of his rash. Liver biopsy on hospital day 30 for severe cholestatic liver injury demonstrated vanishing bile ducts. Alkaline phosphatase peaked to 2278 U/L on hospital day 44 and serum total bilirubin peaked at 49.2 mg/dL on hospital day 61. Fevers, tachypnea, tachycardia, and leukocytosis persisted, and suspected HLH was diagnosed following a bone marrow biopsy. At the time of abstract submission, the patient remains hospitalized receiving HLH treatment with etoposide and high-dose steroids. DISCUSSION: This case report highlights a concurrent case of SJS/TEN and vanishing bile duct syndrome following an episode of acute influenza B. While there have been previously reported cases of VBDS with concurrent SJS/TEN, this appears to be the first reported case of SJS/TEN, VBDS, and subsequent HLH in the same patient. While this is a single case, it raises the possibility of a shared immuno-allergic pathogenesis between these three conditions.Table 1.: Peak hepatic panel lab valuesTable 2.: Hemophagocytic Lymphohistiocytosis diagnostic criteria

Within the spectrum of autoimmune liver diseases, there are patients who manifest features of more than one disease, which was previously identified as having overlap syndrome1,2 and is now referred to as variant syndromes. The most common variant syndrome is between primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). Typically, AIH presents with elevated serum immunoglobulin (Ig) G, whereas PBC is associated with elevated serum IgM.3,4 Previous studies have suggested that plasma cells in liver biopsies of AIH patients are predominantly IgG+, whereas in PBC, there is an abundance of IgM+ cells.5,6 We wanted to determine the immunostaining pattern for IgG and IgM of liver plasma cells among Hispanic patients in Los Angeles with features of both PBC-AIH compared with those with PBC or AIH alone.

Niacin (vitamin B3) is available as a prescription medication and over-the-counter supplement. Although it is well known for its vasodilatory effect, it has also been associated with mild hepatotoxicity and, rarely, acute liver failure. We present the case of a 74-year-old Hispanic woman who developed acute liver failure (anicteric encephalopathy and coagulopathy) after her home dose of immediate-release niacin was replaced with an extended-release formulation during an inpatient hospital stay. This is the first reported case of niacin toxicity associated with a histopathologic finding of diffuse microvesicular steatosis. This unique phenotype strongly implicates mitochondrial impairment as a mechanism of niacin-induced hepatotoxicity.