Single cell-resolved study of advanced murine MASH reveals a homeostatic pericyte signaling module.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.

The prevalence and risk factors for cognitive impairment in obesity and NAFLD.

BACKGROUND: Severe obesity may be accompanied by cognitive dysfunction and NAFLD, but the associations remain unclear. We describe the prevalence and features of cognitive dysfunction and examine the associations between cognitive dysfunction and the presence and severity of NAFLD, and the associations between cognitive dysfunction and signs of other obesity-related comorbidities and neuronal damage. METHODS: A cross-sectional study of patients with a body mass index of 35 kg/m2 underwent evaluation for bariatric surgery. They were screened for adiposity-related comorbidity and underwent a liver biopsy and basic cognitive testing with the Continuous Reaction Time test, the Portosystemic Encephalopathy Syndrome test, and the Stroop Test. A representative subgroup also underwent the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). The primary study outcome was "cognitive impairment," defined as ≥2 abnormal basic cognitive tests and/or an abnormal RBANS. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) served as a biomarker for neuronal damage. RESULTS: We included 180 patients; 72% were women, age 46 ± 12 years, 78% had NAFLD, and 30% with NASH without cirrhosis. 8% were cognitively impaired by the basic tests and 41% by RBANS results. Most impaired were executive and short-time memory functions. There were no associations between cognitive impairment and BMI, NAFLD presence or severity, or metabolic comorbidities. Male sex (OR: 3.67, 95% CI, 1.32-10.27) and using 2 or more psychoactive medications (5.24, 95% CI, 1.34-20.4) were associated with impairment. TREM2 was not associated with cognitive impairment. CONCLUSIONS: Nearly half of this severely obese study cohort exhibited measurable multidomain cognitive impairment. This was not dependent on NAFLD or another adiposity comorbidity.

Stellate cell expression of SPARC-related modular calcium-binding protein 2 is associated with human non-alcoholic fatty liver disease severity.

Background & Aims: Histological assessment of liver biopsies is the gold standard for diagnosis of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), despite its well-established limitations. Therefore, non-invasive biomarkers that can offer an integrated view of the liver are needed to improve diagnosis and reduce sampling bias. Hepatic stellate cells (HSCs) are central in the development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods: We performed RNA-sequencing on liver biopsies from a histologically characterised cohort of obese patients (n = 30, BMI >35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings using single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results: Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared to no-NAFLD (p.adj <0.001). Single-cell RNA-sequencing data indicated that SMOC2 was primarily expressed by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p <0.001), with a predictive accuracy of AUROC 0.88. Conclusions: Increased SMOC2 in plasma appears to reflect HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the most common forms of chronic liver diseases. Currently, liver biopsies are the gold standard for diagnosing NAFLD. Blood-based biomarkers to complement liver biopsies for diagnosis of NAFLD are required. We found that activated hepatic stellate cells, a cell type central to NAFLD pathogenesis, upregulate expression of the secreted protein SPARC-related modular calcium-binding protein 2 (SMOC2). SMOC2 was elevated in blood samples from patients with NASH and may hold promise as a blood-based biomarker for the diagnosis of NAFLD.

The Role of Diagnostic Biomarkers, Omics Strategies, and Single-Cell Sequencing for Nonalcoholic Fatty Liver Disease in Severely Obese Patients.

Liver disease due to metabolic dysfunction constitute a worldwide growing health issue. Severe obesity is a particularly strong risk factor for non-alcoholic fatty liver disease, which affects up to 93% of these patients. Current diagnostic markers focus on the detection of advanced fibrosis as the major predictor of liver-related morbidity and mortality. The most accurate diagnostic tools use elastography to measure liver stiffness, with diagnostic accuracies similar in normal-weight and severely obese patients. The effectiveness of elastography tools are however hampered by limitations to equipment and measurement quality in patients with very large abdominal circumference and subcutaneous fat. Blood-based biomarkers are therefore attractive, but those available to date have only moderate diagnostic accuracy. Ongoing technological advances in omics technologies such as genomics, transcriptomics, and proteomics hold great promise for discovery of biomarkers and increased pathophysiological understanding of non-alcoholic liver disease and steatohepatitis. Very recent developments have allowed for single-cell sequencing and cell-type resolution of gene expression and function. In the near future, we will therefore likely see a multitude of breakthrough biomarkers, developed from a deepened understanding of the biological function of individual cell types in the healthy and injured liver.

Prediction of overt hepatic encephalopathy by the continuous reaction time method and the portosystemic encephalopathy syndrome test in clinically mentally unimpaired patients with cirrhosis.

BACKGROUND AND AIM: Predicting overt hepatic encephalopathy (OHE) is important because the condition is frequent, often requires hospitalization and is potentially preventable. The risk of OHE is related to pre-existing discrete cognitive defects, and for clinical practice it is recommended to apply two different psychometric tests to detect such deficits. We used the continuous reaction time test (CRT) and the portosystemic encephalopathy (PSE) syndrome test and examined their single and combined value for OHE prediction in cirrhosis patients. PATIENTS AND METHODS: We studied 130 clinically mentally unimpaired cirrhosis patients by the two tests and followed them for an average of 38.5 months. The CRT measures velocity and stability of motor reaction times to 150 repeated auditory signals. The PSE is a five sub-set paper-and-pencil test battery evaluating cognitive and psychomotor processing, speed and vision-motor coordination. We collected data on episodes of OHE during follow-up. The clinical course was analysed in patient groups according to the outcome of each test and of both tests together. No anti-HE treatment was initiated except for cases with OHE. RESULTS: At baseline, the CRT test was abnormal in 74 patients and the PSE in 47. During follow-up 35 patients (27%) experienced 74 OHE events. 23 patients with abnormal CRT experienced OHE (prediction sensitivity 65%). The PSE predicted OHE in 14 patients (prediction sensitivity 40%). One or both tests were abnormal in 87/130 (67%) and this predicted OHE in 27 patients (21%) (prediction sensitivity 77%). CONCLUSION: The CRT test was clinically useful in identifying two-thirds of clinically mentally unimpaired cirrhosis patients who later experienced OHE, and the use of both the CRT and PSE showed satisfactory prediction by identifying three-fourths of later OHE cases.