The pathogenesis of enteric viral infections is attributed to both viral replication and the resultant immune-inflammatory response. To recapitulate this complex pathophysiology, we engineer macrophage-augmented organoids (MaugOs) by integrating human macrophages into primary intestinal organoids. Echovirus 1, echovirus 6, rotavirus, seasonal coronavirus OC43 and SARS-CoV-2- known to directly invade the intestine- are used as disease modalities. We demonstrate that these viruses efficiently propagate in MaugOs and stimulate the host antiviral response. However, rotavirus, coronavirus OC43 and SARS-CoV-2, but not the two echoviruses, trigger inflammatory responses. Acetate, a microbial metabolite abundantly present in the intestine, potently inhibits virus-induced inflammatory responses in MaugOs, while differentially affecting viral replication in macrophages and organoids. Furthermore, we provide a proof-of-concept of combining antiviral agent with either anti-inflammatory regimen or acetate to simultaneously inhibit viral infection and inflammatory response in MaugOs. Collectively, these findings demonstrate that MaugOs are innovative tools for studying the complex virus-host interactions and advancing therapeutic development.

INTRODUCTION:Adipose tissue is a key mediator of metabolic dysfunction-associated steatotic liver disease (MASLD) development and progression into metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis. Since direct comparisons of body composition parameters are lacking, we here investigate 12 different body composition parameters. METHODS:Adult participants from National Health and Nutrition Examination Survey 2017-2023 with liver health data were included. Exclusion criteria were age older than 80 years, excessive alcohol (>60 g/d), viral hepatitis, and missing anthropometrics. MASLD was defined as controlled attenuation parameter ≥275 dB/m with metabolic dysfunction, MASH as FibroScan-aspartate aminotransferase ≥0.35, and increased liver stiffness measurement (LSM) as ≥8 kPa. Predictive performance of 12 body composition parameters was assessed using area under the curve analysis. Predicted probabilities of outcomes were visualized for standardized parameters, and nonlinearity was assessed through restricted cubic splines. RESULTS:Among 11,579 participants (age 51 [35-63], 47% male), 41% had MASLD, 6.5% at-risk MASH, and 9.9% increased LSM. Waist circumference (WC) and not BMI or waist-to-height ratio obtained the highest area under the curve for MASLD (0.82), at-risk MASH (0.73), and increased LSM (0.75) outperforming or equaling all other indices across subgroups. Associations between WC and MASLD were nonlinear, with slight risk saturation beyond 100 cm; at-risk MASH was linearly associated across the entire spectrum; and increased LSM risk rose only after WC >100 cm. DISCUSSION:In the general population, MASLD and MASH risk increased even when WC < 100 cm, while increased LSM risk was increasing only >100 cm. Although relatively minor differences, WC consistently demonstrated the highest predictive value for MASLD, at-risk MASH, and increased LSM and therefore most suited for MASLD diagnosis, management, and risk stratification.

Background & aims: Patients with chronic HBV infection and concomitant metabolic dysfunction-associated steatohepatitis (MASH) have been shown to develop more severe liver disease than patients with chronic HBV alone. However, our understanding of the underlying mechanisms is limited. Herein, we study how comorbid MASH impacts immune activity in the livers of patients with chronic HBV infection. Methods: Bulk RNA sequencing was performed on liver biopsies from patients with only MASH (n = 10), only HBeAg-negative chronic HBV (ENEG; n = 11), combined MASH/ENEG (n = 9) and healthy controls (n = 9). Biopsies with no or minimal fibrosis (<= F2) were selected to avoid confounding effects of fibrosis. We compared whole transcriptome data from patients with MASH/ENEG to those with ENEG alone to determine the impact of comorbid MASH on chronic hepatitis B. Results: There is a high degree of overlap of liver gene expression profiles in patients with only ENEG vs. those with only MASH compared to healthy controls, suggesting a largely shared mechanism of liver dysfunction and immune activity for these distinct conditions. In patients with ENEG, comorbid MASH was associated with significantly reduced interferon pathway activity (normalized enrichment score = 2.03, p.adj = 0.0251), the expression of interferon-stimulated genes (e.g., IFIT2, IFI27, IFITM1, IFI6), and macrophage gene signatures (e.g., MARCO, CD163, CD5L, CD63), when compared to patients with ENEG alone. Conclusions: Transcriptomic profiling of the liver suggests that MASH negatively impacts interferon-stimulated gene expression and macrophage gene signatures in the livers of patients with ENEG, which may affect antiviral immune pathways, viral replication and inflammatory responses, resulting in an increased risk of advanced fibrosis in patients with chronic hepatitis B. Our study provides valuable insights for guiding future research aimed at developing effective, tailored strategies for managing patients with both conditions. (c) 2024 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article underthe CC BY license (http://creativecommons.org/licenses/by/4.0/).Introduction

Nucleos(t)ide analogs (NAs) provide prolonged viral suppression with favourable clinical outcomes in chronic hepatitis B (CHB) patients. Characterisation of adverse hepatic events after NA cessation leading to liver transplantation (LT) is vital to the improvement of patient management and safety considerations. This is a retrospective case series of CHB patients who developed hepatic decompensation due to NA discontinuation and were referred for LT. Patients with hepatocellular carcinoma or coinfection were excluded. Of 11 CHB patients included (81.8% clinical jaundice, 63.6% ascites, 54.5% hepatic encephalopathy and 18.2% variceal bleeding), 45.5% underwent LT, 36.4% were waitlisted (1 active, 1 died, 2 delisted of whom 1 died), and 18.2% died after referral during the assessment period. Median age was 55.1 years, 81.8% were male, and 72.7% had cirrhosis at NA cessation. Reasons for NA withdrawal included nonadherence (81.8%) and physician discretion (18.2%). Median time from NA cessation to a decompensating event was 3.2 months, and from the decompensating event to referral was 16.0 days. This study shows that most patients experience decompensations soon after NA cessation and reinforces that patients should not discontinue treatment themselves. Physicians should very carefully select non-cirrhotic, adherent patients for NA withdrawal, after which close monitoring and timely retreatment are crucial.

BACKGROUND AND AIMS:Screening for liver disease in the general population requires accurate non-invasive tests (NITs). A head-to-head comparison of NITs for early detection of clinically relevant liver disease among the target population for screening is lacking. APROACH AND RESULTS:Among the meta-cohort (Rotterdam Study and National Health and Nutrition Examination Survey) with metabolic dysfunction aged 18-80 years, 10 NITs were investigated. The diagnostic accuracy for clinically relevant conditions [increased liver stiffness measurement (LSM), at-risk metabolic dysfunction-associated steatohepatitis, advanced fibrosis, or cirrhosis) was assessed. Subgroup analysis included stratification by age group and diabetes/obesity status.We analysed 11,404 participants. Metabolic dysfunction-associated fibrosis 5 (MAF-5) obtained the highest AUC for increased LSM (≥8 kPa: 0.80; ≥12 kPa: 0.87) and advanced fibrosis (AUC: 0.90). Fibrotic NASH index and MAF-5 performed best for detecting metabolic dysfunction-associated steatohepatitis (AUC: 0.93 and AUC: 0.92, p =ns) and SAFE for cirrhosis (AUC: 0.92). To obtain 80% sensitivity for LSM ≥8 kPa, the corresponding MAF-5 cut-off resulted in fewer referrals (42%) compared to fibrosis-4 index (77%) and higher specificity (62% vs. 24%); MAF-5 was also superior for detection of LSM ≥12 kPa and advanced fibrosis. Age-dependent scores yielded lower sensitivity among younger individuals, for example, by referring 20% of the population with the highest NIT scores, the fibrosis-4 index, steatosis-associated fibrosis estimator, NAFLD fibrosis score, FORNS, and Hepamet fibrosis score yielded <10% sensitivity for LSM ≥8 kPa among individuals aged 18-35 years, while fibrotic NASH index and MAF-5 obtained 40% and 71%. CONCLUSIONS:Of the 10 investigated NITs, MAF-5 discriminated best between all conditions except cirrhosis, for which the steatosis-associated fibrosis estimator yielded the highest accuracy. The performance of the fibrosis-4 index was poor, implying that referral pathways for significant liver disease in low-prevalence populations can be improved when more accurate NITs such as MAF-5 are employed.

Background & Aims: Patients with chronic HBV (CHB) with advanced fibrosis are at high risk for hepatocellular carcinoma (HCC). Liver stiffness measurement (LSM) correlates with fibrosis in untreated patients, and is used to monitor changes in severity of liver disease. However, the association between on-treatment LSM and HCC risk is controversial. Methods: We conducted an international multicenter retrospective cohort study of patients with CHB with advanced fibrosis and assessed the association between on-treatment LSM, HCC development, and decompensation events. Results: We analyzed 562 patients (62.8% F4 LSM measurement, 69.2% Asian). During antiviral therapy, the on-treatment LSM decreased to <6 kPa in 209 (37.2%), to 6–9 kPa in 174 (31.0%), and remained >9 kPa in 179 (31.9%) patients. During a median follow-up of 6.8 years after the on-treatment LSM, 56 patients developed HCC and 18 (32.2%) had an on-treatment LSM <6 kPa. The 5-year cumulative HCC incidence was comparable across on-treatment LSM strata; 4.4% for <6 kPa, 5.5% for 6–9 kPa, and 5.8% for >9 kPa (p = 0.300). In multivariable analysis, older age (adjusted hazard ratio (aHR) 1.058, 95% CI 1.026–1.091, p <0.001) and lower platelet count (aHR 0.992, 95% CI 0.992–0.998, p = 0.005) were associated with HCC development, whereas on-treatment LSM was not (aHR 0.974, p = 0.974). By contrast, patients with an LSM decrease to ≤9 kPa had a negligible risk of decompensation (0% vs. 1.8% at 5 years, p = 0.017). Conclusions: Most patients with CHB with advanced fibrosis experienced a decrease in LSM during antiviral therapy. Although a decrease in liver stiffness was associated with a lower risk of decompensation, an improvement in liver stiffness was not associated with a reduction in HCC risk. Impact and implications: The majority of CHB patients with advanced fibrosis have a decrease in LSM during antiviral therapy. Although a decrease in LSM was associated with a lower risk of subsequent hepatic decompensation, an improvement in LSM was not associated with a reduction in HCC risk. HCC surveillance should therefore be continued in patients with advanced fibrosis at baseline regardless of LSM values obtained during therapy.

Background:The LiverRisk score (LRS) has recently been proposed to predict liver fibrosis and future development of liver-related outcomes in the general population. Here, we performed an external validation of this score. Methods:We used data from National Health and Nutrition Examination Survey 2017-2020, a United States population-based cohort to assess the diagnostic accuracy of the LRS to detect a liver stiffness measurement (LSM) ≥8 and ≥12 kPa. Performance was tested among the entire general population and clinically relevant subgroups. Results:The cohort comprised 7,025 participants (aged 49 [33-63], 49% male), and 9.7% had an LSM ≥8 and 3.2% had an LSM ≥12 kPa. The area under the receiver characteristic operator curve (AUC) in the overall population was 0.73 (95% confidence interval [CI] :0.71-0.75) and 0.78 (95% CI: 0.74-0.81) to detect an LSM ≥8 and ≥ 12 kPa, respectively, significantly outperforming the fibrosis 4 index (FIB-4) but not the nonalcoholic fatty liver disease fibrosis score, steatosis-associated fibrosis estimator (SAFE), or metabolic dysfunction-associated fibrosis 5 (MAF-5). Performance was consistent among most subgroups, but AUC levels to detect an LSM ≥8 kPa decreased to <0.70 among participants aged 18-40 or 60-80 years, blacks, and individuals with diabetes or liver steatosis. The LRS categorized 80.5% as very low risk, 17.7% as low risk, and 1.8% as at risk, prevalence of an LSM ≥8 in these groups was 6.3%, 20.8%, and 50.5%, respectively. The sensitivity to detect an LSM ≥8 kPa was 47.3% in the overall population (but dropped to 21.3% for individuals aged 18-40 years) despite applying the lowest cut-off, which should yield the highest sensitivity. Conclusion:The LRS score is a promising new tool to predict liver fibrosis; however, its diagnostic accuracy attenuates especially among patients aged 18-40 or 60-80 years. The overall sensitivity was only 47.3% at the lowest LRS cut-off. Further studies assessing cost-benefit ratios according to the LRS compared to FIB-4 and other risk scores such as MAF-5 and SAFE are required to determine its usefulness in referral strategies.

Chronic liver injury triggers the activation and recruitment of immune cells, causing antigen-independent tissue damage and liver disease progression. Tissue inflammation can reshape macrophage composition through monocyte replacement. Replacement of tissue macrophages with monocytes differentiating in an inflammatory environment can potentially imprint a phenotype that switches the liver from an immune-tolerant organ to one predisposed to tissue damage. We longitudinally sampled the liver of patients with chronic hepatitis B who had active liver inflammation and were starting antiviral therapy. Antiviral therapy suppressed viral replication and liver inflammation, which coincided with decreased myeloid activation markers. Single-cell RNA-Seq mapped peripheral inflammatory markers to a monocyte-derived macrophage population, distinct from Kupffer cells, with an inflammatory transcriptional profile. The inflammatory macrophages (iMacs) differentiated from blood monocytes and were unique from macrophage found in healthy or cirrhotic liver. iMacs retained their core transcriptional signature after inflammation resolved, indicating inflammation-mediated remodeling of the macrophage population in the human liver that may affect progressive liver disease and immunotherapy.

The pathophysiology of acute viral diseases is complex. It is characterized by strong inflammatory responses driven by immune cells, leading to tissue damage. Currently available in vitro models mainly recapitulate the viral life cycle but fail to model immune cell-mediated pathogenesis. Here we build macrophage-augmented organoids (MaugOs) by integrating macrophages into primary organoids that are cultured from human liver tissues. We test the infections of two RNA viruses, hepatitis E virus and SARS-CoV-2, and one DNA virus, monkeypox virus, which either primarily or secondarily affect the human liver. In all three models of acute viral diseases, MaugOs recapitulate infection and the resulting inflammatory response, although to different levels. We use this system to dissect the multifunctional role of human bile on hepatitis E virus replication and the inflammatory response through distinct mechanisms of action. We also show that MaugOs recapitulate features of inflammatory cell death triggered by hepatitis E virus infection when integrated with pro-inflammatory macrophages. Furthermore, we demonstrate a proof of concept in MaugOs for development of multitarget therapeutics that simultaneously target the virus, inflammatory response and the resultant inflammatory cell death. Macrophage-augmented organoids integrate macrophages into organoids that are cultured from human liver tissue for analysis of immune responses during three different viral infections.