HEPATIC RETICULOENDOTHELIAL SYSTEM CELL IRON DEPOSITION IS INDEPENDENTLY ASSOCIATED WITH ADVANCED FIBROSIS IN NASH: ANALYSIS OF 2833 PATIENTS FROM THE NASH-CRN

Background:
The clinical course of HBV in individuals with HIV coinfection is marked by accelerated disease progression. A tenofovir-containing anti-retroviral regimen is recommended in most people with HIV-1/HBV-coinfection but there have not been randomized studies of TDF vs TAF in treatment-naïve HIV-1/HBV-coinfected individuals. We report primary endpoint results from a phase 3 study comparing B/F/TAF vs DTG+F/TDF at Week (W) 48 in participants initiating treatment for both viruses.
Methods:
Adults with HIV-1/HBV coinfection were randomized 1:1 to initiate blinded treatment with B/F/TAF or DTG+F/TDF (with placebo). Primary endpoints were proportion of participants with HIV-1 RNA <50 copies/mL (FDA Snapshot) and plasma HBV DNA <29 IU/mL (missing=failure) at Week 48. Noninferiority was assessed with 95% CI (12% margin). Secondary and other endpoints included change from baseline CD4 count, proportion with HBsAg and HBeAg loss/seroconversion, and ALT normalization (AASLD criteria).
Results:
243 participants were randomized and treated (121 B/F/TAF, 122 DTG+F/TDF) from 11 countries in Asia, Europe, North and Latin America. Baseline characteristics were median age 32 years, 4.5% female, 88% Asian, 30% HIV-1 RNA >100,000 c/mL, 40% CD4 <200 cells/μL, median HBV DNA 8.1 log10 IU/mL, 78% HBeAg+. At W48, B/F/TAF was noninferior to DTG+F/TDF at achieving HIV-1 RNA <50 copies/mL (95% vs 91%, difference 4.1%; 95% CI -2.5% to 10.8%, p=0.21), with mean CD4 gains of +200 and +175 cells/μL, respectively. B/F/TAF was superior to DTG+F/TDF at achieving HBV DNA <29 IU/mL (63% vs 43%, difference 16.6%; 95% CI 5.9% to 27.3%, p=0.0023). Participants treated with B/F/TAF vs DTG+F/TDF had numerically higher HBsAg loss (13%, 6%, p=0.059), HBeAg loss (26%, 14%, p=0.055), HBeAg seroconversion (23%, 11%, p=0.031), and ALT normalization (73%, 55%, p=0.066). Most frequent AEs were upper respiratory tract infection (17%, 11%), COVID-19 (13%, 11%), pyrexia (9%, 12%), ALT increase (7%, 11%), and nasopharyngitis (11%, 4%). ALT flares (elevations at ≥2 consecutive post-baseline visits) occurred in 11 participants (7 B/F/TAF, 4 DTG+F/TDF) which resolved.
Conclusions:
In adults with HIV-1/HBV-coinfection starting antiviral therapy, both B/F/TAF and DTG+F/TDF had high HIV-1 suppression at year 1, with B/F/TAF resulting in superior HBV DNA suppression and significantly more HBeAg seroconversion. Safety findings were similar between groups.

Background and Aims: Non-alcoholic steatohepatitis (NASH) is a major cause of cirrhosis and the effect of alcohol use on NASH progression and fibrosis/cirrhosis remains to be explored. While common elements in the pathogenesis of NASH and alcohol- associated liver disease (ALD) have been described the combined effects of NASH and ALD remain obscure. We hypothesized that repeated alcohol binges in a high fat-cholesterol-sugar diet (NASH diet) -induced model of NASH accelerates liver damage and fibrosis through inflammatory pathways activation and neutrophil recruitment.
Methods: 8-10 weeks old male C57BL/6 mice received either chow or NASH diet for 3 months with or without weekly alcohol binges. Liver mRNA was analyzed for fibrosis and inflammation markers. Hepatic fibrosis and inflammation were further tested by immunohistochemistry (IHC), qPCR and western blotting. Neutrophil infiltration and neutrophil extracellular traps (NETs) were examined.
Results: Liver transcriptome screen revealed major increase in genes involved in leukocyte migration, inflammatory response and extracellular matrix reorganization in mice that received weekly alcohol binges (EtOH) plus NASH diet compared to either of the diets alone. NASH diet and EtOH binges, respectively, resulted in increased serum ALT compared to chow diet, and EtOH plus NASH diet caused the greatest liver damage compared to all groups. Fibrosis markers, including Sirius red staining, a-SMA and vimentin protein levels, were highest in the NASH diet plus EtOH group compared to NASH diet and EtOH alone. We found an increase in the Ly6G⁺ neutrophils in the liver after EtOH binges that was even higher in mice with NASH plus EtOH. Increased citrullinated histone and neutrophil elastase levels in NASH plus EtOH mice, but not in NASH mice indicated the presence of neutrophil extracellular traps (NETs) in the liver. In vitro, cell-free NETs caused robust hepatic stellate cell activation (HSCs), indicated by increased smooth muscle actin and collagen protein production that was abrogated by DNase treatment. HSCs sensed NETs via NLRP3 inflammasome, as NLRP3 inhibition attenuated their activation. Interestingly, neutrophil infiltration and NETs production were partially regulated by NLRP3. Inhibition of NLRP3 by in vivo administration of MCC950, an NLRP3 inflammasome inhibitor, or NLRP3 KO mice significantly attenuated liver damage, neutrophil infiltration and NETs formation in NASH plus EtOH binge fed mice.
Conclusion: Alcohol binges in NASH accelerate liver damage and fibrosis. NETs directly induce a pro-fibrotic stellate cell phenotype via NLRP3. Inhibition of NLRP3 attenuates NET formation and in vivo NLRP3 inhibition may represent a target to attenuate the pro-fibrotic effect of alcohol in NASH fibrosis.

Objective: To assess trends in liver transplantation (LT) for ACLFin older patients (≥ 65 years old) in comparison to younger patients (18-65 years old). In addition, we also wanted to explore differences in the etiology of underlying cirrhosis between these two age cohorts.

Methods: All adults who underwent LT for ACLF between 2005-2021 were identified using the UNOS database and subdivided into older (≥ 65 years) and younger (18-65 years) age groups. ACLF patients were identified using the EASL-CLIF criteria. We excluded those listed with status 1, 1A, or 1B, multi-organ transplant and living donor transplant. We grouped etiologies of cirrhosis into alcohol-related liver disease (ALD), NASH, HCV, cryptogenic cirrhosis and PBC/PSC. The transplant years (2005-2021) were divided into 3 eras: pre-direct-acting antiviral (DAA) (2005-2013), DAA era (2014-2019), and COVID pandemic era (2020-2021).
Results: Between 2005 and 2021 a total of 2142 patients ≥ 65 years of age underwent LT for ACLF. Of these 26% had NASH, 25% ALD, 10% had HCV and 8% had PBC +PSC. In the year 2005, only 66 older patients underwent LT for ACLF, but this increased by 262% to 239 patients in 2021.
In the pre-DAA era, ALD (20%) was the leading cause of underlying ACLF in the elderly followed by NASH, cryptogenic cirrhosis, and HCV. During DAA era, NASH was the predominant etiology of ACLF followed by ALD, but in the COVID pandemic year of 2020 ALD overtook NASH as the leading cause of ACLF in older adults followed by a reversal in 2021 (Fig 1).
During the same period, 16,931 younger patients with ACLF underwent LT. This increased from 648 in 2005 to 1908 in 2021 (+194%). The most prevalent etiology for underlying cirrhosis in younger patients was ALD (44%) followed by HCV (15%) and NASH (10%). Between 2005 and 2013, HCV was the leading cause of underlying cirrhosis in ACLF were HCV and ALD, however, in the DAA era, HCV started declining swiftly and was replaced by a rapid rise in ALD. In the years 2020 and 2021, ALD leading to ACLF was responsible for 62% and 68% of all LTs in younger patients respectively (Fig 2).

Conclusion: There is considerable variation in the etiology of underlying cirrhosis in ACLF between older and younger patients. LT for ACLF has consistently increased across all age groups from 2005 to 2021 but has been more profound (262% increase) in the older population. Overall NASH is the leading cause of ACLF in this population, but in the pandemic year of 2020 ALD replaced NASH as the predominant etiology. While in younger patients who undergo LT for ACLF, ALD remains the most prevalent etiology and continues to increase at an alarmingly high rate.

Background: Bariatric surgery remains a potent therapy for nonalcoholic fatty liver disease (NAFLD), but its inherent risk and eligibility requirement limit its adoption. Therefore, understanding how bariatric surgery improves NAFLD, which accounts for 40-60% of obesity cases, is paramount to developing novel therapeutics. Here, we show that the microbiome changes induced by sleeve gastrectomy (SG) reduces glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory peptide (GIP), signaling and confer resistance against diet induced obesity (DIO) and NAFLD. We also show that intraperitoneal (IP) injections of a GIP receptor (GIPR) antagonist in vivo confer a decrease in weight gain. Approach and Results: We examined a cohort of NALFD patients undergoing SG and evaluated their microbiome, serum metabolites, and GI hormones. We saw significant changes in Bacteroides, lipid-related metabolites and reduction in GIP. To examine if the changes in the microbiome were causally related to NAFLD, we performed fecal microbial transplant in antibiotic treated mice from patients before and after their surgery who had significant weight loss and improvement of their NAFLD. Mice transplanted with the microbiome of patients after bariatric surgery were more resistant to DIO and NAFLD development compared to mice transplanted with the microbiome of patients before surgery. This resistance to DIO and NAFLD was also associated with a reduction in GIP levels in mice with post-bariatric microbiome. We further show that the reduction in GIP was related to higher levels of Akkermansia and differing levels of indolepropionate, a bacteria derived tryptophan-related metabolite. To examine alterations in weight by blocking GIP signaling, 20 WT C57/Bl6 mice were placed on a nonalcoholic steatohepatitis (NASH) diet, 10 of which received daily intraperitoneal (IP) injections of [Pro³]-GIP, a GIPR antagonist, whereas the other 10 received daily IP injections of a saline vehicle, for 12 weeks to observe weight prevention of DIO. These mice have displayed consistent trends of increased weight regardless of treatment, however, an observable decrease in average weight gain has been observed in mice receiving the [Pro³]-GIP-treatment beginning at the 5-week time point. Conclusions: Overall, this is one of the few studies showing that GIP signaling is altered by the gut microbiome and it supports that the positive effect of bariatric surgery on NAFLD is in part due to microbiome changes. Additionally, the role of GIPR antagonism for improving NAFLD using WT C57/Bl6 mice with diet-induced NAFLD will pave the way for GIPR antagonism and microbiome-based targets as therapeutic targets for NAFLD in future human studies.

Introduction
We previously showed that the pattern of hepatic iron staining is associated with NASH severity and advanced fibrosis. We examined the relationship between hepatic iron deposition and disease severity in a large biopsy-proven cohort enrolled in the Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database Study.

Methods
Patients enrolled in the NASH CRN NAFLD Database, PIVENS or FLINT studies, from November 2004 to May 2022, were included in the analysis. Patient demographics, body weight, height, liver biopsy findings, and laboratory data obtained within six months of the liver biopsy were compared between patients with and without stainable hepatic iron. Multivariate logistic regression analysis was performed to identify the relationship between the pattern of iron deposition and advanced fibrosis. Only variables that met the cut-off of p<0.2 on univariate analysis were included in the study.

Results
A total of 2,833 patients were included in the analysis; 1,166 patients (41%) had positive hepatic iron staining. Of these, 272 (23.2%) patients had iron localized to hepatocytes (HC), 284 patients had iron in reticuloendothelial system (RES) cells, and the remaining had a mixed pattern of iron deposition. Most patients in the iron stain-positive group were Male (55%), middle-aged (40-60 years) (57%), White (77%), and had obesity (66%). Patients with RES iron deposition had higher AST, ALT, insulin, and HOMA-IR levels than other groups. These results are presented in Table 1. The lowest mean iron, ferritin, and transferrin saturation were noted in the iron-stain-negative patients, while the lowest mean TIBC was noted in the patients with mixed iron deposition. A higher proportion of patients with RES iron deposition had grade 3 steatosis (29%), cirrhosis (16%), severe hepatocyte ballooning (47%), severe lobular inflammation (15%), more than mild chronic portal inflammation (30%) and definite NASH (71%) compared to other groups. Patients in the RES group also had higher mean NAS scores and fibrosis scores than other groups. On multivariate regression analysis adjusting for age at biopsy, gender, diabetes status, and BMI, patients with RES iron were more likely to have advanced fibrosis (aOR-1.34, 95% CI- 1.11-1.62, p-0.003), while patients with HC iron had lower odds of advanced fibrosis (aOR-0.68, 95% CI- 0.55-0.83, p<0.001) (Figure 1)


Conclusion
The current study describes the largest cohort of patients with biopsy-proven NASH examining the relationship between the pattern of hepatic iron staining and histological severity. RES iron staining is independently associated with more severe NASH and advanced fibrosis, while hepatocellular iron deposition is associated with less severe disease. These data provide important insights into the role of iron as a co-factor in NASH and may have implications for therapy for this disease.