THE ASSOCIATION BETWEEN MENTAL ILLNESS AND ALL-CAUSE MORTALITY IN PATIENTS WITH CIRRHOSIS: A VETERANS AFFAIRS RETROSPECTIVE COHORT STUDY

Background: Alcoholic hepatitis (AH) is a life-threatening form of alcohol-associated liver disease (ALD), with no effective therapy. Liver neutrophil infiltration is a hallmark of AH, yet the mechanisms by which alcohol promotes neutrophil recruitment and disrupts neutrophil functions remain elusive. Identifying new therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase, plays an important role in neutrophil development and function, however, the mechanism by which BTK regulates neutrophil-mediated liver damage, specifically in AH, is not yet studied.
Methods: Female, C57BL6 mice aged 10-12 weeks received Lieber-deCarli diet with alcohol for 10 days followed by one binge (alcoholic hepatitis-AH-model) or calorie-matched control diet. Some mice received oral gavage of Evobrutinib, a BTK inhibitor (BTKi) or DMSO from day7 to day10. Liver damage was assessed by measuring serum ALT. Serum levels of proinflammatory cytokines were measured by ELISA. BTK phosphorylation (pBTK) was assessed in human or mouse livers by western blotting, and in bone marrow (BM) progenitors or liver neutrophils by flow cytometry. Circulating neutrophils from healthy controls (HC) and AH patients were analyzed for BTK expression by RNA-seq. Tandem mass spectrometry (LC-MS/MS) was performed in mouse liver lysates to identify BTK substrates.
Results: Our results from unbiased RNA-seq in circulating neutrophils showed a significant increase in BTK in AH patients compared to HC. Consistently, pBTK was significantly increased in neutrophils from AH patients compared to HC. We found that pBTK (Y223 and Y551) was upregulated in humans as well as in mouse livers with ALD. We further dissected that acute alcohol binge rather than chronic moderate alcohol use triggers pBTK in mice. In vitro, physiologically relevant doses of alcohol induced rapid pBTK induction in neutrophils that was partially TLR4-mediated. In a preclinical model of AH, administration of Evobrutinib, a small molecule BTK inhibitor, significantly decreased pro-inflammatory cytokines levels, and attenuated liver damage. Importantly, we discovered that alcohol-induced liver neutrophil infiltration correlated with the expansion of BM granulopoiesis, and that pBTK in BM progenitors was essential for alcohol-induced granulopoiesis. In vivo, BTK inhibition significantly reduced granulopoiesis, circulating neutrophils, and liver infiltration in the presence of alcohol administration. Mechanistically, using LC-MS/MS we discovered CD84 as a novel kinase target of BTK which is involved in granulopoiesis.
Conclusion: Our findings define a novel role of BTK in regulating CD84 phosphorylation and granulopoiesis that could be harnessed to address pressing therapeutic needs in AH.

Background: ALD ranges from simple steatosis to alcohol-induced steatohepatitis (ASH), alcoholic hepatitis and liver cirrhosis. Liver steatosis, inflammation, fibrosis, and ductular reaction (DR) and biliary senescence are hallmarks of ALD. Substance P (SP, encoded by Tac1 and degraded by membrane metalloendopeptidase, MME) modulates biliary functions by interaction with NK1R. While activation of the SP/NK1R axis increases DR and liver fibrosis via the release of biliary SASPs, KO of NK1R ameliorates liver phenotypes in cholestatic mice. Women are more susceptible to ALD than men and there is no data regarding the role of NK1R in the progression of ALD in females. We evaluated the role of NK1R on liver damage in an acute model of ALD in female mice. Methods: Wild-type (WT, n=8) and NK1R KO female mice (n=5) were subjected to the chronic-plus-binge ethanol (EtOH) feeding model of ALD (NIAAA model). Mice were fed EtOH diet for 10 days with a single gavage (5 g/kg EtOH) to induce ASH. Control mice were pair-feed an isocaloric control diet for 10 days, followed by a single gavage of isocaloric maltodextrin. On day 10, we collected liver, serum, cholangiocytes and hepatocytes. In mice we measured the levels of ALT in serum, and SP in serum and biliary supernatant. Steatosis was examined by H&E and Oil Red O staining. Fatty acid oxidation in hepatocytes was evaluated by qPCR for PPARa and Acetyl CoA synthetase 1 (ACS1). We evaluated DR by IHC for CK-19 and biliary senescence by IF for p16 in liver sections. Human specimens from male (n=5) and female (n=4) healthy controls (n=9), and patients with alcoholic cirrhosis male (n=15) and female (n=6) were evaluated for serum SP levels and NK1R immunoreactivity of cholangiocytes, hepatocytes and hepatic stellate cells. NK1R and MME protein levels were evaluated in human total liver (n=4) by immunoblots. Results: NK1R was mainly expressed by human cholangiocytes. There were enhanced SP serum levels and increased NK1R biliary immunoreactivity in female/male patients with alcoholic cirrhosis compared to controls. MME protein expression decreased in total liver from female patients with alcoholic cirrhosis compared to controls. There were increased levels of ALT in serum, and SP levels in serum and biliary supernatant from WT EtOH-fed mice compared to controls. There was a moderate (not significant) increase in DR in EtOH-fed mice compared to controls. There was increased hepatic steatosis, lipid accumulation and enhanced p16 immunoreactivity in mouse liver sections from EtOH-fed WT mice (compared to CD-fed WT mice), phenotypes that returned to normal values in EtOH-fed NK1R KO mice. All EtOH-fed mice showed a significant decrease in the expression of PPARa and ACS1 in hepatocytes compared to CD-fed WT mice. Conclusion: Downregulation of NK1R signaling may be important in the management of ALD phenotypes.

Background
There is a huge burden of undiagnosed but clinically significant liver disease in otherwise asymptomatic at risk individuals in community alcohol services. Early identification followed by effective behaviour change interventions are pivotal to change natural history of alcohol related liver disease. We aim to establish the feasibility of conducting a definitive RCT to test the effectiveness of transient elastography (TE) based advice and alcohol recovery video stories (ARVS) in changing high-risk drinking behaviour in community alcohol services.
Methods
RCT was conducted at three community alcohol services. Participants (≥18years) presenting to any of these services with a primary alcohol problem were recruited and randomised (1:1) to either control group and continued routine care, or to intervention group who in addition to routine care had TE, received advice based on results of the liver stiffness measure (LSM) and watched ARVS. Data were collected on demographics, self-reported alcohol intake, AUDIT, and severity of alcohol dependence questionnaire (SADQ) score. Follow up data was collected at six months. Our analysis assessed the feasibility of conducting a RCT, including identifying participant recruitment and retention rates, documenting intervention delivery and acceptability, and evaluating limited efficacy in changing drinking behaviour.
Results
382 participants were screened for eligibility, 184 were randomised (intervention n=93, control n=91), 128 (intervention n=71, control n=59) attended the post-randomisation baseline appointment and were included. Mean age was 43.9years (SD±11.9), majority were male (n=94, 73.4%), white (n=106, 82.8%), and heterosexual (n=115, 90.6%). Over a quarter were poly-drug users (n=25, 27.35), 22.8% (n=29) had co-existing disability, and 75.8% (n=97) had mental health issues. Baseline median daily self-reported alcohol intake was 26units (range 2-73), 92.8% (n=116) were alcohol dependent on AUDIT assessment, 54.3% (n=69) had severe alcohol dependence on SADQ. Six months follow-up was available in 75 (intervention n=47, control n=28). Intervention group compared to control reported greater reduction in daily self-reported alcohol intake (23.0 vs 19.5 units) (Figure 1), AUDIT category (57.8% vs 53.2%), were more likely to attend follow up, and complete the allocated treatment program. Of participants randomised to the intervention group 52 had transient elastography, of whom 21.2% (n=11) had raised LSM (≥8 kilopascal) (Figure 2). None of participants who received transient elastography reported increase in self-reported alcohol intake or AUDIT category.
Conclusion
Integration of transient elastography and ARVS in community alcohol services is feasible, it can stratify clinically significant liver disease, and supplement change in drinking behaviour. Normal LSM does not provide false reassurance.

Background: We showed previously that production of acetaldehyde after acute ethanol causes widespread mtDepo in mouse liver, which stimulates Type 2 mitophagy. In this study, we tested the hypotheses that after CBE, mitophagic overburden causes dysregulated autophagic processing and increased release of mtDAMPs, promoting inflammatory responses and liver injury and that activation of aldehyde dehydrogenase-2, which degrades acetaldehyde, attenuates these alterations. Methods: Female mice were fed a liquid diet containing 5% ethanol for 10 days and then gavaged once with ethanol (5 g/kg) on day 11. Alda-1 (20 mg/kg, sc) or vehicle was injected daily. mtDepo and steatosis were detected by intravital multiphoton microscopy. Results: CBE increased serum ALT to ~340 U/L, and histology showed moderate to severe hepatic steatosis, leukocyte infiltration, and scattered necrotic cell death. After CBE, cleaved caspase-3 (apoptosis marker) increased by 75%, interleukin-1β increased by 81%, and myeloperoxidase (neutrophil marker) increased by ~350%. Alda-1 attenuated each of these increases. After CBE, mtDepo occurred in ~90% hepatocytes. Moreover, numerous fat droplets (micro- and macro-vesicular) appeared in hepatocytes. More and larger fat droplets developed in hepatocytes with mtDepo. We also explored if mtDepo increased mitophagy. Mitophagy-associated proteins PINK1, p62 and LC3 increased, indicating increased mitophagic burden, which Alda-1 also blunted. Using intravital multiphoton microscopy detecting TMRM fluorescence in GFP-LC3 transgenic (Tg) mice, we observed that GFP-LC3 puncta increased after CBE, consistent with increased mitophagy. However, the increase of GFP-LC3 puncta was not as great as observed previously after a single acute ethanol gavage. Although mitophagic burden increased after CBE, TFEB, the master regulator of lysosomal enzyme expression and function, decreased, indicating disrupted lysosomal processing of mitophagosomes. Decreased lysosomal processing may lead to release of cytotoxic, pro-inflammatory, and/or profibrotic mtDAMPs. As evidence of mtDAMP release after CBE, serum mtDNA/nuclear DNA ratios increased ~10-fold, serum succinate doubled, and serum cytochrome c about tripled. TFEB also regulates expression of PGC1α, the master regulator of mitochondrial biogenesis (MB). MB must occur after mitophagy to recover mitochondrial mass and function. PGC1α, TFAM (a target gene of PGC1α that controls mtDNA replication and transcription), and ND3 (an oxidative phosphorylation protein) all decreased after CBE. Alda-1 increased TFEB, decreased mtDAMP release, and increased MB after CBE. Conclusion: CBE increases mtDepo in an acetaldehyde-dependent fashion, which leads to mitophagic overburden, disruption of mitochondrial homeostasis, mtDAMP release, and ultimately development of liver injury and inflammation (NIAAA & NIDDK).

Background & Aims: Patients with cirrhosis have a higher burden of mental health comorbidities than the average population, which is known to result in worsened disease outcomes. Outside of the hepatitis C population, few studies have investigated the potential protective effects of regular outpatient mental healthcare. We used national Veterans Health Administration data to characterize trends in mental health diagnoses and utilization of outpatient mental health care, to explore the association between mental illness and all-cause mortality, and to understand the effects of regular outpatient mental healthcare.
Methods: We conducted a retrospective cohort study of veterans with cirrhosis from 2008-2021. Simple linear regression was used to analyze trends in mental illness and engagement with outpatient care. We used inverse probability treatment weighting (IPTW)-adjusted Cox regression analysis to characterize the association between mental health-related diagnoses and all-cause mortality based on three distinct disease categories of any mental health diagnosis, alcohol use disorder (AUD)/substance use disorder (SUD), and non-AUD/SUD. Using these same methods, we examined the impact of regular outpatient mental health visits in subgroup analyses.
Results: We identified 115,409 patients, 81.7% of whom had any mental health diagnosis. There were statistically significant increases in the prevalence of all non-AUD/SUD diagnoses (p<0.001), but a slight decrease in prevalence of AUD and SUD (p<0.05; figure 1). However, the incidence proportions of all individual mental health diagnoses significantly increased since 2016 (p<0.05), with AUD and SUD representing the highest incidence proportions overall. There was also a significant increase in the number of mental health clinic visits per person-year (p<0.001), but a decrease in AUD/SUD clinic visits (p<0.001) during the study window. There was a 54% increased hazard in all-cause mortality for any mental health diagnosis, 11% for non-AUD/SUD, and 44% for AUD/SUD (each p<0.001). Regular mental health visits resulted in a 21% decreased risk in all-cause mortality for AUD/SUD diagnosis, compared to 3% and 9% for any mental health diagnosis and non-AUD/SUD diagnosis, respectively (each p<0.001; figure 2).
Conclusions: Mental illness is associated with an increased risk of all-cause mortality in veterans with cirrhosis. Regular outpatient mental healthcare is protective against all-cause mortality, particularly among patients with AUD/SUD. These findings suggest that greater attention should be paid to identifying mental health disease in patients with cirrhosis, and more resources should be allocated for mental health treatment, particularly in those with alcohol or substance use disorder. Future studies should focus on relevant clinical practice changes.