Dechun Feng

BACKGROUND: In chronic alcohol consumers, immune cells may drive the progression from mild liver injury to more severe alcohol-associated liver disease (ALD), including alcohol-associated hepatitis (AAH) and cancer. Liver macrophages, both resident and infiltrating, express allograft inflammatory factor 1 (AIF1), which is upregulated during inflammation and enhances immune activation. METHODS: Using serum and urine samples from 868 individuals classified as having alcohol use disorder or not, based on DSM-IV/V criteria, along with serum and liver biopsy tissue from a second cohort of 27 patients diagnosed with AAH, we evaluated the impact of the AIF1 promoter single-nucleotide polymorphism (SNP) (rs3132451; C/C , C/G , G/G ) on liver function markers and immune cell profiles. RESULTS: AIF1 transcript levels were genotype dependent: C/C homozygotes expressed 5.2% of the levels observed in G/G individuals, while C/G heterozygotes expressed 46%. Unlike most SNPs associated with harmful effects, the G/G genotype is highly prevalent, present in about 70% of patients. Among chronic alcohol users, G/G individuals exhibited elevated markers of liver injury and a more than 3-fold increase in hepatic immune cells, including infiltrating AIF1 ⁺ macrophages and neutrophils. Despite similar durations of alcohol misuse, G/G individuals had higher Model for End-Stage Liver Disease scores compared with C/G individuals, indicating a significantly greater 90-day mortality risk. Notably, some immune abnormalities, such as elevated neutrophils, persisted in G/G males even after alcohol abstinence. CONCLUSION: These findings suggest that functional genetic variation in AIF1 may contribute to the severity and persistence of ALD. TRIAL REGISTRATION: ClinicalTrials.gov NCT02231840. FUNDING: Research support was provided from the National Institute on Alcohol Abuse and Alcoholism of the NIH under grants 1ZIAAA000440-02 and R24AA025017.

Patients with alcohol-associated cirrhosis (AC) may develop severe alcohol-associated hepatitis (sAH), a disease with high short-term mortality. Our previous studies demonstrated that sAH, but not AC livers, are infiltrated with a high number of self-sustaining IL-8⁺ neutrophils that likely drive the transition from AC to sAH. Monocyte-derived macrophages (MoMFs) also infiltrate the liver in sAH, but their roles remain largely obscure. In the present study, we characterized liver macrophages in human liver explants from sAH and AC patients. Our data revealed a marked reduction in Kupffer cells, whereas MoMFs were increased in sAH and AC. Single-cell RNA-Seq analyses revealed several populations in both AC and sAH, including C1Q ⁺, S100A8 ⁺ , APOE ⁺, TNF ⁺ and VSIG4 ⁺ macrophages, with sAH containing unique C1Q ⁺ macrophages potentially playing a role in removing apoptotic neutrophils in sAH. C1Q ⁺ macrophages also express many genes involved in phagocytosis and proinflammatory and anti-inflammatory functions, suggesting that C1Q ⁺ macrophages have diverse functions in sAH. The roles of C1Q, S100A8, and APOE were further examined in experimental models of alcohol-induced liver injury. Our data revealed that C1q KO mice and macrophage-specific S100a8 KO mice presented similar alcohol-induced liver injury and hepatic neutrophil infiltration, while Apoe KO mice developed much more severe liver injury than did WT mice following chronic-plus-binge ethanol challenge. Taken together, sAH and AC are infiltrated with multiple populations of macrophages that perform diverse functions to drive chronic disease progression. Unique C1Q ⁺ macrophages in sAH play a compensatory role in removing dead cells but may also promote inflammation in sAH.

Background: Healthy livers contain a large number of resident macrophages named Kupffer cells (KCs), which are partially replaced by infiltrating monocyte-derived macrophages (MoMFs) during acute or chronic liver injury. Despite extensive research, understanding macrophage heterogeneity, spatial distribution and interactions with other cells within the liver remains challenging. Methods: This study employs sequential multiplex immunofluorescence staining, advanced image analysis and single-cell RNA sequencing (scRNA-seq) analysis to characterise macrophages in both healthy and diseased livers in mice. Results: Our data revealed that liver KCs made up more than 80% of total immune cells in healthy mouse livers, while massive amounts of MoMFs infiltrated into the livers after acute and chronic liver injury. KCs were more abundant and larger in Zones 1 and 2 compared with Zone 3 in healthy livers. Zone 1 KCs exhibited higher phagocytic activity than Zone 2/3 KCs and MoMFs. We simultaneously evaluated cell proliferation and apoptosis on one slide and found that proliferation and apoptosis of KCs and MoMFs significantly increased in acutely injured livers. We also performed scRNA-seq to investigate liver macrophage gene expression in naïve and concanavalin A (ConA)-treated mice. MoMF clusters expanded following ConA treatment, while KCs remained stable. Macrophages were divided into distinct subtypes, including C1q⁺ MoMFs, with differential expression of genes like Trem2, Spp1, Fabp5 and Gpnmb. Newly recruited C1q⁻ MoMFs expressed high levels of Lyz and Ccr2, while Itgax (Cd11c)⁺ MoMFs expressed endothelin converting enzyme 1 (Ece1), a gene encoding ECE1 enzyme that activates endothelin to promote hepatic stellate cell contraction and necrotic lesion resolution. By immunostaining analysis of the proteins encoded by these signature genes, we identified several populations of MoMFs that were mainly located surrounding the necrotic lesion area and expressed various proteins that are involved in dead cell debris clearance. Conclusion: We developed a robust framework for studying liver macrophages in vivo, offering insights into their roles in host defence and liver injury/repair. We identified several populations of MoMFs that surround necrotic lesion areas and express proteins that promote dead cell debris clearance. These necrotic lesion-associated macrophages likely play key roles in promoting necrotic lesion resolution.

Alcohol-associated liver disease (ALD) is one of the leading chronic liver diseases worldwide, encompassing a spectrum of conditions from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma.1 Histologically, alcohol-associated steatohepatitis (ASH) is characterised by lipid accumulation in hepatocytes, hepatocellular damage, ballooning of hepatocytes, formation of Mallory-Denk bodies, inflammation and pericellular fibrosis.1 Over the past several decades, research has elucidated key molecular mechanisms underlying ALD, such as ethanol-induced hepatotoxicity, oxidative stress, endoplasmic reticulum stress and organ–organ crosstalk pathways like the gut-liver and adipose tissue-liver axes.2 Patients with underlying chronic ALD, particularly those with recent episodes of excessive drinking, are at risk of developing acute alcohol-associated hepatitis (AH), a condition characterised by severe clinical symptoms, including jaundice.3 Severe AH (sAH) is commonly linked to alcohol-associated cirrhosis (AC) and carries a high short-term mortality rate. Due to the limited availability of effective pharmacological therapies, early liver transplantation is frequently required as a life-saving intervention.3 Identifying molecular mechanisms driving AH progression and liver failure is thus essential for developing targeted therapies for this life-threatening disease. AH, first described in 1961, is recognised as an acute inflammatory liver disease characterised by extensive inflammatory cell infiltration.4 Currently, AH is recognised as a form of acute-on-chronic liver injury involving various types of immune cells, including macrophages, neutrophils, T cells and B cells, as evidenced by immunohistochemical analysis.5 Recent RNA-sequencing analyses have highlighted that AH is associated with the upregulation of numerous inflammatory mediators, including pro-inflammatory cytokines, chemokines and adhesion molecules.5–7 However, the key mechanisms that drive the transition from ALD to sAH remain unclear.

Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver–gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD.

The nature of activation signals is essential in determining T cell subset differentiation; however, the features that determine T cell subset preference acquired during intrathymic development remain elusive. Here we show that naive CD4⁺ T cells generated in the mouse thymic microenvironment lacking Scd1, encoding the enzyme catalyzing oleic acid (OA) production, exhibit enhanced regulatory T (T_reg) cell differentiation and attenuated development of experimental autoimmune encephalomyelitis. Scd1 deletion in K14⁺ thymic epithelia recapitulated the enhanced T_reg cell differentiation phenotype of Scd1-deficient mice. The dearth of OA permitted DOT1L to increase H3K79me2 levels at the Atp2a2 locus of thymocytes at the DN2–DN3 transition stage. Such epigenetic modification persisted in naive CD4⁺ T cells and facilitated Atp2a2 expression. Upon T cell receptor activation, ATP2A2 enhanced the activity of the calcium–NFAT1–Foxp3 axis to promote naive CD4⁺ T cells to differentiate into T_reg cells. Therefore, OA availability is critical for preprogramming thymocytes with T_reg cell differentiation propensities in the periphery.