Cao, Sheng

Background: Alcohol-associated hepatitis (AH) is a severe inflammatory liver condition driven by dysregulated immune responses. CD8 T cells accumulate in the liver during AH, yet their functional role remains poorly defined. Methods: A murine NIAAA chronic-binge model with lipopolysaccharide (LPS) challenge was used. Single-cell RNA sequencing of liver and blood immune cells defined CD8 T cell subsets, followed by pathway analysis and reanalysis of human AH datasets. Spatial relationships were validated by immunohistochemistry of human AH liver tissue with CD8 T RM and liver sinusoidal endothelial cell (LSEC) co-staining. LSEC–CD8 T cell interactions were examined using in vitro co-culture, and signaling pathways were assessed by bulk RNA sequencing and phospho-protein analyses. IL15 function was evaluated by in vivo neutralization. Results: Single-cell RNA sequencing in the murine AH model revealed a distinct population of CD8 tissue-resident memory T cells (T RM ) with heightened activation and proinflammatory cytokine production. Analysis of human AH liver single-cell RNA sequencing data, along with immunohistochemistry validation, supported CD8 T RM enrichment in diseased tissue. IL15 emerged as a prominent pathway linked to T RM activation, and IL15 blockade reduced T RM abundance and attenuated EtOH/ lipopolysaccharide-induced liver injury. Mechanistically, LSECs not only provided a structural niche for T RM retention but also amplified IL15 signalling. In vitro, coculture experiments demonstrated that LSECs intensified activation in pre-stimulated CD8 T cells in a stimulus-dependent manner: under IL15 stimulation, LSECs boosted effector function without inducing cell death, whereas under T-cell receptor stimulation, LSECs drove hyperactivation and activation-induced cell death. Bulk RNA-seq and phospho-protein analysis identified the PI3K-AKT pathway as a shared pathway enhanced by LSEC coculture in activated CD8 T cells. These findings define a context-dependent mechanism in which LSECs promote IL15-driven signaling through AKT pathway amplification, promoting T RM persistence and inflammatory activity in AH. Conclusions: IL15-associated signaling within the hepatic microenvironment, shaped by LSEC-CD8 T-cell interactions, promotes activation and persistence of CD8 T RM cells in AH. Targeting the IL15-LSEC-AKT axis may disrupt pathogenic T RM niches and represent a promising therapeutic strategy for severe AH.

Cellular and molecular heterogeneity in the liver has been increasingly recognized to drive liver fibrosis progression, but the particular events that occur initially in response to liver injury and trigger immune cell recruitment remain unclear. Here, we identify epigenetically aberrant liver sinusoidal endothelial cells (LSECs) as key players in this process. Mechanistically, the epigenetic readers like bromodomain-containing protein 4–dependent (BRD4-dependent) super enhancers (SEs) activate proinflammatory genes, including promyelocytic leukemia (PML). The PML protein, in turn, binds BRD4 and amplifies proinflammatory angiocrine signaling through phase separation–dependent SE activation via PML/BRD4 condensate formation. In mouse models, LSEC-specific depletion of the PML/BRD4 complex mitigates liver inflammation and fibrosis. Single-cell RNA-seq reveals that epigenetically aberrant LSECs exhibit a reprogrammed proinflammatory angiocrine landscape in mouse fibrotic livers. TIMP1 + LSECs promote the recruitment of CD63 + monocyte–derived macrophages (MoMFs) during liver fibrosis progression. Thereby, PML/BRD4 in LSECs governs inflammatory immune cell recruitment in liver fibrosis. Pharmacological BRD4 inhibition or epigenetic PML-SE repression alleviates liver inflammation and fibrosis. In conclusion, PML/BRD4-mediated SE activation via phase separation drives proinflammatory angiocrine signaling in LSECs, initiating the inflammatory cascade and subsequent immune cell recruitment during liver fibrosis.

Abstract Rationale: The club cell secretory protein (CC16), encoded by the SCGB1A1 gene, has an anti-inflammatory role in airways diseases, including asthma (Li, AJRCCM 2023; Voraphani, AJRCCM 2023). We hypothesize that additional pathway genes regulated or co-expressed with CC16 have cumulative effects on asthma severity outcomes by modulating airway inflammation and maintaining lung homeostasis. Methods: As published previously, four genes were downregulated in CC16 knockout experiments and correlated with CC16 levels: BPIFA1, SFTPD, LTF, and LYZ (Iannuzo, Front. Immunol 2023). We developed a four-gene biomarker score in 94 patients with asthma from the NHLBI-sponsored Severe Asthma Research Program (SARP3) who underwent research bronchoscopy for lower airway epithelial brushings, RNA isolation, and bulk RNA sequencing. We log-transformed the raw values of each transcript; obtained standardized residuals from regression models that included sex, age, and batch effects; and summed the standardized residuals of all four proteins to estimate a standardized sum score (Zhai, JACI Allergy 2024). Logistic regression models tested the four-gene score, three-gene score without LTF, and individual transcripts for associations with exacerbations during 12-month follow-up and zero-inflated negative binomial regression for number of exacerbations. Results: 30.85% percent of 94 patients experienced at least one exacerbation at the end of follow-up. The CC16-associated four-gene biomarker score showed protective effects on asthma exacerbation risk with a 54% percent reduction in exacerbation risk for every one-fold higher score (OR=0.46, 95% CI: 0.27-0.78, p =0.0040, Figure 1). Transcript expression of BPIFA1 (OR=0.87, 95% CI:0.66-1.16, p=0.337), SFTPD (OR=0.33, 95% CI:0.04-2.52, p=0.283), LYZ (OR=0.62, 95%CI:0.28-1.39, p=0.248), and LTF (OR=0.36, 95% CI:0.18-0.74, p=0.0053) all indicated protective directionality with respect to exacerbation risk. However, only LTF demonstrated a statistically significant association. When we excluded LTF, the remaining three-gene score showed a trend toward association with risk (OR=0.64, 95% CI:0.40-1.03, p=0.068) and was significantly associated with number of exacerbations (RR=0.49, 95% CI:0.26-0.95, p=0.034). Compared to LTF alone (OR=0.48, PPV=0.50, NPV=0.71, Sensitivity=0.14, AUC=0.65), the combined four-gene score (OR=0.46, PPV=0.60, NPV= 0.73, Sensitivity=0.21, AUC=0.68) showed improved predictive performance. Conclusions: We identified a CC16-associated transcriptomic predictive score for asthma exacerbations, with LTF showing the strongest effect. Although the other three genes showed individually weak and non-significant associations with exacerbations, the three gene score was significant for number of exacerbations.We demonstrate the potential of this transcriptomic score as a predictive biomarker for asthma severity-related outcomes. Further research investigating these genes and their interaction with other CC16-associated immune pathways could provide insight into therapeutic targets.

BET bromodomain inhibitors demonstrate significant promise as anti-inflammatory agents targeting a variety of inflammatory disorders. However, clinical data has demonstrated that the administration of non-selective BET bromodomain inhibitors has led to significant dose-limiting toxicity in clinical settings. More selective inhibitors of the second BET bromodomains, referred to as pan-D2 inhibitors, are better tolerated, however their efficacy varies significantly depending on disease indication. Here, we use three orally bioavailable inhibitors, 1–3, that are either selective for the first N-terminal bromodomain of BRD4 (BRD4-D1 selective), or pan-D1 biased, for assessing their cellular and in vivo efficacy and safety profile in two models of inflammatory liver disease. We compare their effects to known pan-D2 and pan-BET inhibitors. Our results show that pan-D1 biased inhibitor, 3, is as efficacious as the pan-BET inhibitor I-BET151 in reducing inflammation from LPS-induced and drug-induced toxicity, whereas pan-D2 inhibitors are less effective. BRD4-D1 selective inhibitors, 1 and 2, are also efficacious; however, new inhibitors with improved cellular target engagement will be necessary to more thoroughly assess their effects. Finally, BRD4-D1 selective inhibitors are better tolerated in a preclinical model of thrombocytopenia than pan-D1 biased inhibitors, while gastrointestinal (GI) toxicity may be a BRD4-driven effect. These results highlight the importance of assessing specific BET protein and bromodomain functions due to their varying roles in diverse disease models.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB–dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

GTP Cyclohydrolase‐I (GTPCHI) is the rate‐limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), which is an essential cofactor for the enzymatic activity of endothelial nitric oxide synthase (eNOS). To better understand the role of GTPCHI in maintaining eNOS function, we analyzed GTPCHI subcellular localization and activity in human umbilical cord vein endothelial cells (HUVEC). Western blot analysis showed similar levels of GTPCHI protein expression in both Triton X‐100 ‐soluble and ‐insoluble fractions, suggesting membrane targeting of GTPCHI. Further analysis of membrane‐associated proteins using sucrose gradient ultracentrifugation revealed that GTPCHI protein expression was localized to caveolar microdomains along with caveolin‐1(Cav‐1) and eNOS. HPLC analysis for GTPCHI activity confirmed the localization of GTPCHI to caveolar microdomains as well as golgi‐associated membrane fractions. Using an in vitro GST pull‐down assay with recombinantGST‐caveolin‐1 fusion protein incubated with the lysate of HUVEC, we demonstrated specific binding of GTPCHI to caveolin‐1. To determine if caveolin‐1 may regulate GTPCHI activity in vivo, we analyzed aortas from Cav‐1 −/ − mice and were able to detect a two‐fold increase in BH4 production and GTPCHI activity in the aortas of Cav‐1 −/ − mice when compared to eNOS −/ − mice or wild type mice. These studies reveal that BH4 synthesis can occur in specialized membrane compartments in close proximity to eNOS and may be subjected to posttranslational regulation by caveolin‐1 which has an inhibitory effect on GTPCHI enzyme activity. Funded by NIH grant HL‐53524.

Mechanisms that mediate migration, homing and integration of blood outgrowth endothelial cells (BOEC) to sites of vascular injury are incompletely understood, although matrix metalloproteinases (MMPs) have been implicated in this process. Farnesoid X receptor (FXR) signaling pathway has recently been recognized to regulate vascular wall signaling. The aim of this study was to characterize the expression of FXR in human BOEC and examine signaling pathways relevant to BOEC migration. FXR mRNA and protein levels were detected by reverse‐transcriptase PCR and Western blot. Microarray analysis using a focused human endothelial cell biology array (Superarray) suggested that natural (Chenodeoxycholic acid (CDCA), 0.1–100μM) and synthetic (6‐ethylchenodeoxycholic acid (6‐ECDCA) 0.01–10μM) FXR ligands significantly increased MMP9 transcription which was confirmed by real time PCR and gelatin zymography. SiRNA based silencing of FXR prevented CDCA induced increases in MMP9 mRNA, while retroviral over‐expression of FXR in BOEC enhanced MMP9 mRNA levels. CDCA induced expression of Small Heterodimeric Partner (SHP), a downstream target of FXR. Retroviral over‐expression of SHP increased, whereas siRNA silencing of SHP inhibited CDCA induced MMP9 mRNA levels. Migration analysis using Boyden assay demonstrated that CDCA stimulated migration of BOEC which was blocked by transfection with FXR or MMP9 siRNA (p<0.05; n=5). These studies identify a novel signaling pathway by which a key protein important in cell homing and migration, MMP9, is regulated through a previously unrecognized FXR‐SHP pathway in BOEC. Funded by NIH Grant # DK59615

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.