Genetic Ancestry, Race, and Severity of Acutely Decompensated Cirrhosis in Latin America.

BACKGROUND & AIMS: Genetic ancestry or racial differences in health outcomes exist in diseases associated with systemic inflammation (eg, COVID-19). This study aimed to investigate the association of genetic ancestry and race with acute-on-chronic liver failure (ACLF), which is characterized by acute systemic inflammation, multi-organ failure, and high risk of short-term death. METHODS: This prospective cohort study analyzed a comprehensive set of data, including genetic ancestry and race among several others, in 1274 patients with acutely decompensated cirrhosis who were nonelectively admitted to 44 hospitals from 7 Latin American countries. RESULTS: Three hundred ninety-five patients (31.0%) had ACLF of any grade at enrollment. Patients with ACLF had a higher median percentage of Native American genetic ancestry and lower median percentage of European ancestry than patients without ACLF (22.6% vs 12.9% and 53.4% vs 59.6%, respectively). The median percentage of African genetic ancestry was low among patients with ACLF and among those without ACLF. In terms of race, a higher percentage of patients with ACLF than patients without ACLF were Native American and a lower percentage of patients with ACLF than patients without ACLF were European American or African American. In multivariable analyses that adjusted for differences in sociodemographic and clinical characteristics, the odds ratio for ACLF at enrollment was 1.08 (95% CI, 1.03-1.13) with Native American genetic ancestry and 2.57 (95% CI, 1.84-3.58) for Native American race vs European American race CONCLUSIONS: In a large cohort of Latin American patients with acutely decompensated cirrhosis, increasing percentages of Native American ancestry and Native American race were factors independently associated with ACLF at enrollment.

Mesenchymal stem cell-regulated miRNA-mRNA landscape in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a major challenge in the field of hepatology. While mesenchymal stem cell (MSC) therapy can improve the prognosis of patients with ACLF, the molecular mechanisms through which MSCs attenuate ACLF remain poorly understood. We performed global miRNA and mRNA expression profiling via next-generation sequencing of liver tissues from MSC-treated ACLF mice to identify important signaling pathways and major factors implicated in ACLF alleviation by MSCs. METHODS: Carbon tetrachloride-induced ACLF mice were treated with saline or mouse bone marrow-derived MSCs. Mouse livers were subjected to miRNA and mRNA sequencing. Related signal transduction pathways were obtained through Gene Set Enrichment Analysis. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed for the differentially expressed miRNA target genes (DETs). Hub miRNA and mRNA associated with liver injury were analyzed using LASSO regression. The expression levels of hub genes were subjected to Pearson's correlation analysis and verified using RT-qPCR. The biological functions of hub genes were verified in vitro. RESULTS: The tricarboxylic acid cycle and peroxisome proliferator-activated receptor pathways were activated in the MSC-treated groups. The proportions of liver-infiltrating NK resting cells, M2 macrophages, follicular helper T cells, and other immune cells were altered after MSC treatment. The expression levels of six miRNAs and 10 transcripts correlated with the degree of liver injury. miR-27a-5p was downregulated in the mouse liver after MSC treatment, while its target gene E2f2 was upregulated. miR-27a-5p inhibited E2F2 expression, suppressed G1/S phase transition and proliferation of hepatocytes, in addition to promoting their apoptosis. CONCLUSIONS: This is the first comprehensive analysis of miRNA and mRNA expression in the liver tissue of ACLF mice after MSC treatment. The results revealed global changes in hepatic pathways and immune subpopulations. The miR-27a-5p/E2F2 axis emerged as a central regulator of the MSC-induced attenuation of ACLF. The current findings improve our understanding of the molecular mechanisms through which MSCs alleviate ACLF.

Hypermethylation of thymosin ß4 predicts a poor prognosis for patients with acute-on-chronic hepatitis B liver failure.

BACKGROUND: It has been demonstrated that thymosin ß4 (Tß4) could inflect the severity of acute-on-chronic hepatitis B liver failure (ACHBLF), but the relationship between its methylation status and the prognosis of liver failure is not clear. This study aimed to determine Tß4 promoter methylation status in patients with ACHBLF and to evaluate its prognostic value. METHODS: The study recruited 115 patients with ACHBLF, 80 with acute-on-chronic hepatitis B pre-liver failure (pre-ACHBLF), and 86 with chronic hepatitis B (CHB). In addition, there were 36 healthy controls (HCs) from the Department of Hepatology, Qilu Hospital of Shandong University. The 115 patients with ACHBLF were divided into three subgroups: 33 with early stage ACHBLF (E-ACHBLF), 42 with mid-stage ACHBLF (M-ACHBLF), and 40 with advanced stage ACHBLF (A-ACHBLF). Tß4 promoter methylation status in peripheral blood mononuclear cells (PBMCs) was measured by methylation-specific polymerase chain reaction, and mRNA was detected by quantitative real-time polymerase chain reaction. RESULTS: Methylation frequency of Tß4 was significantly higher in patients with ACHBLF than in those with pre-ACHBLF, CHB or HCs. However, expression of Tß4 mRNA showed the opposite trend. In patients with ACHBLF, Tß4 promoter methylation status correlated negatively with mRNA levels. The 3-month mortality of ACHBLF in the methylated group was significantly higher than that in the unmethylated group. Also, Tß4 promoter methylation frequency was lower in survivors than in non-survivors. When used to predict the 1-, 2-, and 3-month incidence of ACHBLF, Tß4 methylation status was better than the model for end-stage liver disease (MELD) score. The predictive value of Tß4 methylation was higher than that of MELD score for the mortality of patients with E-ACHBLF and M-ACHBLF, but not for A-ACHBLF. CONCLUSIONS: Tß4 methylation might be an important early marker for predicting disease incidence and prognosis in patients with ACHBLF.

Gene profiling of Toll-like receptor signalling pathways in neutrophils of patients with acute-on-chronic liver failure.

OBJECTIVES: Toll-like receptors (TLRs) on neutrophils play a crucial role in detecting pathogens and organ/tissue injury in acute-on-chronic liver failure (ACLF). However, little is known about the exact mechanisms and potential signalling pathways. The aim of this study was to investigate alterations in TLR signalling pathways in neutrophils of ACLF patients. METHODS: Twenty-seven patients with compensated cirrhosis (n = 9), decompensated cirrhosis (n = 9) and ACLF (n = 9) were enrolled in the study. Neutrophils were isolated, and alterations in TLR signalling pathways were evaluated using an RT2 Profiler™ PCR Array. The fold change for each gene (2(-∆∆CT)) was compared among the groups. Genes with a fold change ratio of ≥ 2 or ≤ 0.5 along with a p value of < 0.05 were considered to be differentially expressed. RESULTS: A total of 17 genes were upregulated in neutrophils from patients with compensated cirrhosis and were mainly distributed in adaptors, TLR-interacting proteins and downstream pathways. Six genes were detected in patients with decompensated cirrhosis. A trend of downregulation of genes in the TLR signalling pathway was observed in neutrophils of patients with cirrhosis and ACLF. TLR3, IFNG, IL1B, TBK1, CCL2 and LTA were downregulated in neutrophils. Moreover, CD14 and IL10 were upregulated in neutrophils of ACLF patients. CONCLUSIONS: TLR signalling pathway genes were differentially regulated in neutrophils between cirrhosis and ACLF. In ACLF patients, defective expression of TLR3 and IFN, along with enhanced CD14 and IL10 expression, was characterized by transcriptional alterations of neutrophils.

Keratin 8 Mutations Were Associated With Susceptibility to Chronic Hepatitis B and Related Progression.

BACKGROUND: Keratin 8 and 18 (K8/K18) are the exclusively expressed keratins intermediate filaments pair in hepatocytes that protect against liver injuries and viral infection. We aimed to explore the genetic link between keratin variants and chronic hepatitis B virus (CHB) infection in a large cohort from a high-epidemic area. METHODS: Genomic deoxyribonucleic acid was isolated from patients, and Sanger sequencing was applied to analyze variations in exon regions of K8/18. Biochemical and functional analysis of novel mutations was also performed. RESULTS: The 713 participants comprised 173 healthy controls and 540 patients, which covered chronic hepatitis (n = 174), decompensated cirrhosis (n = 192), and primary liver carcinoma (n = 174). The frequency of mutations in K8/18 was significantly higher among patients than among controls (8.15% vs 0.58%, P < .001). Significant differences were found between the chronic hepatitis subgroup and controls in multiple comparisons (6.32% vs 0.58%, P = .006). All 21 missense mutations (3.89%) were detected in the keratin 8 (K8), including 4 novel conserved missense variants (R469C, R469H, A447V, and K483T). Multivariate logistic regression analysis demonstrated a higher risk of acute-on-chronic liver failure (ACLF) and missense variants (odds ratio = 4.38, P = .035). Transfection of these variants caused keratin network disruption in vivo. CONCLUSIONS: Novel K8 cytoskeleton-disrupting variants predispose toward ACLF in CHB.

Genetic variants of innate immunity receptors are associated with mortality in cirrhotic patients with bacterial infection.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by acute decompensation of cirrhosis (AD), organ failure(s) and high risk of short-term mortality with bacterial infection frequently as precipitating event. Innate immune pattern recognition receptors and members of the lectin pathway of complement activation are crucial to the innate immune response to pathogens. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) of innate immune components are associated with the occurrence of bacterial infections or mortality in patients with cirrhosis hospitalized for AD or ACLF. METHODS: Twenty-one innate immunity SNPs with known functional implications were genotyped in 826 AD/ACLF patients included in the CANONIC study. Associations between baseline characteristics of the patients, the occurrence of bacterial infections and survival rate at 90 days of follow-up in relation to the innate immunity genetic variants were analysed. RESULTS: The NOD2-G908R genetic variant was associated with mortality (HR 2.25, P = .004) independently of age and MELD Score. This association was also found in a predefined subgroup analysis in patients with bacterial infections (HR 2.78, P < .001) along with MBL_Yx (HR 1.72, P = .008) and MASP2_371 (HR 1.67, P = .012) genetic variants. None of the analysed SNPs were significantly associated with the occurrence of acute bacterial infections or spontaneous bacterial peritonitis in particular. CONCLUSIONS: Innate immune system-specific NOD2-G908R, MBL_Yx and MASP2_371 genetic variants were independently associated with increased risk of short-term mortality in AD/ACLF patients with bacterial infection.

[Correlation between interleukin-6 single nucleotide polymorphism and the occurrence and prognosis of hepatitis B virus-associated acute-on-chronic liver failure].

Objective: To investigate the correlation between interleukin-6 (IL-6) single nucleotide polymorphism (SNP) and the occurrence and prognosis of hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF). Methods: Patients with chronic hepatic diseases diagnosed as HBV infection in the Hepatology Center of the First Affiliated Hospital of Fujian Medical University from July 2012 to March 2018 were divided into HBV-ACLF and non-ACLF group. SNP genotyping of eight loci in IL-6 gene (rs1524107, rs1800795, rs1800797, rs2069827, rs2069830, rs2069837, rs2069840 and rs2069845) was determined by the improved multi-temperature ligase detection reaction (imLDRTM) technique. Simultaneously, case data were reviewed with the 3-months followed up survival condition of the ACLF group. Normally distributed data were expressed as arithmetic means and SDs, and t-test was adopted. Data with skewed distribution were expressed as medians with interquartile range, and were measured by non-parametric test. Multivariate logistic regression analysis was used to analyze the relative risk of genetic polymorphism and HBV-ACLF as well as the relationship between IL-6 SNPs with the occurrence and prognosis of HBV-ACLF. Results: Four hundred patients were included in the study, with 122 (30.5%) in the HBV-ACLF and 278 (69.5%) in the non-ACLF group. There were significant differences in total bilirubin, albumin, and white blood cell count, percentage of neutrophils, platelet count, alanine aminotransferase, aspartate aminotransferase, prothrombin time and international standardized ratio, creatinine and the model for end-stage liver disease score between the two groups (P < 0.001). The genotype of IL-6 genes (rs1800795, rs1800797, rs2069827, and rs2069830) of all subjects showed no mutation or the mutation rate under 1%. There was no significant difference in the genotype of IL-6 (rs1524107, rs2069837, rs2069840 and rs2069845) between the two groups (P > 0.05). Multivariate logistic regression analysis showed that the SNPs in the above four loci of IL-6 gene was not associated with HBV-ACLF risk, nor had significant correlation with the 3-months prognosis. Conclusion: The SNP genotyping of eight loci in IL-6 gene (rs1524107, rs1800795, rs1800797, rs2069827, rs2069830, rs2069837, rs2069840 and rs2069845) is unrelated to the occurrence and short-term prognosis of HBV-ACLF.

Genome-wide association study identifies HLA-DR variants conferring risk of HBV-related acute-on-chronic liver failure.

OBJECTIVE: Acute-on-chronic liver failure (ACLF) is an extreme condition after severe acute exacerbation of chronic hepatitis B; however, the underlying genetic factors involved in its onset and progression are currently unclear. DESIGN: We carried out a genome-wide association study among 399 HBV-related ACLFs (cases) and 401 asymptomatic HBV carriers (AsCs, as controls) without antiviral treatment. The initial findings were replicated in four independent case-control sets (a total of 901 ACLFs and 1686 AsCs). The roles of risk variants on clinical traits of ACLF were also analysed. RESULTS: Among 1300 ACLFs and 2087 AsCs, we identified rs3129859 at human leucocyte antigen (HLA) class II region (chromosome 6p21.32) associated with HBV-related ACLF (combined P dominant =2.64×10-20, OR=1.83). Analysis identified HLA-DRB1*12:02 as the top susceptible HLA allele associated with ACLF (p=3.94×10-6, OR=2.05). The association of rs3129859 was robust in ACLF subgroups (ACLFs with liver cirrhosis, p=1.36×10-16; ACLFs without liver cirrhosis, p=1.52×10-7), and patients at low-replicative phase (p=6.36×10-11, OR=2.29) or HBV e antigen-negative chronic hepatitis B phase (p=1.51×10-14, OR=1.86). Clinical traits analysis in patients with ACLF showed that the risky rs3129859*C allele was also associated with prolonged prothrombin time, faster progression to ascites development and higher 28-day mortality. CONCLUSIONS: Our genome-wide association study identified HLA-DR as the major locus for susceptibility to HBV-related ACLF. Our findings highlight the importance of HLA class II restricted CD4+ T-cell pathway on the immunopathogenesis of HBV-related ACLF.

Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) in cirrhosis is an increasingly recognized syndrome characterized by acute decompensation, organ failure(s) and high short-term mortality. Recent findings suggest that an overexuberant systemic inflammation plays a primary role in ACLF progression. In this study, we examined whether genetic factors shape systemic immune responses in patients with decompensated cirrhosis. Six single-nucleotide polymorphisms (SNPs) in inflammation-related genes (interleukin [IL]-1 beta [IL-1ß], rs1143623; IL-1 receptor antagonist [IL-1ra], rs4251961; IL-10, rs1800871; suppressor of cytokine signaling-3, rs4969170; nucleotide-binding oligomerization domain-containing protein 2, rs3135500; and chemerin chemokine-like receptor 1, rs1878022) were genotyped in 279 patients with cirrhosis with (n = 178) and without (n = 101) ACLF from the CANONIC study of the CLIF consortium. Among these SNPs, we identified two polymorphisms belonging to the IL-1 gene cluster (IL-1ß and IL-1ra) in strong association with ACLF. Both SNPs were protective against ACLF; IL-1ß (odds ratio [OR], 0.34, 95% confidence interval [CI], 0.13-0.89; P < 0.05) and IL-1ra (OR, 0.58; 95% CI, 0.35-0.95; P < 0.05) under the recessive and overdominant inheritance models, respectively. These protective SNPs translated into reduced circulating levels of IL-1ß, IL-1α, IL-6, granulocyte-colony stimulating factor, granulocyte-macrophage colony-stimulating factor, and C-reactive protein at enrollment as well as after 7-14 days of admission. These findings were confirmed in vitro in leukocytes incubated with plasma from patients with decompensated cirrhosis carrying the protective SNP genotypes. Notably, a higher frequency of the protective genotypes was observed in patients without (80%) than in those with (20%) ACLF. Consistently, patients carrying the combined protective genotypes showed a lower 28-day mortality rate. CONCLUSION: These data identify two common functional polymorphisms in the IL-1 gene cluster, which are associated with the inflammatory process related to development of ACLF. (Hepatology 2017;65:202-216).

Pentraxin-3 modulates lipopolysaccharide-induced inflammatory response and attenuates liver injury.

Acute-on-chronic liver injury is characterized by an important inflammatory response frequently associated with endotoxemia. In this context, acute-phase proteins such as Pentraxin-3 (PTX3) are released; however, little is known about their role in chronic liver disease. The aim of this study was to elucidate the role of PTX3 in liver injury. The role of PTX3 was evaluated in cultured human cells, liver tissue slices, and mice with acute-on-chronic liver injury. PTX3 expression was assessed in tissue and serum samples from 54 patients with alcoholic hepatitis. PTX3 expression was up-regulated in animal models of liver injury and strongly induced by lipopolysaccharide (LPS). Liver cell fractionation showed that macrophages and activated hepatic stellate cells were the main cell types expressing PTX3 in liver injury. Ex vivo and in vivo studies showed that PTX3 treatment attenuated LPS-induced liver injury, inflammation, and cell recruitment. Mechanistically, PTX3 mediated the hepatic stellate cell wound-healing response. Moreover, PTX3 modulated LPS-induced inflammation in human primary liver macrophages and peripheral monocytes by enhancing a TIR domain-containing adapter-inducing interferon-dependent response and favoring a macrophage interleukin-10-like phenotype. Additionally, hepatic and plasma PTX3 levels were increased in patients with alcoholic hepatitis, a prototypic acute-on-chronic condition; and its expression correlated with disease severity scores, endotoxemia, infections, and short-term mortality, thus suggesting that expression of PTX3 found in patients could be a counterregulatory response to injury. CONCLUSION: Experimental and human evidence suggests that, in addition to being a potential biomarker for alcoholic hepatitis, PTX3 participates in the wound-healing response and attenuates LPS-induced liver injury and inflammation; therefore, administration of PTX3 could be a promising therapeutic strategy in acute-on-chronic conditions, particularly those associated with endotoxemia. (Hepatology 2017;66:953-968).

Combination of MicroRNAs and Cytokines: a Method for Better Evaluation of Acute-on-Chronic Liver Failure.

BACKGROUND: The aim was to establish expression profiles of microRNAs (miRNAs) in Peripheral Blood Mononuclear Cells (PBMC) and of plasma cytokines from the patients with hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF) and high-risk of acute-on-chronic liver failure (ACLF) patients as well as healthy people and to examine the relationships between these profiles and clinical features. METHODS: Herein, we collected PBMCs and plasma from peripheral blood of HBV-ACLF and ACLF patients as well as healthy people. Microarray, real-time qPCR, and ELISA assays were used. RESULTS: In this study, we found 39 miRNAs including miR-146a, miR-150, and miR-29a downregulated and 5 miRNAs elevated in PBMCs from HBV-ACLF patients compared to healthy controls. However, elevated plasma levels of cytokines such as TNF-α, IFN-α, and TGF-ß were found in PBMCs from HBV-ACLF patients compared with the controls, but no significant difference was found between the high-risk and control groups. MiR-146a, miR-150, miR-29a, PTA, and anti-HBc were positively correlated with the survival of ACLF patients, while TNFα, IFN-α, INR, and TBiL were negatively correlated with the survival of these patients. CONCLUSIONS: Combined examination of miRNAs in PBMCs and cytokines in plasma together is a better method for monitoring and evaluating HBV-ACLF patients.

Serum levels of miRNA in patients with hepatitis B virus-associated acute-on-chronic liver failure.

BACKGROUND: Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF) is a life-threatening condition and its exact pathophysiology and progression remain unclear. The present study aimed to assess the role of serum miRNAs in the evaluation of HBV-ACLF and to develop a model to predict the outcomes for ACLF. METHODS: Serum was collected from 41 chronic hepatitis B and 55 HBV-ACLF patients in addition to 30 chronic asymptomatic HBV carriers as controls. The miRNAs expressions were measured by real-time quantitative PCR (q-PCR). Statistical analyses were conducted to assess the ability of differentially expressed miRNAs and other prognostic factors in identifying ACLF prognosis and to develop a new predictive model. RESULTS: Real-time q-PCR indicated that serum miR-146a-5p, miR-122-3p and miR-328-3p levels were significantly upregulated in ACLF patients compared to chronic hepatitis B and chronic asymptomatic HBV carriers patients. In addition, multivariate regression analyses indicated that Na+, INR, gastrointestinal bleeding and miR-122-3p are all independent factors that are reliable and sensitive to the prognosis of HBV-ACLF. Therefore, we developed a new model for the prediction of HBV-ACLF disease state: Y = 0.402 × Na+ - 1.72 × INR - 4.963 × gastrointestinal bleeding (Yes = 0; No = 1)-0.278 × (miR-122-3p) + 50.449. The predictive accuracy of the model was 95.3% and the area under the receiver operating characteristic curve (AUROC) was 0.847. CONCLUSIONS: Expression levels of these miRNAs (miR-146a-5p, miR-122-3p and miR-328-3p) positively correlate with the severity of liver inflammation in patients with ACLF and may be useful to predict HBV-ACLF severity.

Methylation status of the estrogen receptor 1 promoter predicts poor prognosis of acute-on-chronic hepatitis B liver failure.

BACKGROUND: Acute-on-chronic hepatitis B liver failure (ACHBLF) is an acute deteriorating liver disease and rapidly progresses to multiple organ failure. There is currently no adequate accurate predictive models of ACHBLF prognosis. AIMS: To identify the methylation frequency of the estrogen receptor 1 (ESR1) promoter in ACHBLF and analyze the associated prognostic significance. METHODS: Methylation-specific PCR (MSP) was used to determine the methylation frequency of the ESR1 promoter in peripheral blood mononuclear cells from a training and validation cohort of patients. The training cohort included 113 patients with ACHBLF, 73 with chronic hepatitis B (CHB) and 40 healthy controls (HCs). The validation cohort consisted of 37 patients with ACHBLF. Another 18 patients with pre-ACHBLF who progressed to ACHBLF were used to dynamically evaluate ESR1 promoter methylation changes associated with a severe clinical condition. RESULTS: Death from ACHBLF was associated with hyperbilirubinemia, a higher score in the model for end-stage liver disease (MELD), a higher incidence of hepatic encephalopathy (HE) and an increased frequency of ESR1 promoter methylation during the 28 day follow-up. HE, MELD score and ESR1 promoter methylation were the independent risk factors associated with 28-day mortality from ACHBLF. The frequency of ESR1 promoter methylation was significantly higher than in patients with CHB and HCs. Albumin and the MELD score were significantly associated with ESR1 promoter methylation. Moreover, ESR1 promoter methylation frequency increased with ACHBLF progression. More importantly, ESR1 promoter methylation was an independent risk factor and had a high value to predict 28-day mortality from ACHBLF. CONCLUSIONS: Abnormal ESR1 methylation could be a prognostic biomarker for ACHBLF.

Predisposing Factors in Acute-on-Chronic Liver Failure.

Acute-on-chronic liver failure (ACLF) is a syndrome with high short-term mortality in patients with chronic liver disease. The definition of ACLF has been addressed recently in many publications, and despite regional differences the number and severity of organ failures are decisive for the presence and severity of ACLF. However, the predisposition of patients to develop ACLF has not been addressed in prospective studies. Several predisposing factors for ACLF have been mentioned, but still clear-cut analysis and the sequential processes have not been explored. One reason is that different factors might predispose in one setting and might be the precipitating event in another, thus rendering the generalization of predisposing factors difficult. However, genetic factors, lifestyle, past medical history, aging, latent chronic infections, and the severity of the liver disease and portal hypertension might predispose for the development of ACLF after proper injury and response.

Genomewide Histone H3 Lysine 9 Acetylation Profiling in CD4+ T Cells Revealed Endoplasmic Reticulum Stress Deficiency in Patients with Acute-on-chronic Liver Failure.

Acute-on-chronic liver failure (ACLF) displayed 'sepsis-like' immune paralysis. Little is known about the role of CD4+ T lymphocytes, the primary regulator of innate and adopted immune system, played in ACLF. Acetylation of histone H3 lysine 9 (H3K9ac), a key epigenetic modification, tightly controls gene transcription. Whether and how does H3K9ac modification regulate CD4+ T cells in ACLF remains unclear. PBMCs were isolated from patients with ACLF, immune tolerance of chronic hepatitis B (CHB-T) and immune active of chronic hepatitis B (CHB-A). Then, CD4+ T lymphocytes were purified by magnetic microbeads, and the purity was confirmed by flow cytometry. H3K9ac variations were analysed in CD4+ T cells using chromatin immunoprecipitation microarray and then confirmed by quantitative PCR. Whole-genome H3K9 acetylation analyses were conducted by bioinformatics. A total of 70 genes were differently modified in H3K9ac between CHB-A and ACLF groups, while 44 genes were differently modified in H3K9ac between CHB-T and ACLF groups. Clustering algorithm analysis showed patients with ACLF displayed 'sepsis-like' immune paralysis. Functional analysis showed endoplasmic reticulum (ER) stress, or downstream pathway-related genes, such as BIP, ATF4, PER1, CSNK1D, IRF3, BNIP1, AKT1 and UBC, were differentially modified in ACLF. We profiled H3K9 acetyl modification in CD4+ T lymphocytes from HBV-infected patients with three different immune states, that is ACLF, immune tolerance and immune active phases. ACLF displayed 'sepsis-like' immune paralysis. ER stress in CD4+ T lymphocytes attributed to ACLF. This study provides some useful clues for revealing the mechanisms underlying ACLF.

Genome-wide microarray-based analysis of miRNAs expression in patients with acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe clinical syndrome that may cause a high mortality. However, the mechanism is still not clear. Characterization of the microRNA (miRNA) profiles in ACLF patients may provide new clues to the pathogenesis and management of this syndrome. METHODS: Genome-wide microarray was performed to compare the different miRNA expression profiles in peripheral blood mononuclear cells of a pair of monozygotic twins, an ACLF patient and an HBV asymptomatic carrier (AsC). The case-control miRNA profiles were compared and confirmed by quantitative reverse transcription-polymerase chain reaction in 104 ACLF patients and 96 AsCs. A combined computational prediction algorithm was used to predict the potential target genes. RESULTS: Forty-five miRNAs were increased and eight miRNAs were decreased in the ACLF group. The expressions of hsa-let-7a and hsa-miR-16 were increased by 8.58- and 8.63-fold in ACLF patients compared with that in AsCs, respectively (P<0.001). CARD8, BCL2, IL1RAPL1, LTB, FZD10 and EDA were identified as the target genes of hsa-miR-16; MAP4K3, OPRM1, IGF2BP1 and CERCAM were verified as the target genes of hsa-let-7a. CONCLUSIONS: Our results showed that there is a close relationship between specific miRNAs of peripheral blood mononuclear cells and ACLF. hsa-miR-16 and hsa-let-7a may contribute to the development of ACLF.

Prognostic value of genome-wide methylation in acute-on-chronic hepatitis B liver failure.

AIM: Methylation status of genome varies between pre-acute-on-chronic hepatitis B liver failure (pre-ACHBLF), acute-on-chronic hepatitis B liver failure (ACHBLF), and chronic hepatitis B patients. This study aimed to find better prognostic indicators for acute-on-chronic liver failure. METHODS: The level of global genome methylation in peripheral blood mononuclear cells (PBMCs) was detected. The overall genome methylation rate was determined using MethylFlash™ Methylated DNA Quantification Kit(Colorimetric). DNMT activity were measured using DNA Methyltransferase Activity/Inhibition Assay Kit. Gene expression of DNA methyltransferases (DNMT)，methyl-CpG-binding domain (MBD) were detected by qRT-PCR. RESULTS: The global genome methylation level in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). There was also obvious difference of the global genome methylation level between pre-ACHBLF group and CHB group (P<0.001). Meanwhile, the activity of DNMT in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). The mRNA expression level of DNMT1 was higher than that in pre-ACHBLF group (P<0.01) and CHB group (PP<0.001). The mRNA expression level of MBD1 in ACHBLF group was also higher than that in CHB group (P<0.001) and healthy controls (HCs) (P<0.01). And the mRNA expression level of MBD3 and MBD4 in ACHBLF, pre-ACHBLF and CHB group were lower than that in HCs (P<0.001). Meanwhile we observed an opposite change in the mRNA expression level of MECP2. The ROC curve suggested that global genome methylation level was a better prognostic predictor than MELD score in ACHBLF (AUC 0.950, SE 0.0237, 95%CI 0.874-0.986 VS AUC 0.863, SE 0.0439, 95%CI 0.765-0.931, P=0.0429). CONCLUSIONS: Genome methylation level can be a good biomarker in predicting the severity and prognosis of ACHBLF.

Genetic landscape and immune mechanism of monocytes associated with the progression of acute-on-chronic liver failure.

OBJECTIVE: Acute-on-chronic liver failure (ACLF) has a high prevalence and short-term mortality. Monocytes play an important role in the development of ACLF. However, the monocyte subpopulations with unique features and functions in ACLF and associated with disease progression remain poorly understood. We investigated the specific monocyte subpopulations associated with ACLF progression and their roles in inflammatory responses using the single-cell RNA sequencing (scRNA-seq). METHODS: We performed scRNA-seq on 17,310 circulating monocytes from healthy controls and ACLF patients and genetically defined their subpopulations to characterize specific monocyte subpopulations associated with ACLF progression. RESULTS: Five monocyte subpopulations were obtained, including pro-inflammatory monocytes, CD16 monocytes, HLA monocytes, megakaryocyte-like monocytes, and NK-like monocytes. Comparisons of the monocytes between ACLF patients and healthy controls showed that the pro-inflammatory monocytes had the most significant gene changes, among which the expressions of genes related to inflammatory responses and cell metabolism were significantly increased while the genes related to cell cycle progression were significantly decreased. Furthermore, compared with the ACLF survival group, the ACLF death group had significantly higher expressions of pro-inflammatory cytokines (e.g., IL-6) and their receptors, chemokines (e.g., CCL4 and CCL5), and inflammation-inducing factors (e.g., HES4). Additionally, validation using scRNA-seq and flow cytometry revealed the presence of a cell type-specific transcriptional signature of pro-inflammatory monocytes THBS1, whose production might reflect the disease progression and poor prognosis. CONCLUSIONS: We present the accurate classification, molecular markers, and signaling pathways of monocytes associated with ACLF progression. Therapies targeting pro-inflammatory monocytes may be a promising approach for blocking ACLF progression.

Knockdown of CXCL1 improves ACLF by reducing neutrophil recruitment to attenuate ROS production and hepatocyte apoptosis.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is an acute decompensated syndrome based on chronic liver disease, while neutrophil recruitment is the most critical early step. C-X-C motif chemokine ligand 1 (CXCL1), a cytokine that recruits neutrophils, was significantly upregulated in both ACLF mice and patients with ACLF. This present study aims to explore the role of CXCL1 in the pathogenesis of ACLF. METHODS: We established an ACLF mouse model induced by carbon tetrachloride, lipopolysaccharide, and D-galactosamine, and used adeno-associated virus to achieve overexpression and knockdown of Cxcl1. We employed mass cytometry, flow cytometry, multiplex cytokine and chemokine analysis, Western blot, and reactive oxygen species (ROS) detection in mice blood and liver. ACLF patients (n = 10) and healthy controls (n = 5) were included, and their liver samples were stained using multiplex immunohistochemistry techniques. RESULTS: CXCL1 was significantly elevated in both ACLF mice and patients. CXCL1 recruits neutrophils by binding to the C-X-C motif chemokine receptor 2 on the surface of neutrophils, affects ACLF prognosis by generating ROS and mitochondrial depolarization and modulating caspase3-related apoptotic pathways. We found that the knockdown of CXCL1 attenuated the infiltration of neutrophils in the mouse liver, reduced the expression of inflammatory cytokines, and also significantly downregulated ROS production and caspase3-related hepatocyte apoptosis, thereby ameliorating the liver injury of ACLF. CONCLUSIONS: CXCL1 is a core player in the mobilization of neutrophils in ACLF, and the knockdown of Cxcl1 improves neutrophil infiltration, reduces ROS levels, and reduces hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. Our results revealed a previously unknown link between CXCL1-induced neutrophil recruitment and ACLF, providing evidencing for potential therapies targeting ACLF.

Identification of oxidative stress-related biomarkers associated with the development of acute-on-chronic liver failure using bioinformatics.

Acute-on-chronic liver failure (ACLF) is a serious stage of chronic liver disease with high short-term mortality and no definitely effective treatment. Oxidative stress (OS) is involved in the development of ACLF. OS-related genes targeted therapy may provide additional assistance for the treatment of ACLF. ACLF related gene sets and oxidative stress-related genes (OSGs) were respectively downloaded from gene expression omnibus (GEO) database and GeneCards database for integrated bioinformatics analyses (functional enrichment, weighted gene co-expression network and immune cells infiltration). Immune-related differentially expressed oxidative stress-related genes (DEOSGs) in ACLF were used for construction of protein-protein interaction (PPI) network in which hub genes were screened out. Hub genes with consistently good diagnostic or prognostic value for ACLF in four gene sets were named as key genes. DEOSGs were significantly enriched in biological process and signaling pathways related to inflammation, immune response and oxidative stress. Six key genes (MPO, CCL5, ITGAM, TLR2, TLR4, and TIMP1) were identified and found to be highly correlated with immune response and metabolic process. This study deepened our understanding of the impact of oxidative stress on the pathogenesis and prognosis of ACLF and provided more insights into the prediction of prognosis and molecular targeted therapy in ACLF.