In vivo Bruton's tyrosine kinase inhibition attenuates alcohol-associated liver disease by regulating CD84-mediated granulopoiesis.

Severe alcohol-associated hepatitis (AH) is a life-threatening form of alcohol-associated liver disease. Liver neutrophil infiltration is a hallmark of AH, yet the effects of alcohol on neutrophil functions remain elusive. Identifying therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton's tyrosine kinase (BTK) plays an important role in neutrophil development and function; however, the role of BTK in AH is unknown. Using RNA sequencing of circulating neutrophils, we found an increase in Btk expression (P = 0.05) and phosphorylated BTK (pBTK) in patients with AH compared with healthy controls. In vitro, physiologically relevant doses of alcohol resulted in a rapid, TLR4-mediated induction of pBTK in neutrophils. In a preclinical model of AH, administration of a small-molecule BTK inhibitor (evobrutinib) or myeloid-specific Btk knockout decreased proinflammatory cytokines and attenuated neutrophil-mediated liver damage. We found that pBTK was essential for alcohol-induced bone marrow granulopoiesis and liver neutrophil infiltration. In vivo, BTK inhibition or myeloid-specific Btk knockout reduced granulopoiesis, circulating neutrophils, liver neutrophil infiltration, and liver damage in a mouse model of AH. Mechanistically, using liquid chromatography-tandem mass spectrometry, we identified CD84 as a kinase target of BTK, which is involved in granulopoiesis. In vitro, CD84 promoted alcohol-induced interleukin-1ß and tumor necrosis factor-α in primary human neutrophils, which was inhibited by CD84-blocking antibody treatment. Our findings define the role of BTK and CD84 in regulating neutrophil inflammation and granulopoiesis, with potential therapeutic implications in AH.

MASLD/MetALD and mortality in individuals with any cardio-metabolic risk factor: A population-based study with 26.7 years of follow-up.

BACKGROUND AND AIMS: A new term, metabolic dysfunction-associated steatotic liver disease (MASLD), has been proposed by a multi-society expert panel. However, it remains unclear whether hepatic steatosis per se in MASLD contributes to an increased risk of mortality in individuals with any cardio-metabolic risk factor (CMRF), which is also a significant risk factor for increased mortality. This study aimed to compare all-cause and cause-specific mortality between the "MASLD/MetALD" and "no steatotic liver disease (SLD)" groups in individuals with any CMRF. APPROACH AND RESULTS: A population-based cohort study was conducted using 10,750 participants of the Third National Health and Nutrition Examination Survey. All-cause and cause-specific (cardiovascular, cancer, diabetes, and liver) mortality risks were compared between the "MASLD," "MetALD," and "no SLD" groups using the Cox proportional hazards model with complex survey design weights, adjusted for confounders. Over 26 years, the "MASLD" group did not show significantly increased all-cause (adjusted HR 1.04[95% CI: 0.95-1.14], p = 0.413), cardiovascular (0.88 [0.75-1.04], p = 0.139), or cancer (1.06[0.84-1.33], p = 0.635) mortality risk compared to the "no SLD" group in individuals with any CMRF. The MetALD group was associated with increased all-cause (1.41 [1.05-1.89], p = 0.022), cancer (2.35 [1.33-4.16], p = 0.004), and liver (15.04 [2.96-76.35], p = 0.002) mortality risk compared with the no SLD group. This trend was more pronounced in the MetALD group with advanced fibrosis assessed by Fibrosis-4 (FIB-4). CONCLUSIONS: In individuals with CMRF, the presence of steatotic liver disease (MASLD) alone did not increase the risk of mortality, except in cases with more alcohol consumption (MetALD). Therefore controlling metabolic risk factors and reducing alcohol consumption in people with MASLD or MetALD will be crucial steps to improve long-term health outcomes.

Elevated Plasma Levels of Ketone Bodies Are Associated With All-Cause Mortality and Incidence of Heart Failure in Older Adults: The CHS.

Background Chronic disease, such as heart failure, influences cellular metabolism and shapes circulating metabolites. The relationships between key energy metabolites and chronic diseases in aging are not well understood. This study aims to determine the relationship between main components of energy metabolism with all-cause mortality and incident heart failure. Methods and Results We analyzed the association between plasma metabolite levels with all-cause mortality and incident heart failure among US older adults in the CHS (Cardiovascular Health Study). We followed 1758 participants without heart failure at baseline with hazard ratios (HRs) of analyte levels and metabolic profiles characterized by high levels of ketone bodies for all-cause mortality and incident heart failure. Multivariable Cox analyses revealed a dose-response relationship of 50% increase in all-cause mortality between lowest and highest quintiles of ketone body concentrations (HR, 1.5 [95% CI, 1.0-1.9]; P=0.007). Ketone body levels remained associated with incident heart failure after adjusting for cardiovascular disease confounders (HR, 1.2 [95% CI, 1.0-1.3]; P=0.02). Using K-means cluster analysis, we identified a cluster with higher levels of ketone bodies, citrate, interleukin-6, and B-type natriuretic peptide but lower levels of pyruvate, body mass index, and estimated glomerular filtration rate. The cluster with elevated ketone body levels was associated with higher all-cause mortality (HR, 1.7 [95% CI, 1.1-2.7]; P=0.01). Conclusions Higher concentrations of ketone bodies predict incident heart failure and all-cause mortality in an older US population, independent of metabolic and cardiovascular confounders. This association suggests a potentially important relationship between ketone body metabolism and aging.

Selective deletion of ENTPD1/CD39 in macrophages exacerbates biliary fibrosis in a mouse model of sclerosing cholangitis.

Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39-/-), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-ß1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39fl/fl and Cd39fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.

Lipoprotein metabolism in liver diseases.

PURPOSE OF REVIEW: The liver is the central hub of lipoprotein metabolism. A complex relationship exists between dyslipidemia and chronic liver diseases (CLDs). Recent advances in the genetics of nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) exemplify the pivotal role of lipoprotein metabolism in the pathogenesis of CLD. We review these relationships in four quintessential forms of CLD: NAFLD, ALD, cholestatic liver disease and cirrhosis, with a focus on recent discoveries. RECENT FINDINGS: An I148 M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3) and an E167K variant in transmembrane 6 superfamily 2 (TM6SF2) are major genetic risk factors for the development and progression of NAFLD. These genetic variants also increase the risk of ALD. Both PNPLA3 and TM6SF2 are involved in the hepatic assembly of very low-density lipoprotein. The discovery of these two genetic variants highlights the risk of CLD when environmental factors are combined with functional modifications in the lipoprotein metabolism pathway. SUMMARY: The relationship between CLD and lipoprotein metabolism is reciprocal. On the one hand, the progression of CLD impairs lipoprotein metabolism; on the other hand, modifications in lipoprotein metabolism can substantially increase the risk of CLD. These relationships are at play among the most common forms of CLD affecting a significant proportion of the population.

Purinergic signaling during intestinal inflammation.

Inflammatory bowel disease (IBD) is a devastating disease that is associated with excessive inflammation in the intestinal tract in genetically susceptible individuals and potentially triggered by microbial dysbiosis. This illness markedly predisposes patients to thrombophilia and chronic debility as well as bowel, lymphatic, and liver cancers. Development of new therapies is needed to re-establish long-term immune tolerance in IBD patients without increasing the risk of opportunistic infections and cancer. Aberrant purinergic signaling pathways have been implicated in disordered thromboregulation and immune dysregulation, as noted in the pathogenesis of IBD and other gastrointestinal/hepatic autoimmune diseases. Expression of CD39 on endothelial or immune cells allows for homeostatic integration of hemostasis and immunity, which are disrupted in IBD. Our focus in this review is on novel aspects of the functions of CD39 and related NTPDases in IBD. Regulated CD39 activity allows for scavenging of extracellular nucleotides, the maintenance of P2-receptor integrity and coordination of adenosinergic signaling responses. CD39 together with CD73, serves as an integral component of the immunosuppressive machinery of dendritic cells, myeloid cells, T and B cells. Genetic inheritance and environental factors closely regulate the levels of expression and phosphohydrolytic activity of CD39, both on immune cells and released microparticles. Purinergic mechanisms associated with T regulatory and supressor T helper type 17 cells modulate disease activity in IBD, as can be modeled in experimental colitis. As a recent example, upregulation of CD39 is dependent upon ligation of the aryl hydrocarbon receptor (AHR), as with natural ligands such as bilirubin and 2-(1' H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Decreased expression of CD39 and/or dysfunctional AHR signaling, however, abrogates the protective effects of immunosuppressive AHR ligands. These factors could also serve as biomarkers of disease activity in IBD. Heightened thrombosis, inflammation, and immune disturbances as seen in IBD appear to be associated with aberrant purinergic signaling. Ongoing development of therapeutic strategies augmenting CD39 ectonucleotidase bioactivity via cytokines or AHR ligands offers promise for management of thrombophilia, disordered inflammation, and aberrant immune reactivity in IBD.

CD39 mediated regulation of Th17-cell effector function is impaired in juvenile autoimmune liver disease.

BACKGROUND & AIMS: T-helper-type 17 (Th17) cells are involved in autoimmune tissue damage. CD39 is an ectonucleotidase that catalyzes extracellular ATP/ADP hydrolysis, culminating in the generation of immunosuppressive adenosine. Functional CD39 expression confers immunosuppressive properties upon immune cells. As the proportion of CD39 lymphocytes is decreased in juvenile autoimmune liver disease (AILD), we have explored whether decreased CD39 expression is present on Th17 cells and whether this phenomenon is associated with heightened effector function and inflammation. METHODS: Thirty-eight patients with juvenile AILD (22 autoimmune hepatitis and 16 autoimmune sclerosing cholangitis), 8 disease controls (DC) and 16 healthy subjects (HS) were studied. Peripheral blood cell phenotype was determined by flow cytometry; ability to suppress by inhibition of cell proliferation/effector cytokine production; ectoenzymatic activity by thin layer chromatography; expression of adenosine receptor, adenosine deaminase (ADA) and phosphodiesterases (PDE) by quantitative real-time PCR or by Western Blot. RESULTS: CD39(+) Th17 (Th17(CD39+)) cells from HS appear activated and contain high frequencies of lymphocytes producing regulatory cytokines. In AILD, however, Th17(CD39+) cells are markedly diminished and fail to generate AMP/adenosine, thereby limiting control of both target cell proliferation and IL-17 production. When compared to HS, Th17 cells from AILD patients also show lower A2A adenosine receptor expression while displaying similar levels of PDE4A, PDE4B and ADA. Only rare Th17(CD39+) cells are observed by liver immunohistochemistry. CONCLUSIONS: Th17(CD39+) cells in juvenile AILD are both quantitatively decreased and qualitatively deficient. Low levels CD39 and A2A expression may contribute to the perpetuation of Th17 cell effector properties and unfettered inflammation in this disease.

A Standardized Assessment of Functional Disability Predicts 1-year Mortality in Patients Undergoing Transjugular Intrahepatic Portosystemic Shunt for Refractory Ascites.

GOALS: To determine the association between functional disability and mortality after transjugular intrahepatic portosystemic shunt (TIPS). BACKGROUND: TIPS is a common therapeutic procedure for cirrhotic patients with refractory ascites. The conventional metric for periprocedure risk stratification is the model for end-stage liver disease (MELD), which uses biochemical parameters to predict post-TIPS mortality. It does not account for functional disability. STUDY: This is a retrospective cohort study of 83 patients admitted at an academic liver transplant center with cirrhosis and refractory ascites for the purpose of TIPS placement. We assessed the association of patients' reported activities of daily living (ADL) on a scale of 1 to 21 before TIPS with a primary outcome of 1-year mortality. Multivariable regression to adjust for MELD and Child class was performed. RESULTS: A higher ADL score or functional independence, was associated with decreased 1-year mortality when modeled as both a continuous variable [odds ratio (OR), 0.80; 95% confidence interval (CI), 0.66-0.97; P=0.02) and a dichotomous variable (ADL 21 vs. <21; OR, 0.21; 95% CI, 0.05-0.70; P=0.01). After adjusting for MELD and Child class, functional independence was associated with decreased 1-year transplant-free mortality (OR, 0.22; 95% CI, 0.05-0.77; P=0.02). An ADL score consistent with dependence (<21) was significantly associated with a 3.40-day (95% CI, 1.76-5.04) longer hospital stay, adjusting for MELD and Child class (P<0.0001). CONCLUSIONS: Functional disability is a predictor of post-TIPS mortality and length of stay after controlling for MELD.

Low LDL-C and high HDL-C levels are associated with elevated serum transaminases amongst adults in the United States: a cross-sectional study.

BACKGROUND: Dyslipidemia, typically recognized as high serum triglyceride, high low-density lipoprotein cholesterol (LDL-C) or low high-density lipoprotein cholesterol (HDL-C) levels, are associated with nonalcoholic fatty liver disease (NAFLD). However, low LDL-C levels could result from defects in lipoprotein metabolism or impaired liver synthetic function, and may serve as ab initio markers for unrecognized liver diseases. Whether such relationships exist in the general population has not been investigated. We hypothesized that despite common conception that low LDL-C is desirable, it might be associated with elevated liver enzymes due to metabolic liver diseases. METHODS AND FINDINGS: We examined the associations between alanine aminotransferase (ALT), aspartate aminotransferase (AST) and major components of serum lipid profiles in a nationally representative sample of 23,073 individuals, who had no chronic viral hepatitis and were not taking lipid-lowering medications, from the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2010. ALT and AST exhibited non-linear U-shaped associations with LDL-C and HDL-C, but not with triglyceride. After adjusting for potential confounders, individuals with LDL-C less than 40 and 41-70 mg/dL were associated with 4.2 (95% CI 1.5-11.7, p = 0.007) and 1.6 (95% CI 1.1-2.5, p = 0.03) times higher odds of abnormal liver enzymes respectively, when compared with those with LDL-C values 71-100 mg/dL (reference group). Surprisingly, those with HDL-C levels above 100 mg/dL was associated with 3.2 (95% CI 2.1-5.0, p<0.001) times higher odds of abnormal liver enzymes, compared with HDL-C values of 61-80 mg/dL. CONCLUSIONS: Both low LDL-C and high HDL-C, often viewed as desirable, were associated with significantly higher odds of elevated transaminases in the general U.S. adult population. Our findings underscore an underestimated biological link between lipoprotein metabolism and liver diseases, and raise a potential need for liver evaluation among over 10 million people with particularly low LDL-C or high HDL-C in the United States.

Lipoprotein metabolism in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD), an escalating health problem worldwide, covers a spectrum of pathologies characterized by fatty accumulation in hepatocytes in early stages, with potential progression to liver inflammation, fibrosis, and failure. A close, yet poorly understood link exists between NAFLD and dyslipidemia, a constellation of abnormalities in plasma lipoproteins including triglyceride-rich very low density lipoproteins. Apolipoproteins are a group of primarily liver-derived proteins found in serum lipoproteins; they not only play an extracellular role in lipid transport between vital organs through circulation, but also play an important intracellular role in hepatic lipoprotein assembly and secretion. The liver functions as the central hub for lipoprotein metabolism, as it dictates lipoprotein production and to a significant extent modulates lipoprotein clearance. Lipoprotein metabolism is an integral component of hepatocellular lipid homeostasis and is implicated in the pathogenesis, potential diagnosis, and treatment of NAFLD.

Interfacial properties of apolipoprotein B292-593 (B6.4-13) and B611-782 (B13-17). Insights into the structure of the lipovitellin homology region in apolipoprotein B.

The N-terminal sequence of apolipoprotein B (apoB) is critical in triacylglycerol-rich lipoprotein assembly. The first 17% of apoB (B17) is thought to consist of three domains: B5.9, a beta-barrel, B6.4-13, a series of 17 alpha-helices, and B13-17, a putative beta-sheet. B5.9 does not bind to lipid, while B6.4-13 and B13-17 contain hydrophobic interfaces that can interact with lipids. To understand how B6.4-13 and B13-17 might interact with triacylglycerol during lipoprotein assembly, the interfacial properties of both peptides were studied at the triolein/water interface. Both B6.4-13 and B13-17 are surface active. Once bound, the peptides can be neither exchanged nor pushed off the interface. Some residues of the peptides can be ejected from the interface upon compression but readsorb on expansion. B13-17 binds to the interface more strongly. The maximum pressure the peptide can withstand without being partially ejected (Pi(max)) is 19.2 mN/m for B13-17 compared to 16.7 mN/m for B6.4-13. B13-17 is purely elastic at the interface, while B6.4-13 forms a viscous-elastic film. When they are spread at an air/water interface, the limiting area and the collapse pressures are 16.6 A(2)/amino acid and 31 mN/m for B6.4-13 and 17.8 A(2)/amino acid and 35 mN/m for B13-17, respectively. The alpha-helical B6.4-13 contains some hydrophobic helices that stay bound and prevent the peptide from leaving the surface. The beta-sheets of B13-17 bind irreversibly to the surface. We suggest that during lipoprotein assembly, the N-terminal apoB starts recruiting lipid as early as B6.4, but additional sequences are essential for formation of a lipid pocket that can stabilize lipoprotein emulsion particles for secretion.

Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B.

Apolipoproteins play a central role in lipoprotein metabolism, and are directly implicated in cardiovascular diseases, but their structural characterization has been complicated by their structural flexibility and heterogeneity. Here we describe the structural characterization of the N-terminal region of apolipoprotein B (apoB), the major protein component of very low-density lipoprotein and low-density lipoprotein, in the presence of phospholipids. Specifically, we focus on the N-terminal 6.4-17% of apoB (B6.4-17) complexed with the phospholipid dimyristoylphosphatidylcholine in vitro. In addition to circular dichroism spectroscopy and limited proteolysis, our strategy incorporates nanogold-labeling of the protein in the reconstituted lipoprotein complex followed by visualization and molecular weight determination with scanning transmission electron microscopy imaging. Based on the scanning transmission electron microscopy imaging analysis of approximately 1300 individual particles where the B6.4-17 is labeled with nanogold through a six-His tag, most complexes contain either two or three B6.4-17 molecules. Circular dichroism spectroscopy and limited proteolysis of these reconstituted particles indicate that there are no large conformational changes in B6.4-17 upon lipoprotein complex formation. This is in contrast to the large structural changes that occur during apolipoprotein A-I-lipid interactions. The method described here allows a direct measurement of the stoichiometry and molecular weight of individual particles, rather than the average of the entire sample. Thus, it represents a useful strategy to characterize the structure of lipoproteins, which are not structurally uniform, but can still be defined by an ensemble of related patterns.

Defining lipid-interacting domains in the N-terminal region of apolipoprotein B.

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted alpha-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogeneous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC-dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous with lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.

A phosphorylation-induced conformation change in dematin headpiece.

Dematin is an actin binding protein from the junctional complex of the erythrocyte cytoskeleton. The protein has two actin binding sites and bundles actin filaments in vitro. This actin bundling activity is reversibly regulated by phosphorylation in the carboxyl terminal "headpiece" domain (DHP). DHP is a typical villin-type headpiece actin binding motif and contains a flexible N-terminal loop and an alpha-helical C-terminal subdomain that is phosphorylated at Ser74. The NMR structure of a Ser74-to-Glu mutant (DHPs74e) closely mimics the conformation of phosphorylated DHP. The negative charge at Ser74 does not alter the conformation of the C-terminal subdomain, but attracts the N-terminal loop toward the C terminus, changing the orientation of the N-terminal subdomain. NMR relaxation studies also indicate reduced mobility in the N-terminal loop in DHPs74e. Thus, phosphorylation in DHP serves as a switch controlling the conformational state of DHP and the actin bundling activity of dematin.

Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B.

Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.