Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.

BACKGROUND & AIMS: Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF. METHODS: The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array. RESULTS: Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism. CONCLUSIONS: Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF. LAY SUMMARY: Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.

Assessing the role of amino acids in systemic inflammation and organ failure in patients with ACLF.

BACKGROUND & AIMS: Systemic inflammation and organ failure(s) are the hallmarks of acute-on-chronic liver failure (ACLF), yet their pathogenesis remains uncertain. Herein, we aimed to assess the role of amino acids in these processes in patients with ACLF. METHODS: The blood metabolomic database of the CANONIC study (comprising 137 metabolites, with 43% related to amino acids) - obtained in 181 patients with ACLF and 650 with acute decompensation without ACLF (AD) - was reanalyzed with a focus on amino acids, in particular 9 modules of co-regulated metabolites. We also compared blood metabolite levels between ACLF and AD. RESULTS: The main findings in ACLF were: i) Metabolite modules were increased in parallel with increased levels of markers of systemic inflammation and oxidative stress. ii) Seventy percent of proteinogenic amino acids were present and most were increased. iii) A metabolic network, comprising the amino acids aspartate, glutamate, the serine-glycine one-carbon metabolism (folate cycle), and methionine cycle, was activated, suggesting increased purine and pyrimidine nucleotide synthesis. iv) Cystathionine, L-cystine, glutamate and pyroglutamate, which are involved in the transsulfuration pathway (a methionine cycle branch) were increased, consistent with increased synthesis of the antioxidant glutathione. v) Intermediates of the catabolism of 5 out of the 6 ketogenic amino acids were increased. vi) The levels of spermidine (a polyamine inducer of autophagy with anti-inflammatory effects) were decreased. CONCLUSIONS: In ACLF, blood amino acids fueled protein and nucleotide synthesis required for the intense systemic inflammatory response. Ketogenic amino acids were extensively catabolized to produce energy substrates in peripheral organs, an effect that was insufficient because organs failed. Finally, the decrease in spermidine levels may cause a defect in autophagy contributing to the proinflammatory phenotype in ACLF. LAY SUMMARY: Systemic inflammation and organ failures are hallmarks of acute-on-chronic liver failure (ACLF). Herein, we aimed to characterize the role of amino acids in these processes. The blood metabolome of patients with acutely decompensated cirrhosis, and particularly those with ACLF, reveals evidence of intense skeletal muscle catabolism. Importantly, amino acids (along with glucose), are used for intense anabolic, energy-consuming metabolism in patients with ACLF, presumably to support de novo nucleotide and protein synthesis in the activated innate immune system.

Untargeted lipidomics uncovers lipid signatures that distinguish severe from moderate forms of acutely decompensated cirrhosis.

BACKGROUND & AIMS: Acute decompensation (AD) of cirrhosis is a heterogeneous clinical entity associated with moderate mortality. In some patients, this condition develops quickly into the more deadly acute-on-chronic liver failure (ACLF), in which other organs such as the kidneys or brain fail. The aim of this study was to characterize the blood lipidome in a large series of patients with cirrhosis and identify specific signatures associated with AD and ACLF development. METHODS: Serum untargeted lipidomics was performed in 561 patients with AD (518 without and 43 with ACLF) (discovery cohort) and in 265 patients with AD (128 without and 137 with ACLF) in whom serum samples were available to perform repeated measurements during the 28-day follow-up (validation cohort). Analyses were also performed in 78 patients with AD included in a therapeutic albumin trial (43 patients with compensated cirrhosis and 29 healthy individuals). RESULTS: The circulating lipid landscape associated with cirrhosis was characterized by a generalized suppression, which was more manifest during AD and in non-surviving patients. By computing discriminating accuracy and the variable importance projection score for each of the 223 annotated lipids, we identified a sphingomyelin fingerprint specific for AD of cirrhosis and a distinct cholesteryl ester and lysophosphatidylcholine fingerprint for ACLF. Liver dysfunction and infections were the principal net contributors to these fingerprints, which were dynamic and interchangeable between patients with AD whose condition worsened to ACLF and those who improved. Notably, blood lysophosphatidylcholine levels increased in these patients after albumin therapy. CONCLUSIONS: Our findings provide insights into the lipid landscape associated with decompensation of cirrhosis and ACLF progression and identify unique non-invasive diagnostic biomarkers of advanced cirrhosis. LAY SUMMARY: Analysis of lipids in blood from patients with advanced cirrhosis reveals a general suppression of their levels in the circulation of these patients. A specific group of lipids known as sphingomyelins are useful to distinguish between patients with compensated and decompensated cirrhosis. Another group of lipids designated cholesteryl esters further distinguishes patients with decompensated cirrhosis who are at risk of developing organ failures.

The PREDICT study uncovers three clinical courses of acutely decompensated cirrhosis that have distinct pathophysiology.

BACKGROUND & AIMS: Acute decompensation (AD) of cirrhosis is defined as the acute development of ascites, gastrointestinal hemorrhage, hepatic encephalopathy, infection or any combination thereof, requiring hospitalization. The presence of organ failure(s) in patients with AD defines acute-on-chronic liver failure (ACLF). The PREDICT study is a European, prospective, observational study, designed to characterize the clinical course of AD and to identify predictors of ACLF. METHODS: A total of 1,071 patients with AD were enrolled. We collected detailed pre-specified information on the 3-month period prior to enrollment, and clinical and laboratory data at enrollment. Patients were then closely followed up for 3 months. Outcomes (liver transplantation and death) at 1 year were also recorded. RESULTS: Three groups of patients were identified. Pre-ACLF patients (n = 218) developed ACLF and had 3-month and 1-year mortality rates of 53.7% and 67.4%, respectively. Unstable decompensated cirrhosis (UDC) patients (n = 233) required ≥1 readmission but did not develop ACLF and had mortality rates of 21.0% and 35.6%, respectively. Stable decompensated cirrhosis (SDC) patients (n = 620) were not readmitted, did not develop ACLF and had a 1-year mortality rate of only 9.5%. The 3 groups differed significantly regarding the grade and course of systemic inflammation (high-grade at enrollment with aggravation during follow-up in pre-ACLF; low-grade at enrollment with subsequent steady-course in UDC; and low-grade at enrollment with subsequent improvement in SDC) and the prevalence of surrogates of severe portal hypertension throughout the study (high in UDC vs. low in pre-ACLF and SDC). CONCLUSIONS: Acute decompensation without ACLF is a heterogeneous condition with 3 different clinical courses and 2 major pathophysiological mechanisms: systemic inflammation and portal hypertension. Predicting the development of ACLF remains a major future challenge. CLINICALTRIALS. GOV NUMBER: NCT03056612. LAY SUMMARY: Herein, we describe, for the first time, 3 different clinical courses of acute decompensation (AD) of cirrhosis after hospital admission. The first clinical course includes patients who develop acute-on-chronic liver failure (ACLF) and have a high short-term risk of death - termed pre-ACLF. The second clinical course (unstable decompensated cirrhosis) includes patients requiring frequent hospitalizations unrelated to ACLF and is associated with a lower mortality risk than pre-ACLF. Finally, the third clinical course (stable decompensated cirrhosis), includes two-thirds of all patients admitted to hospital with AD - patients in this group rarely require hospital admission and have a much lower 1-year mortality risk.

Albumin Lipidomics Reveals Meaningful Compositional Changes in Advanced Cirrhosis and Its Potential to Promote Inflammation Resolution.

Albumin infusions are therapeutically used to revert hypoalbuminemia and to replace the extensively oxidized albumin molecule circulating in patients with acutely decompensated (AD) cirrhosis. Because albumin has high affinity for lipids, here we characterized the albumin lipidome in patients with AD and explored the albumin effects on the release of fatty acid (FA)-derived lipid mediators by peripheral leukocytes. Lipids and lipid mediators were measured by liquid chromatography-tandem mass spectrometry in albumin-enriched and albumin-depleted plasma fractions separated by affinity chromatography and in leukocyte incubations from 18 patients with AD and 10 healthy subjects (HS). Lipid mediators were also measured in 41 patients with AD included in an albumin therapy trial. The plasma lipidome associated with AD cirrhosis was characterized by generalized suppression of all lipid classes except FAs. In contrast to HS, albumin from patients with AD had lower content of polyunsaturated FAs (PUFAs), especially of the omega-3-PUFA docosahexaenoic acid. Consistent with this, the PUFA-derived lipid mediator landscape of albumin from patients with AD was dominated by lower content of monohydroxy FA precursors of anti-inflammatory/pro-resolving lipid mediators (i.e., 15-hydroxyeicosatetraenoic acid [15-HETE]). In addition, albumin from patients with AD was depleted in prostaglandin (PG) E2 , suggesting that this proinflammatory PG primarily travels disassociated to albumin in these patients. Incubation of leukocytes with exogenous albumin reduced PG production while inducing 15-lipoxygenase expression and 15-HETE release. Similar effects were seen under lipopolysaccharide plus N-formylmethionyl-leucyl-phenylalanine-stimulated conditions. Finally, PG levels were lower in patients with AD receiving albumin therapy, whereas 15-HETE was increased after albumin treatment compared with baseline. Conclusion: Our findings indicate that the albumin lipid composition is severely disorganized in AD cirrhosis and that administration of exogenous albumin has the potential to redirect leukocyte biosynthesis from pro-inflammatory to pro-resolving lipid mediators.