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Background and aim Extracellular matrix (ECM) components released during excessive fat mass expansion are considered potential endogenous danger/alarm signals contributing to innate immune system activation. The aim of the current study was to specifically measure plasma levels of low molecular weight (LMW) hyaluronan (HA) and to evaluate its role as pro-inflammatory damage-associated molecular pattern (DAMP) on leukocyte response in the context of human obesity. Subjects and methods Participants were selected according to their body mass index (BMI, kg/m2) as non-obese (BMI n = 18) and obese (BMI > 29.9, n = 33). Plasma samples were size-dependent fractionated using ion-exchange chromatography to specifically obtain LMW HA fractions that were subsequently quantified by ELISA. Cell incubation experiments with synthetic HA molecules were performed on freshly Ficoll-isolated neutrophils (PMN) and peripheral blood monocytes (PBMC). Leukocyte and adipose tissue gene expression was assessed by real-time PCR and NF-κB activation by western blot. Plasma cytokine levels were measured by fluorescent bead-based (Luminex) immunoassay. Results We observed a statistically significant increase in the circulating levels of HA fragments of LMW in individuals with obesity which were consistent with significant up-regulated expression of the LMW HA synthesizing enzyme hyaluronan synthase-1 (HAS-1) in obese adipose tissue. Gene expression assessment of HA receptors revealed up-regulated levels for TLR2 in both obese PMN and PBMC. Synthetic HA molecules of different sizes were tested on leukocytes from healthy donors. LMW HA fragments (15–40 kDa) and not those from intermediate molecular sizes (75–350 kDa) induced a significant up-regulation of the expression of major pro-inflammatory cytokines such as IL-1β, MCP-1 and IL-8 in PBMC. Importantly, LMW HA was able to induce the phosphorylation of IKK α/β complex supporting its pro-inflammatory role through NF-κB activation. Conclusion Circulating LMW HA molecules are elevated in obesity and may play an important role in triggering low-grade inflammation and the development of metabolic complications.

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.

Background and aim: Injury to hepatocyte mitochondria is common in metabolic dysfunction-associated fatty liver disease. Here, we investigated whether changes in the content of essential fatty acid-derived lipid autacoids affect hepatocyte mitochondrial bioenergetics and metabolic efficiency. Approach and results: The study was performed in transgenic mice for the fat-1 gene, which allows the endogenous replacement of the membrane omega-6-polyunsaturated fatty acid (PUFA) composition by omega-3-PUFA. Transmission electron microscopy revealed that hepatocyte mitochondria of fat-1 mice had more abundant intact cristae and higher mitochondrial aspect ratio. Fat-1 mice had increased expression of oxidative phosphorylation complexes I and II and translocases of both inner (translocase of inner mitochondrial membrane 44) and outer (translocase of the outer membrane 20) mitochondrial membranes. Fat-1 mice also showed increased mitofusin-2 and reduced dynamin-like protein 1 phosphorylation, which mediate mitochondrial fusion and fission, respectively. Mitochondria of fat-1 mice exhibited enhanced oxygen consumption rate, fatty acid β-oxidation, and energy substrate utilization as determined by high-resolution respirometry, [1-14 C]-oleate oxidation and nicotinamide adenine dinucleotide hydride/dihydroflavine-adenine dinucleotide production, respectively. Untargeted lipidomics identified a rich hepatic omega-3-PUFA composition and a specific docosahexaenoic acid (DHA)-enriched lipid fingerprint in fat-1 mice. Targeted lipidomics uncovered a higher content of DHA-derived lipid autacoids, namely resolvin D1 and maresin 1, which rescued hepatocytes from TNFα-induced mitochondrial dysfunction, and unblocked the tricarboxylic acid cycle flux and metabolic utilization of long-chain acyl-carnitines, amino acids, and carbohydrates. Importantly, fat-1 mice were protected against mitochondrial injury induced by obesogenic and fibrogenic insults. Conclusion: Our data uncover the importance of a lipid membrane composition rich in DHA and its lipid autacoid derivatives to have optimal hepatic mitochondrial and metabolic efficiency.

Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.

Inflammation is a characteristic feature of virtually all acute and chronic liver diseases. It intersects different liver pathologies from the early stages of liver injury, when the inflammatory burden is mild-to-moderate, to very advanced stages of liver disease, when the inflammatory response is very intense and drives multiple organ dysfunction and failure(s). The current review describes the most relevant features of the inflammatory process in two different clinical entities across the liver disease spectrum, namely non-alcoholic steatohepatitis (NASH) and acute-on-chronic liver failure (ACLF). Special emphasis is given within these two disease conditions to gather the most relevant data on the specialized pro-resolving mediators that orchestrate the resolution of inflammation, a tightly controlled process which dysregulation commonly associates with chronic inflammatory conditions.

Significance Fatty liver, which is an initial step in the development of more severe complications such as liver cirrhosis, is prevalent worldwide in our society. This study demonstrates that stimulation of soluble guanylate cyclase (sGC), an enzyme producing the second messenger cGMP, protects against the most common features of fatty liver, namely inflammation and fibrosis, in animal models of the disease. Our study also provides an explanation for this protection and describes how sGC stimulation blocks the inflammasome (a protein complex responsible for the production of the potent proinflammatory cytokine interleukin-1β) in liver macrophages. The results of this study support the investigation of sGC stimulators, which are already approved for treatment in other conditions, in patients with fatty liver disease., Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine triphosphate into cyclic guanosine-3′,5′-monophosphate, a key second messenger in cell signaling and tissue homeostasis. It was recently demonstrated that sGC stimulation is associated with a marked antiinflammatory effect in the liver of mice with experimental nonalcoholic steatohepatitis (NASH). Here, we investigated the mechanisms underlying the antiinflammatory effect of the sGC stimulator praliciguat (PRL) in the liver. Therapeutic administration of PRL exerted antiinflammatory and antifibrotic actions in mice with choline-deficient l-amino acid-defined high-fat diet-induced NASH. The PRL antiinflammatory effect was associated with lower F4/80- and CX3CR1-positive macrophage infiltration into the liver in parallel with lower Ly6CHigh- and higher Ly6CLow-expressing monocytes in peripheral circulation. The PRL antiinflammatory effect was also associated with suppression of hepatic levels of interleukin (IL)-1β, NLPR3 (NACHT, LRR, and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), and active cleaved-caspase-1, which are components of the NLRP3 inflammasome. In Kupffer cells challenged with the classical inflammasome model of lipopolysaccharide plus adenosine triphosphate, PRL inhibited the priming (expression of Il1b and Nlrp3) and blocked the release of mature IL-1β. Mechanistically, PRL induced the protein kinase G (PKG)-mediated phosphorylation of the VASP (vasodilator-stimulated phosphoprotein) Ser239 residue which, in turn, reduced nuclear factor-κB (NF-κB) activity and Il1b and Nlrp3 gene transcription. PRL also reduced active cleaved-caspase-1 levels independent of pannexin-1 activity. These data indicate that sGC stimulation with PRL exerts antiinflammatory actions in the liver through mechanisms related to a PKG/VASP/NF-κB/NLRP3 inflammasome circuit.

Decompensated cirrhosis is characterized by exuberant systemic inflammation. Although the inducers of this feature remain unknown, the presence of circulating forms of oxidized albumin, namely human nonmercaptalbumin 1 (HNA1) and HNA2, is a common finding in cirrhosis. The aim of this study was to explore the ability of these oxidized albumin forms to induce systemic inflammation by triggering the activation of peripheral leukocytes. We observed significantly higher plasma levels of HNA1 and HNA2 in patients with cirrhosis (n = 256) compared to healthy volunteers (n = 48), which gradually increased during the course from compensated to decompensated to acute-on-chronic liver failure. Plasma HNA1 and HNA2 levels significantly correlated with inflammatory markers (i.e., interleukin-6 [IL-6], IL-1β, tumor necrosis factor-alpha [TNF-α] and IL-8) in patients with cirrhosis. To directly test the inflammatory effects of HNA1 and HNA2 on leukocytes, these oxidized albumin forms were prepared ex vivo and their posttranslational modifications monitored by liquid chromatography (LC)-quadrupole time-of-flight/mass spectrometry (MS). HNA1, but not HNA2, increased IL-1β, IL-6, and TNF-α mRNA and protein expression in leukocytes from both healthy volunteers and patients with cirrhosis. Moreover, HNA1 up-regulated the expression of eicosanoid-generating enzymes (i.e., cyclooxygenase-2 [COX-2] and microsomal prostaglandin E [PGE] synthase 1) and the production of inflammatory eicosanoids (PGE2 , PGF2α , thromboxane B2 , and leukotriene B4 ), as determined by LC-electrospray ionization-MS/MS. The inflammatory response to HNA1 was more pronounced in peripheral blood mononuclear cells (PBMCs) and marginal in polymorphonuclear neutrophils. Kinome analysis of PBMCs revealed that HNA1 induced the phosphorylation of p38 mitogen-activated protein kinase, the inhibition of which blocked HNA1-induced cytokine and COX-2 induction. Conclusion: HNA1 triggers an inflammatory response in PBMCs, providing a rationale for its removal and replacement by reduced albumin in the prevention of systemic inflammation in patients with advanced liver disease.

Background and Purpose Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation and fibrosis. Soluble guanylate cyclase (sGC) stimulation reduces inflammation and fibrosis in experimental models of lung, kidney and heart disease. Here, we tested whether sGC stimulation is also effective in experimental NASH. Experimental Approach NASH was induced in mice by feeding a choline-deficient, l-amino acid-defined, high-fat diet. These mice received either placebo or the sGC stimulator IW-1973 at two different doses (1 and 3 mg·kg−1·day−1) for 9 weeks. IW-1973 was also tested in high-fat diet (HFD)-induced obese mice. Steatosis, inflammation and fibrosis were assessed by Oil Red O, haematoxylin–eosin, Masson's trichrome, Sirius Red, F4/80 and α-smooth muscle actin staining. mRNA expression was assessed by quantitative PCR. Levels of IW-1973, cytokines and cGMP were determined by LC-MS/MS, Luminex and enzyme immunoassay respectively. Key Results Mice with NASH showed reduced cGMP levels and sGC expression, increased steatosis, inflammation, fibrosis, TNF-α and MCP-1 levels and up-regulated collagen types I α1 and α2, MMP2, TGF-β1 and tissue metallopeptidase inhibitor 1 expression. IW-1973 restored hepatic cGMP levels and sGC expression resulting in a dose-dependent reduction of hepatic inflammation and fibrosis. IW-1973 levels were ≈40-fold higher in liver tissue than in plasma. IW-1973 also reduced hepatic steatosis and adipocyte hypertrophy secondary to enhanced autophagy in HFD-induced obese mice. Conclusions and Implications Our data indicate that sGC stimulation prevents hepatic steatosis, inflammation and fibrosis in experimental NASH. These findings warrant further evaluation of IW-1973 in the clinical setting.

Human serum albumin (HSA) is an emerging treatment for preventing excessive systemic inflammation and organ failure(s) in patients with acutely decompensated (AD) cirrhosis. Here, we investigated the molecular mechanisms underlying the immunomodulatory properties of HSA. Administration of HSA to patients with AD cirrhosis with elevated circulating bacterial DNA rich in unmethylated cytosine-phosphate-guanine dideoxynucleotide motifs (CpG-DNA) was associated with reduced plasma cytokine concentrations. In isolated leukocytes, HSA abolished CpG-DNA-induced cytokine expression and release independently of its oncotic and scavenging properties. Similar anti-inflammatory effects were observed with recombinant human albumin. HSA exerted widespread changes on the immune cell transcriptome, specifically in genes related to cytokines and type I interferon responses. Our data revealed that HSA was taken up by leukocytes and internalized in vesicles positively stained with early endosome antigen 1 and colocalized with CpG-DNA in endosomes, where the latter binds to Toll-like receptor 9 (TLR9), its cognate receptor. Furthermore, HSA also inhibited polyinosinic:polycytidylic acid- and lipopolysaccharide-induced interferon regulatory factor 3 phosphorylation and TIR domain-containing adapter-inducing interferon-β-mediated responses, which are exclusive of endosomal TLR3 and TLR4 signaling, respectively. The immunomodulatory actions of HSA did not compromise leukocyte defensive mechanisms such as phagocytosis, efferocytosis, and intracellular reactive oxygen species production. The in vitro immunomodulatory effects of HSA were confirmed in vivo in analbuminemic humanized neonatal Fc receptor transgenic mice. These findings indicate that HSA internalizes in immune cells and modulates their responses through interaction with endosomal TLR signaling, thus providing a mechanism for the benefits of HSA infusions in patients with cirrhosis.

We investigated the effect of albumin treatment (20% solution) on hypoalbuminemia, cardiocirculatory dysfunction, portal hypertension, and systemic inflammation in patients with decompensated cirrhosis with and without bacterial infections., SCOPUS: ar.j, info:eu-repo/semantics/published

Obesity comorbidities are closely associated with chronic low-grade adipose tissue inflammation. A number of SNPs associated with inflammation has been identified, underscoring the impact of genetic determinants on this process. Here, we screened SNPs in genes with pro-inflammatory (IL-1β, IL-6, STAT3 and JAK2), anti-inflammatory (IL-10 and SOCS3) and pro-resolving (ERV1/ChemR23) properties in 101 obese and 99 non-obese individuals. Among the SNPs analyzed, we identified that individuals carrying a C allele in the rs1878022 polymorphism of the ERV1/ChemR23 gene, which encodes for the receptor of the pro-resolving mediator RvE1, had increased ERV1/ChemR23 protein expression and reduced levels of the inflammatory cytokine IL-6 in adipose tissue. Moreover, patients carrying the C allele in homozygosity had lower plasma levels of IL-6, IFN-α2, IL-15, IL-1ra, IL-10, GM-CSF, G-CSF and VEGF and enhanced leukocyte responsiveness to RvE1. C-carriers also exhibited decreased TAG to HDL ratio, a surrogate marker of insulin resistance and a predictor of incident fatty liver. Finally, we confirmed in vivo that the ERV1/ChemR23 receptor regulates systemic and tissue inflammation since mice lacking ERV1/ChemR23 expression showed increased IL-6 levels in adipose tissue and peritoneal macrophages. Together, our study identified an ERV1/ChemR23 variant that protects patients with obesity from excessive inflammatory burden.

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are hallmarks of nonalcoholic fatty liver disease (NAFLD), which is the hepatic manifestation of the metabolic syndrome associated with obesity. The specialized proresolving lipid mediator maresin 1 (MaR1) preserves tissue homeostasis by exerting cytoprotective actions, dampening inflammation, and expediting its timely resolution. Here, we explored whether MaR1 protects liver cells from lipotoxic and hypoxia-induced ER stress. Mice were rendered obese by high-fat diet feeding, and experiments were performed in primary hepatocytes, Kupffer cells, and precision-cut liver slices (PCLSs). Palmitate-induced lipotoxicity increased ER stress and altered autophagy in hepatocytes, effects that were prevented by MaR1. MaR1 protected hepatocytes against lipotoxicity-induced apoptosis by activating the UPR prosurvival mechanisms and preventing the excessive up-regulation of proapoptotic pathways. Protective MaR1 effects were also seen in hepatocytes challenged with hypoxia and TNF-α-induced cell death. High-throughput microRNA (miRNA) sequencing revealed that MaR1 actions were associated with specific miRNA signatures targeting both protein folding and apoptosis. MaR1 also prevented lipotoxic-triggered ER stress and hypoxia-induced inflammation in PCLSs and enhanced Kupffer cell phagocytic capacity. Together, these findings describe the ability of MaR1 to oppose ER stress in liver cells under conditions frequently encountered in NAFLD.-Rius, B., Duran-Güell, M., Flores-Costa, R., López-Vicario, C., Lopategi, A., Alcaraz-Quiles, J., Casulleras, M., Lozano, J. J., Titos, E., Clària, J. The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.