title: Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes.
abstract: OBJECTIVE
  Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype.
  
  
  METHODS
  Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction.
  
  
  RESULTS
  Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis.
  
  
  CONCLUSIONS
  Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Petra
authors.initials: P
authors.lastname: Hirsova
authors.affiliation: Division of Pediatric Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Samar H
authors.initials: SH
authors.lastname: Ibrahim
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Krishnan
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Vikas K
authors.initials: VK
authors.lastname: Verma
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Bronk
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Werneburg
authors.affiliation: Transplant Center, Intermountain Medical Center, Murray, Utah.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Charlton
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Vijay H
authors.initials: VH
authors.lastname: Shah
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Harmeet
authors.initials: H
authors.lastname: Malhi
authors.affiliation: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota. Electronic address: gores.gregory@mayo.edu.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D000067128:Extracellular Vesicles / Q000187:drug effects* / Q000378:metabolism; D057809:HEK293 Cells; D006505:Hepatitis / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000378:metabolism / Q000473:pathology; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D065626:Non-alcoholic Fatty Liver Disease / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D019308:Palmitic Acid / Q000494:pharmacology*; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D017207:Rats, Sprague-Dawley; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D018836:Inflammation Mediators; D008244:Lysophosphatidylcholines; D047428:Protein Kinase Inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; C505639:TNFRSF10B protein, human; C505640:Tnfrsf10b protein, mouse; D019308:Palmitic Acid; C515851:ROCK1 protein, human; C515854:Rock1 protein, mouse; D054460:rho-Associated Kinases; D020169:Caspases
keywords: Cell Death; Exosomes; Lipotoxic; Microvesicles