Laboratory Investigation (2009) 89, 549–561 & 2009 USCAP, Inc All rights reserved 0023-6837/09 $32.00 Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study Eizo Watanabe1,2, Jared T Muenzer3, William G Hawkins2, Christopher G Davis1, David J Dixon2, Jonathan E McDunn1, Daniel J Brackett4,5, Megan R Lerner4,5, Paul E Swanson6 and Richard S Hotchkiss1,2,7 Autophagy is the regulated process cells use to recycle nonessential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its function in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of six septic patients and four control patients as well as liver specimens from mice with surgical sepsis (by cecal ligation and puncture) or sham operation. EM demonstrated increased autophagic vacuoles in septic vs nonseptic patients. Randomly selected fields (3000 mm2) from control and septic patients contained 1.2±1.5 vs 5.3±3.3 (mean±s.d.) complex lysosomal/autophagolysosomal structures per image respectively (Po0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles, and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2±1.9 vs 38.7±3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P ¼ 0.002). Quantitative RT-PCR demonstrated that sepsis induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death. Laboratory Investigation (2009) 89, 549–561; doi:10.1038/labinvest.2009.8; published online 2 February 2009 KEYWORDS: cell death; inflammation; cytokines; electron microscopy; gene expression Autophagy was first used to describe electron microscopic is subjected to lysosomal acid hydrolases and other enzymes observations of novel single- or double-membrane vesicles that digest the engulfed material. Autophagy is thought to be that contained cytoplasmic components including organelles an adaptive response during times of cell stress, such as in various stages of disintegration.1,2 Autophagy is now nutrient limitation. For example, autophagy may occur known to be a process by which a cell degrades and recycles between meals within the liver to maintain its metabolic its own nonessential, redundant, or inefficient components. functions, supplying amino acids through catabolism.5 In The autophagic pathway is distinct from the ubiquitin– addition to its role in enabling the cell to gain vital nutrients proteosome system that degrades individual proteins. In during times of stress, autophagy can also be involved in eukaryotic cells, autophagy starts with the formation of tumor suppression, deletion of misfolded aggregate-prone a double-membrane vesicle, an autophagosome, that proteins, and antigen presentation.6–8 Autophagy has encompasses part of the cell cytoplasm or organelle.3,4 also been found to have a function in innate immunity, The autophagosome subsequently fuses with a lysosome and neutralizing intracellular pathogens including Streptococcus 1Department of Anesthesiology, Washington University School of Medicine, St Louis, MO, USA; 2Department of Surgery, Washington University School of Medicine, St Louis, MO, USA; 3Department of Pediatrics, Washington University School of Medicine, St Louis, MO, USA; 4Department of Surgery, Oklahoma University Health Sciences Center, Oklahoma City, OK, USA; 5Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA; 6Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA and 7Department of Medicine, Washington University School of Medicine, St Louis, MO, USA Correspondence: Dr RS Hotchkiss, MD, Department of Anesthesiology, Washington University School of Medicine, 660 S Euclid, St Louis, MO 63110, USA. E-mail [email protected] Received 15 November 2008; revised 22 December 2008; accepted 23 December 2008 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 89 May 2009 549 Hepatocyte autophagy in sepsis E Watanabe et al pyogenes9 and Mycobacterium tuberculosis.10 Although au- three had primary liver neoplasms (hepatic adenoma, tophagy is generally considered to be an adaptive mechanism, hepatocellular carcinoma, and hemangioma) and one had unbridled autophagy may result in cell death distinct from metastatic breast carcinoma. Samples from these control necrosis or classical apoptosis.11 patients were obtained from the uninvolved margins of the A particularly profound cell stress occurs during sepsis, the resected liver segments. None of the control patients received host response that results during severe infection.12 Sepsis is chemotherapy or radiation therapy in the 2 months before characterized by a constellation of signs and symptoms that resection. The protocol for tissue harvesting was approved by often include fever, hypotension, increased muscle protein the Human Studies Committee at University of Washington breakdown (cachexia), hypermetabolism, and the develop- School of Medicine. ment of multiple organ dysfunction.13 Given that autophagy is a well-established cellular response to stress, we hypothe- sized that autophagy would be accelerated during sepsis. The Mouse Cecal Ligation and Puncture Model of Sepsis and purpose of the present study was to determine the extent of Tissue Sampling hepatic autophagy during severe sepsis. The liver was elected Male C57BL/6J mice aged 6–8 weeks, 22–26 g body weight, as an organ of interest because autophagy has been described were purchased from Jackson Laboratories (Bar Harbor, ME, in this organ5 and because of reports implicating autophagy USA). Mice were housed for at least 1 week before utilization. in hepatocytes during animal models of sepsis.14 Liver CLP or sham surgery was performed by the methods of Baker specimens were removed rapidly post mortem from patients et al15 as previously described. Anesthesia was induced with who died of sepsis or from patients having elective liver 5% isoflurane followed by maintenance with 2% isoflurane. resections and were evaluated by electron microscopy (EM). A small midline abdominal incision was performed, and the The findings in patients were compared to results obtained in cecum was exteriorized and ligated immediately distal to the a clinically relevant mouse peritonitis model of sepsis, ie, ileocecal valve, in a manner that did not result in intestinal the cecal ligation and puncture (CLP) model.15 In animals, obstruction. The cecum was then punctured two times with a the spleen was also examined because of the documented 25-gauge hollow-bore needle. The abdominal wall was closed increase in apoptotic death in the spleen during sepsis16,17 in two layers and each animal received a subcutaneous and the link between apoptosis and autophagy.18–20 Organ- injection of 2 ml 0.9% NaCl to compensate for third-space and cell-type-specific quantitative RT-PCR (qRT-PCR) was fluid loss. Sham mice were treated identically except that the performed in mouse liver, CD4 þ and B splenocytes 8 and cecum was neither ligated nor punctured. Tissue samples 18 h after sepsis or sham surgery to determine whether were obtained 24 h after surgery. All experiments were con- known mediators of autophagic and apoptotic cell death ducted in accordance with the National Institutes of Health were transcriptionally regulated. Guidelines for the Care and Use of Laboratory Animals and with approval of the Washington University Animal Studies MATERIALS AND METHODS Committee. Clinical Samples The present work represents a continuation of previous studies from this laboratory in which we reported findings in Transmission Electron Microscopy patients who died of sepsis and multiple organ dysfunction.21 Specimens were excised and immediately immersion-fixed Patients were classified as septic based on one of the three overnight in a modified Karnovsky’s fixative containing 3% following criteria: (1) positive blood, abdominal fluid, or glutaraldehyde and, 2% paraformaldehyde in 1 M sodium tissue cultures for bacteria or fungi; (2) intraoperative evi- cacodylate buffer (pH 7.4). Tissue samples were then rinsed dence of infection, eg, perforated large bowel with peritoneal in 1 M sodium cacodylate buffer, post-fixed in 2% osmium contamination, ischemic bowel with purulent abdominal tetroxide, dehydrated through graded ethanols and propylene fluid; or (3) a histopathologic diagnosis of infection at oxide, and embedded in Embed 812 (Electron Microscopy post-mortem examination (eg, bronchopneumonia, intra- Sciences, Hatfield, PA, USA). Sections were cut 90-nm thick abdominal abscess).