Eizo Watanabe

IL-7 Promotes T Cell Viability, Trafficking, and Functionality and Improves Survival in Sepsis

Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-γ production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-γ– and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.

Mechanisms of Cardiac and Renal Dysfunction in Patients Dying of Sepsis

RATIONALE The mechanistic basis for cardiac and renal dysfunction in sepsis is unknown. In particular, the degree and type of cell death is undefined. OBJECTIVES To evaluate the degree of sepsis-induced cardiomyocyte and renal tubular cell injury and death. METHODS Light and electron microscopy and immunohistochemical staining for markers of cellular injury and stress, including connexin-43 and kidney-injury-molecule-1 (Kim-1), were used in this study. MEASUREMENTS AND MAIN RESULTS Rapid postmortem cardiac and renal harvest was performed in 44 septic patients. Control hearts were obtained from 12 transplant and 13 brain-dead patients. Control kidneys were obtained from 20 trauma patients and eight patients with cancer. Immunohistochemistry demonstrated low levels of apoptotic cardiomyocytes (<1-2 cells per thousand) in septic and control subjects and revealed redistribution of connexin-43 to lateral membranes in sepsis (P < 0.020). Electron microscopy showed hydropic mitochondria only in septic specimens, whereas mitochondrial membrane injury and autophagolysosomes were present equally in control and septic specimens. Control kidneys appeared relatively normal by light microscopy; 3 of 20 specimens showed focal injury in approximately 1% of renal cortical tubules. Conversely, focal acute tubular injury was present in 78% of septic kidneys, occurring in 10.3 ± 9.5% and 32.3 ± 17.8% of corticomedullary-junction tubules by conventional light microscopy and Kim-1 immunostains, respectively (P < 0.01). Electron microscopy revealed increased tubular injury in sepsis, including hydropic mitochondria and increased autophagosomes. CONCLUSIONS Cell death is rare in sepsis-induced cardiac dysfunction, but cardiomyocyte injury occurs. Renal tubular injury is common in sepsis but presents focally; most renal tubular cells appear normal. The degree of cell injury and death does not account for severity of sepsis-induced organ dysfunction.

Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

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 μm2) 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 (P<0.001). 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=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.

Management of intra-abdominal hypertension in patients with severe acute pancreatitis with continuous hemodiafiltration using a polymethyl methacrylate membrane hemofilter

Abstract:  To evaluate, with a prospective observational study, whether continuous hemodiafiltration using a polymethyl methacrylate membrane hemofilter (PMMA‐CHDF) is effective for prevention and treatment of  intra‐abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) on patients with severe acute pancreatitis (SAP). The study was carried out in the general intensive care unit (ICU) of a university hospital. Seventeen consecutive patients with SAP were treated in the intensive care unit and underwent PMMA‐CHDF whether or not they had renal failure. Blood level of interleukin (IL)‐6, as an indicator of cytokine network activation, and intra‐abdominal pressure (IAP) were measured daily to investigate their time‐course of changes and the correlation between the two. The blood level of IL‐6 was high at 1350 ± 1540 pg/mL on admission to the ICU. However, it significantly decreased to 679 ± 594 pg/mL 24 h after initiation of PMMA‐CHDF (P < 0.05), and thereafter decreased rapidly. Mean intra‐abdominal pressure (IAP) on admission was high, at 14.6 ± 5.3 mm Hg, with an IAP of 20 mm Hg or over in 2 of 17 patients, showing that they had already developed IAH. The IAP was significantly lower (P < 0.05) 24 h after initiation of PMMA‐CHDF, and subsequently decreased. There was a significant positive correlation between blood level of IL‐6 and IAP, suggesting that PMMA‐CHDF improved vascular permeability through elimination of cytokines, and that it thereby decreased interstitial edema to lower IAP. Sixteen of the 17 patients were discharged from the hospital in remission from SAP without development of complications. Continuous hemodiafiltration using a polymethyl methacrylate membrane hemofilter appears to be effective for prevention and treatment of IAH in patients with SAP through the removal of causative cytokines of hyperpermeability.

Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis

IntroductionIt is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis.MethodsWe examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes.ResultsAutophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality.ConclusionsAll autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals.

Mechanisms and consequences of ebolavirus-induced lymphocyte apoptosis.

Ebolavirus (EBOV) is a member of the filovirus family and causes severe hemorrhagic fever, resulting in death in up to 90% of infected humans. EBOV infection induces massive bystander lymphocyte apoptosis; however, neither the cellular apoptotic pathway(s) nor the systemic implications of lymphocyte apoptosis in EBOV infection are known. In this study, we show data suggesting that EBOV-induced lymphocyte apoptosis in vivo occurs via both the death receptor (extrinsic) and mitochondrial (intrinsic) pathways, as both Fas-associated death domain dominant negative transgenic mice and mice overexpressing bcl-2 were resistant to EBOV-induced lymphocyte apoptosis. Surprisingly, inhibiting lymphocyte apoptosis during EBOV infection did not result in improved animal survival. Furthermore, we show for the first time that hepatocyte apoptosis likely occurs in EBOV infection, and that mice lacking the proapoptotic genes Bim and Bid had reduced hepatocyte apoptosis and liver enzyme levels postinfection. Collectively, these data suggest that EBOV induces multiple proapoptotic stimuli and that blocking lymphocyte apoptosis is not sufficient to improve survival in EBOV infection. These data suggest that hepatocyte apoptosis may play a role in the pathogenesis of EBOV infection, whereas lymphocyte apoptosis appears to be nonessential for EBOV disease progression.

Targeted delivery of siRNA to cell death proteins in sepsis

Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder.

Cytokine-related Genotypic Differences in Peak Interleukin-6 Blood Levels of Patients with Sirs and Septic Complications

BACKGROUND The aim of the present study was to investigate whether tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6-related genotypic differences affect IL-6 blood levels in patients with systemic inflammatory response syndrome (SIRS) in an intensive care unit (ICU). METHODS Seven polymorphisms of TNF, IL-1, and IL-6-related polymorphisms were studied with an allele-specific polymerase chain reaction. One hundred and thirteen patients diagnosed with SIRS whose sequential organ failure assessment scores were > or =5 at the time when their daily measured IL-6 blood level peaked during the ICU stay (IL-6 max) were examined. IL-6 max, survival, and septic complications were compared between carriers and non-carriers of less frequent alleles, indicated as allele*2, in each polymorphism. RESULTS In single nucleotide polymorphism (SNP) at position -238 site of TNF-alpha (TNF-alpha-238*G/A), IL-6-596*G/A, and IL-6-174*C/T, allele*2 frequencies were much lower in the Japanese than in the Caucasian population. IL-6 max was significantly higher in allele*2 carriers of IL-1beta-511*C/T. Associations were found between susceptibility to septic shock and allele*2 carriage for both IL-1beta-511*C/T and TNF-alpha-308*G/A, and also between poor prognosis and allele*2 carriage in both IL-1 receptor antagonist second intron various number of tandem repeats polymorphism (IL-1raRN*1-5) and TNF-alpha-308*G/A. IL-1beta-511*C/T and IL-1raRN*1-5 were in linkage disequilibrium in this study population. CONCLUSIONS Carriers of less frequent alleles in IL-1-related polymorphisms appear to have significant vulnerability to production of excessive IL-6 blood levels and to deterioration in septic shock.

Treatment of Severe Sepsis and Septic Shock by CHDF Using a PMMA Membrane Hemofilter as a Cytokine Modulator

It has been reported that various types of blood purification intended for the removal of humoral mediators, such as cytokines, were performed in patients with severe sepsis/septic shock. While high-volume hemofiltration, hemofiltration using high cut-off membrane filters, and direct hemoperfusion with a polymyxin-B immobilized column are widely used in the treatment of severe sepsis/septic shock, we perform continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF), which shows an excellent cytokine-adsorbing capacity, for the treatment of severe sepsis/septic shock. In our previous study, it was found that PMMA-CHDF could efficiently remove various pro-inflammatory cytokines such as TNFalpha, IL-6 and IL-8 from the bloodstream, resulting in early recovery from septic shock. Furthermore, PMMA-CHDF could remove anti-inflammatory cytokines such as IL-10 from bloodstream, suggesting that it might improve immunoparalysis as well. These findings suggest that PMMA-CHDF is useful for the treatment of patients with severe sepsis/septic shock as a cytokine modulator.

Association between lymphotoxin-α (tumor necrosis factor-β) intron polymorphism and predisposition to severe sepsis is modified by gender and age.

Objective:To investigate the significance of functional polymorphisms of inflammatory response genes by analysis of a large population of patients, both with and without severe sepsis, and representative of the diverse populations (geographic diversity, physician diversity, clinical treatment diversity) that would be encountered in critical care clinical practice. Design:Collaborative case-control study conducted from July 2001 to December 2005. Setting:A heterogeneous population of patients from 12 U.S. intensive care units represented by the Genetic Predisposition to Severe Sepsis archive. Patients:A total of 854 patients with severe sepsis and an equal number of mortality, age, gender, and race-matched patients also admitted to the intensive care unit without evidence of any infection (matched nonseptic controls). Measurements and Main Results:We developed assays for six functional single nucleotide polymorphisms present before the first codon of tumor necrosis factor at −308, IL1B at −511, IL6 at −174, IL10 at −819, and CD14 at −159, and in the first intron of LTA (also known as tumor necrosis factor-B) at +252 (LTA[+252]). The Project IMPACT critical care clinical database information management system developed by the Society of Critical Care Medicine and managed by Tri-Analytics and Cerner Corporation was utilized. Template-directed dye-terminator incorporation assay with fluorescence polarization detection was used as a high-throughput genotyping strategy. Fifty-three percent of the patients were male with 87.3% and 6.4% of Caucasian and African American racial types, respectively. Overall mortality was 35.1% in both severe sepsis and matched nonseptic control patients group. Average ages (standard deviation) of the severe sepsis and matched nonseptic control patients were 63.0 (16.05) and 65.0 (15.58) yrs old, respectively. Among the six single nucleotide polymorphisms, LTA (+252) was most overrepresented in the septic patient group (% severe sepsis; AA 45.6: AG 51.1: GG 56.7, p = .005). Furthermore, the genetic risk effect was most pronounced in males, age >60 yrs (p = .005). Conclusions:LTA(+252) may influence predisposition to severe sepsis, a predisposition that is modulated by gender and age. Although the genetic influences can be overwhelmed by both comorbid factors and acute illness in individual cases, population studies suggest that this is an influential biological pathway modulating risk of critical illnesses.