Cynthia L. Tannahill

Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor α (TNFα) and abrogates endotoxin-induced lethality

Excessive tumor necrosis factor α (TNFα) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNFα production rely on monoclonal antibodies or immunoadhesins that bind TNFα and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNFα processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 μg/mL endotoxin, GM-6001 at concentrations <5 μg/mL blocked release of TNFα, but did not affect the release of either IL-1β or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNFα. To confirm the role of secreted TNFα in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 μg/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNFα response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1β and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNFα processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNFα both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

TNF-α Receptor Signaling and IL-10 Gene Therapy Regulate the Innate and Humoral Immune Responses to Recombinant Adenovirus in the Lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-α and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. β-Galactosidase expression in mice receiving intratracheal instillation of Adv/β-gal (adenovirus construct expressing β-galactosidase) was transient (less than 14 days), but a significant early increase of β-galactosidase expression was seen in mice lacking either or both TNF-α receptors. Absence of TNF-α or the p55 receptor significantly attenuated the Ab response to both adenovirus and β-galactosidase. Human IL-10 expression in the lung suppressed local TNF-α production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with β-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-α signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with β-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with β-galactosidase, and is nonimmunogenic in the lung.

Increased Survival in Sepsis by In Vivo Adenovirus-Induced Expression of IL-10 in Dendritic Cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (105) yields only local expression, while transduction with higher particle numbers (107 and 1010) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (105) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.

Stored Packed Red Blood Cell Transfusion Up-regulates Inflammatory Gene Expression in Circulating Leukocytes

Summary Background Data:The transfusion of more than 6 units of packed red blood cells (PRBCs) within the first 12 hours of injury is the strongest independent predictor of multiple organ failure (MOF). This suggests that stored blood contains bioactive factors that may modify the immunoinflammatory response. Methods:To simulate postinjury major transfusions ex vivo, we obtained whole blood from 4 healthy adults and divided it into four 7-mL groups (I–IV). Group I was not diluted. Group II had 7 mL of 0.9% sterile saline (SS) added. Group III received 3.5 mL each of leuko-reduced stored PRBC and SS (simulating a major transfusion). Group IV received 3.5 mL each of SS and a hemoglobin-based oxygen carrier (PolyHeme) to evaluate the effects of hemoglobin alone. The hemoglobin content in groups III and IV was measured to be equal. Total leukocyte RNA was purified, and its gene array profiles were obtained. Results:Of the 56,475 oligonucleotide probe sets interrogated, 415 were statistically different (P < 0.001). Fourteen of the 415 probe sets were inflammatory-related. The PRBC group had a significantly different expression profile compared with the others and included up-regulation of the interleukin-8, toll-like receptor 4, cryropyrin, prostaglandin-endoperoxide synthase-2, and heparinase genes. Conclusions:PRBCs activate inflammatory genes in circulating leukocytes, which may be central to the pathogenesis of the adverse inflammatory responses that lead to postinjury MOF.

Adenoviral Delivery of Human and Viral IL-10 in Murine Sepsis

Adenovirus (Ad) gene therapy has been proposed as a drug-delivery system for the targeted administration of protein-based therapies, including growth factors and biological response modifiers. However, inflammation associated with Ad transduction has raised concern about its safety and efficacy in acute inflammatory diseases. In the present report, intratracheal and i.v. administration of a first-generation adenoviral recombinant (E1,E3 deleted) either containing an empty cassette or expressing the anti-inflammatory cytokines viral or human IL-10 (IL-10) was administered to mice subjected to zymosan-induced multisystem organ failure or to acute necrotizing pancreatitis. Pretreatment of mice with the intratracheal instillation of Ad expressing human IL-10 or viral IL-10 reduced weight loss, attenuated the proinflammatory cytokine response, and reduced mortality in the zymosan-induced model, whereas pretreatment with a control adenoviral recombinant did not significantly exacerbate the response. Pretreatment of mice with pancreatitis using adenoviral vectors expressing IL-10 significantly reduced the degree of pancreatic and liver injury and liver inflammation when administered systemically, but not intratracheally. We conclude that adenoviral vectors can be administered prophylactically in acute inflammatory syndromes, and expression of the anti-inflammatory protein IL-10 can be used to suppress the underlying inflammatory process.

Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

BACKGROUND Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-α

Dendritic cells (DCs) play a key role in the interface between the innate and acquired immune systems. In response to both exogenous as well as endogenous signals, DCs undergo a programmed maturation to become an efficient, antigen-presenting cell. Yet little is known regarding the differential responses by endogenous versus exogenous stimuli on DC maturation. In the present report, we have compared the phenotypic, functional, and genome-wide expression responses associated with maturation by bone marrow derived DCs to either an endogenous danger signal, tumor necrosis factor-alpha (TNF-alpha), or a microbial product, bacterial lipopolysaccharide (LPS). Examination of the cell surface expression of DCs as well as cytokine production demonstrated that patterns of DC maturation varied dramatically depending upon the stimulus. Whereas LPS was highly effective in terms of inducing phenotypic and functional maturation, TNF-alpha exposure produced a phenotypically distinct DC. Gene expression patterns in DCs 6 and 24 h after LPS and TNF-alpha exposure revealed that these activation signals produce fundamentally different genomic responses. Supervised analysis revealed that the expression of 929 probe sets discriminated among the treatment groups, and the patterns of gene expression in TNF-alpha stimulated DCs were more similar to unstimulated cells at both 6 and 24 h post-stimulation than to LPS-stimulated cells at the same time points. These findings reveal that DCs are capable of a varying phenotypic response to different antigens and endogenous signals.

Discordant tumor necrosis factor-α superfamily gene expression in bacterial peritonitis and endotoxemic shock

BACKGROUND Tumor necrosis factor-alpha (TNF-alpha) is a member of a large family of predominantly homotrimeric type II membrane-associated proteins with both proinflammatory and apoptosis-inducing properties. Although TNF-alpha expression has been studied extensively, little is known about the expression of other members of the TNF-alpha superfamily during acute inflammatory processes. METHODS TNF-alpha, Fas ligand (FasL), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) messenger RNA (mRNA) expression were examined in liver, lung, spleen, and kidney after either a cecal ligation and puncture or endotoxemic shock with use of semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS Cecal ligation and puncture increased TNF-alpha mRNA in lung and liver (both P < .05) within 3 hours, which was paralleled by increased FasL mRNA. In the spleen TNF-alpha and FasL mRNA significantly declined (both P < .05). In contrast to TNF-alpha and FasL, TRAIL mRNA levels were unchanged in all organs except lung, where it was reduced at 24 hours (P < .05). Endotoxemic shock also increased lung TNF-alpha and FasL mRNA levels (both P < .05). CONCLUSIONS In acute inflammatory processes TNF-alpha and FasL mRNA increase concordantly in several solid organs. In contrast, TRAIL mRNA levels do not consistently change during these acute inflammatory processes, suggesting that its expression is under independent and discordant regulatory control.

Induction and immunolocalization of manganese superoxide dismutase in acute acetic acid-induced colitis in the rat

BACKGROUND & AIMS Oxygen radicals and reactive oxygen species play an important role in inflammatory episodes in the bowel. Nonetheless, little is known about the regulation of colonic superoxide dismutases and key antioxidant enzymes with cytoprotective and radical detoxifying properties. The aim of this study was to examine the regulation of manganese superoxide dismutase (SOD) in acute acetic acid-induced colitis. METHODS Colitis was induced in adult rats by the rectal administration of 5% acetic acid. Total RNA and protein were isolated from the inflamed colon from 1 to 24 hours after the induction of colitis. MnSOD messenger RNA and protein levels were evaluated by Northern and Western analyses, respectively. MnSOD protein was localized in cross sections of the colon by immunocytochemistry. RESULTS MnSOD messenger RNA levels showed a rapid 14-96-fold induction in response to acetic acid administration. Western analysis showed a 22-49-fold induction in MnSOD protein levels. Immunocytochemistry showed induction of MnSOD protein, specifically in smooth muscle cells, epithelial cells at the base of the glands, and myenteric plexus neurons. CONCLUSIONS MnSOD messenger RNA and protein levels are rapidly induced following the inflammatory insult, implicating a role for MnSOD in the acute phase of colonic inflammation. We suggest that induction of MnSOD in specific cell types may have a cytoprotective function.