Caroline Oberholzer

Sepsis syndromes : Understanding the role of innate and acquired immunity

ABSTRACT— An intact innate and acquired immune response are essential for defeating systemic microbial infections. Recognition molecules, inflammatory cells, and the cytokines they produce are the principal means for host tissues to recognize invading microbes and to initiate intercellular communication between the innate and acquired immune systems. However, activation of host innate immunity may also occur in the absence of microbial recognition, through expression of internal “danger” signals produced by tissue ischemia and necrosis. When activation of the innate immune system is severe enough, the host response itself can propel the patient into a systemic inflammatory response syndrome (SIRS), or even multiple system organ failure (MSOF) and shock. Although most patients survive the initial SIRS insult, these patients remain at increased risk of developing secondary or opportunistic infections because of the frequent onset of a compensatory anti‐inflammatory response syndrome (CARS). The initial activation of the innate immune response often leads to macrophage deactivation, T‐cell anergy, and the rapid apoptotic loss of lymphoid tissues, which all contribute to the development of this CARS syndrome and its associated morbidity and mortality. Initial efforts to treat the septic patient with anticytokine therapies directed at the SIRS response have been disappointing, and therapeutic efforts to modify the immune response during sepsis syndromes will require a more thorough understanding of the innate and acquired immune responses and the increased apoptosis in the lymphoid tissue.

Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug.

Interleukin (IL)-10 is a pleiotropic cytokine produced by both T cells and macrophages and possesses both anti-inflammatory and immunosuppressive properties. IL-10 circulates in the blood of patients with sepsis syndromes, and increased concentrations of IL-10 have been associated with an adverse clinical outcome. Experimental studies in rodents and primates have demonstrated that endogenously produced and exogenously administered IL-10 can reduce the magnitude of the inflammatory response and improve outcome, primarily in models of endotoxemic and bacteremic shock. However, endogenous IL-10 production and systemic administration can also exacerbate T-cell dysfunction, decrease T-cell apoptosis, reduce antimicrobial function, and increase mortality in other less acute bacterial models of sepsis or after thermal injury. Targeted delivery of IL-10 to individual tissues may obviate the adverse effects of systemic delivery. The potential anti-inflammatory properties of IL-10 will have to be carefully weighed against its immunosuppressive properties when considering its use in patients with acute inflammation and sepsis syndromes.

Cytokine signaling--regulation of the immune response in normal and critically ill states.

Cytokines are produced during the activation of innate and acquired immunity, and are the principal means for intercellular communication of a microbial invasion. Cytokines serve to initiate the inflammatory response and to define the magnitude and the nature of the acquired immune response. The response of critically ill patients to their injury and/or invading pathogens is dependent, in large part, on the pattern of cytokines which are produced. The immunologic response of critically ill patients can vary from a strongly proinflammatory response, characterized by increased production of tumor necrosis factor-alpha, interleukin (IL)-1, interferon (IFN)-gamma, and IL-12 to one predominantly of anergy, characterized by increased production of T(H)2 cytokines, like IL-10 and to IL-4. Therapeutic efforts to modify the host immune response in critical illness will require a more thorough understanding of the cytokine milieu and the factors that determine their production.

Molecular Characterization of the Acute Inflammatory Response to Infections with Gram-Negative versus Gram-Positive Bacteria

ABSTRACT Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo with Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1β, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killed S. aureus markedly increased IL-1β and IL-18 levels more than E. coli lipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at the P < 0.001 level whose expression discriminated among gram-negative-organism-stimulated, gram-positive-organism-stimulated, and unstimulated whole-blood leukocytes. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression.

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.

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.

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.

Considering immunomodulatory therapies in the septic patient: should apoptosis be a potential therapeutic target?

Treatment of sepsis and septic shock remains a clinical conundrum. Recent prospective trials with anti-cytokine and anti-inflammatory therapies have shown only modest clinical benefit. The successful treatment of the patient with sepsis syndrome will likely require multi-modal therapies aimed at several of the immunological and physiological disturbances which are occurring simultaneously. Recent studies in experimental animals and critically ill patients have suggested that increased apoptosis of lymphoid organs and some parenchymal tissues may contribute to the immune suppression, anergy and organ system dysfunction. Therapies aimed at inhibiting lymphoid cell apoptosis may contribute to improved outcome, and should be considered in the treatment of hospitalized patients with sepsis syndromes. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis may be an appropriate therapeutic target for the septic patient.

Genetic determinants of lipopolysaccharide and D-galactosamine-mediated hepatocellular apoptosis and lethality.

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent upon endogenously produced TNF-α. Unlike the response to high dose lipopolysaccharide alone, death in this model is a direct result of hepatocyte apoptosis. In a series of recent studies, we have demonstrated that mortality and hepatic injury following lipopolysaccharide administration in D-galactosamine-sensitized mice is dependent upon secreted 17 kDa TNF-α acting primarily through the p55 TNF receptor. Transgenic mice expressing null forms of TNF-α, the p55 receptor, or expressing only a cell-associated form of TNF-α exhibited no mortality and only modest liver injury when challenged with 8 mg of D-galactosamine and 100 ng of lipopolysaccharide. Although Fas ligand expression is increased in the liver, it appeared to play no significant role in outcome, since mice expressing a mutant form of Fas ligand are still sensitive to LPS- and D-galactosamine-induced lethality. Finally, we have seen significant variation in LPS- and D-galactosamine-mediated lethality among different strains of mice. The non-obese diabetic or NOD mouse is highly resistant to LPSand D-galactosamine-induced lethality, and this appears to be secondary to a post-receptor defect in p55 TNF receptor signaling. The studies confirm an essential role for TNF-α and p55 TNF receptor signaling in the hepatocyte apoptosis and lethality associated with lipopolysaccharide and D-galactosamine administration.

Functional modification of dendritic cells with recombinant adenovirus encoding interleukin 10 for the treatment of sepsis.

Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.