Table 31-2. Impact of apoptosis on immune function

Decreased presentation of antigen to T cells

Decreased macrophage activation and depressed phagocytic capability

Impaired adaptive immune response

Disrupted cross-talk between adaptive and innate immune systems

Release of anti-inflammatory cytokines T-cell anergy

and increased survival in murine models of sepsis,34,35 a finding that has been recapitulated by others.43

The antiapoptotic Bcl-2 family members, of which Bcl-2 is a member, contain four Bcl-2 homology (BH) domains with sequence and structural homology. The hallmark of this protein family is the BH4 domain, a conserved N-terminal helical domain that is required for these proteins to perform their antiapoptotic functions.33 The remaining Bcl-2 family members have proapoptotic activity. These proteins comprise two subfamilies classified by their BH domain composition. A handful of proteins contain BH1-BH3 domains, and the remainder (the largest Bcl-2 subfamily) contains only the BH3 domain. The BH1-BH3 containing family members have been shown to oligomerize and permeabilize the mitochondrial outer membrane, which leads to cytochrome c release and ultimately apoptosis.33 Under basal conditions, these proteins are kept in an inactive reservoir, primarily through interactions with BH4 containing Bcl-2 family members. Although it is accepted that the BH3-only proteins trigger the activation of Bax and Bak, there is currently a controversy regarding the precise mechanism BH3 proteins employ to activate Bax/Bak.44 Regardless of the mechanism, supporters of both models have demonstrated that Bim, Bid, and p53upregulated modulator of apoptosis (PUMA) are the most potent BH-3 only proteins.33

In CLP, knockout of Bim prevented apoptotic loss of lymphocytes in sepsis and improved survival, whereas knockout of PUMA provided modest protection against apoptosis but no survival advantage.39 Bid is unique among the BH3-only proteins in that it mediates cross-talk between the extrinsic and intrinsic pathways. Caspase-8 cleaves Bid into truncated (t)Bid, which reinforces death receptor signaling by activating the intrinsic apoptotic pathway. Knockout of Bid in mice rendered lymphocytes resistant to sepsis-induced apoptosis and increased survival.45

Examination of the apoptotic pathways in sepsis reveals that there is no single mediator or pathway that is responsible for lymphocyte apoptosis. Indeed, these

data reveal that lymphocytes have extensive apoptotic machinery and that sepsis creates an environment rich in death stimuli that can activate these processes. The type of cell, its activation state and phase of the cell cycle, and type of microorganism all appear to influence whether a lymphocyte will undergo apoptosis. On the basis of findings in animal models of sepsis, the BCL-2 family plays a central role in sepsis-induced apoptosis.

5. THE EFFECT OF APOPTOSIS ON THE IMMUNE SYSTEM

Lymphocyte apoptosis may play a role in the evolution of the inflammatory response that is seen in septic patients by modulating cellular function directly and also by disrupting the network of immune cell interactions that are necessary to mount an effective immune response (Table 31-2).

5.1. Cellular effects of an increased apoptotic burdens

Apoptotic cells themselves are immunosuppressive. Macrophages and dendritic cells that take up and eliminate apoptotic cells release anti-inflammatory (Th2inducing) cytokines such as IL-10 and transforming growth factor beta (TGF-β) and suppress proinflammatory cytokines.46 T cells that come into contact with these macrophages and dendritic cells become anergized or undergo apoptosis themselves. A systemic burden of apoptotic cells has been found to worsen survival in septic mice. Mice receiving adoptive transfer of apoptotic lymphocytes before CLP have worse survival in sepsis, whereas transfer of necrotic cells improves survival.47 This differential may be due in part to how macrophages clear cellular debris and the resulting effect on IFN- γ production; phagocytic uptake of apoptotic cells by macrophages leads to a decrease IFN-γ production, whereas uptake of necrotic cells increases it.

5.2. Network effects of selective loss of immune cell types

Apoptosis of dendritic cells cripples the innate immune system by reducing the capacity to process and present antigen to the adaptive system. Loss of T and B cells disrupts the adaptive immune response and impairs the communication between the adaptive and innate arms of the immune system. The role of the adaptive immune system in sepsis has been described in studies using mice that lack T and B cells (Rag 1−/– mice). Adoptive transfer of lymphocytes over-expressing Bcl-2 to Rag−/– mice attenuates mortality owing to

sepsis.48 Survival of immune cells in apoptosis improves survival in sepsis and may also promote the survival of other types of cells, such as neutrophils.43 Adoptive transfer of myeloid (CD11b+) cells from transgenic mice over-expressing Bcl-2 in myeloid cells was found to improve survival, decrease gut epithelial apoptosis, and increase peritoneal neutrophil counts in Rag 1−/– mice when compared with Rag 1−/– receiving cells from wild-type C57/BL6 mice. These data suggest that survival of lymphocytes and myeloid cells may exert a “bystander” effect by releasing some sort of cytoprotective molecule that inhibits apoptosis of gut epithelium and other immune effector cells.

5.3. Studies of immunomodulation by apoptotic cells in other fields

The impact of apoptotic cells in sepsis is consistent with data from the transplant literature. The immunomodulatory effects of apoptotic cells were determined in a study using infusion of apoptotic cells in mice that later received bone marrow transplants.49 The investigators found that apoptotic cell infusion caused a TGF-β dependent T-regulatory cell expansion, which in turn caused immunosuppressive effects via a cell contact mediated mechanism. In their model, the downregulation of the immune system had beneficial effects of decreasing graft-versus-host disease and increasing engraftment of donor bone marrow cells. Although beneficial in the case of bone marrow transplant, in an animal model of sepsis or in patients dying from sepsis, downregulation of the immune system could be catastrophic.

6. DEVELOPING THERAPIES TO AMELIORATE

SEPSIS-INDUCED LYMPHOCYTE APOPTOSIS

As discussed previously, research over the last decade has established the important role that immune cell apoptosis plays in the response to sepsis. These findings have encouraged the development and application of several technologies to modulate apoptosis. Two different approaches to antiapoptotic therapies are under investigation – rational therapies using biologic agents (RNA, peptides, cytokines, and proteins) and screeningbased discovery of novel cytoprotective agents. Rational therapies have been based on the knockout and transgenic mice that have well-documented survival advantages in animal models of sepsis.

Therapeutic recapitulation of the survival advantage observed in transgenic mice has required detailed molecular mechanistic understanding of apoptosis

pathways. For example, mice that over-express Bcl-2 or Bcl-xL in lymphocytes have a profound survival advantage in sepsis. Bcl-2 and Bcl-xL are cytoplasmic proteins that undergo BH4 domain-dependent trafficking from the endoplasmic reticulum to the mitochondrial surface with the help of FKBP38.50 Unfortunately, these proteins and their BH4 effector domain are unable to cross cell membranes, making them unlikely therapeutic candidates.

However, intracellular delivery of membrane-imper- meant materials was enabled by the discovery of cell permeation peptides including the Antennapedia homeodomain peptide and a short, polycationic peptide from the HIV protein Tat.51 Previous work revealed that Tat-mediated delivery of the BH4 domain of either Bcl-2 or Bcl-xL could protect cells from diverse apoptogenic stimuli, including chemotherapy,52 ischemiareperfusion injury,53,54 and radiation injury.55 Administration of antiapoptotic proteins such as BCL-2 and BCL-xl or their BH4 domains conjugated to the Tat peptide has been shown to be effective in preventing sepsisinduced lymphocyte apoptosis.56

Therapies aimed at recapitulating the phenotypes of knockout mice require inhibiting the synthesis of proapoptotic proteins. Small interfering RNA is one exciting modality that has been shown to be effective in preventing lymphocyte apoptosis and improving survival in sepsis models. siRNA to Fas, caspase-8, and Bim have all been reported to decrease sepsis-induced apoptosis and mortality after CLP.57,58 Although the basic principles of RNA interference are well worked out, clinical applications will require additional technology. Most importantly, tissue-specific delivery agents that target the biodistribution and uptake of therapeutic nucleic acids must be developed to prevent undesirable side effects. At the writing of this chapter, two approaches appear to hold promise, biologic targeting via ligand (e.g., transferrin59,60) or antibody (e.g., anti-CD461) and materials-based targeting using different polymer compositions.62,63

An alternative approach to rational biologic therapy is to develop pharmacological (small-molecule) modulators of essential apoptosis proteins. Previous work has shown that small-molecule caspase inhibitors can reduce sepsis-induced apoptosis48; however, these molecules have not entered clinical study. One potential difficulty with caspase inhibition to prevent apoptosis is that caspases have recently been found to play a key role in the cell cycle.64 Another potential challenge is that once executioner caspases are activated, cells have accumulated enough damage to no longer respond appropriately to their environment.

Cytoprotection can be accomplished in other ways besides caspase activation; however, with the exception of Bcl-2 family members and FADD, molecular mechanisms that contravene sepsis-induced lymphocyte apoptosis are not well-established. Instead of targeting a single enzyme or protein–protein interaction, high throughput screening of compound libraries using a cell phenotype assay can be employed to discover novel cytoprotective agents that can serve as lead compounds for drug development.

Immunotherapy is also emerging as a potential therapeutic strategy for the treatment of sepsis. Administration of cytokines, such as IL-7 or IL-15, or antibodies to negative co-stimulatory molecules (Programmed Death receptor-1) have been shown to prevent apoptosis, reverse immunosupression, and improve survival in mice after CLP. Through modulation of BCL-2 and the prevention of lymphocyte apoptosis, these therapies may improve immune dysfunction seen in sepsis. 65,66,67

Although encouraging preclinical data have been developed for peptide, RNA, and small-molecule therapies, translating these findings from the laboratory to the clinic remains a significant challenge. It is likely that at least three key areas will need further research before clinical trials of antiapoptotic therapies. First, further development of lead compounds will need to be pursued. Second, we need a better understanding of how sepsis alters drug metabolism, pharmacokinetics, and pharmacodynamics. And finally, any antiapoptotic agent, even if administered a single time, has the theoretical risk of facilitating cellular transformation. Therefore, there will need to be a demonstration that shortterm treatment with a candidate antiapoptotic therapy does not significantly increase the risk of cancer.

7. CONCLUSION

From premature infants to elderly patients requiring elective surgeries, the risk of sepsis continues to be a significant cause of morbidity and mortality around the world. Over the last decade, our understanding of the pathophysiology of sepsis has grown tremendously. Most patients dying from sepsis are now thought to die during a state of “immunoparalysis,” not the initial inflammatory response. One of the key findings in the pathophysiology of sepsis has been the connection between immune effector cell apoptosis and prolonged immune suppression. Several research groups have reported dramatic improvements in survival in animal models of sepsis when apoptosis is prevented. These findings represent a significant impetus for pursuing clinical trials with antiapoptotic, immuno-

supportive agents to determine whether prevention of sepsis-induced apoptosis can change the course of disease in human patients. Hopefully, strategies to block sepsis-induced apoptosis will able to decrease the morbidity and mortality from this highly lethal disorder.

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