Apoptosis In Sepsis Research Articles

Inhibition of enterocyte NFκB exacerbates intestinal barrier dysfunction in a murine model of sepsis

Inhibition of enterocyte NFκB augments sepsis‐induced gut apoptosis and increases mortality. It is unknown if enterocyte NFκB contributes to alterations in intestinal barrier function.We hypothesized that inhibition of enterocyte NFκB exacerbates intestinal permeability in sepsis.Mice lacking enterocyte IKKβ (resultant NFκB inhibition) were created via Cre/lox recombination under control of the villin promoter. Wildtype (WT) and IKKβ null (Vil‐Cre/IkkβF/Δ) mice underwent cecal ligation and puncture or sham laparotomy (n=5‐10/group). At 24 hr, intestinal permeability was measured in vivo with FD4. Claudin‐2, occludin, and ZO‐1 were evaluated by qRT‐PCR and fluorescent staining.Septic WT mice had increased permeability compared to shams (495.1±46.5 vs. 297.8±15.7 ng/ml; p<0.01), while septic Vil‐Cre/IkkβF/Δ mice had augmented permeability (665.0±95.1 ng/ml; p<0.01). Claudin‐2 mRNA was increased in septic WT mice compared to shams (1.9±0.3 vs. 1.0±0.2; p<0.05) and septic Vil‐Cre/IkkβF/Δ mice had further increased claudin‐2 (3.22± 0.5; p<0.05). Claudin‐2 staining revealed a similar pattern. There were no differences in occludin or ZO‐1.Inhibition of enterocyte NFκB exacerbates permeability and is associated with parallel changes in claudin‐2. Since inhibition of enterocyte NFκB augments gut apoptosis in sepsis, changes in permeability may be apoptosis‐dependent.

Nanomedicine for the management of lung and blood diseases.

Nanotechnology provides a broad range of opportunities to develop new solutions for clinical problems. For the pulmonary field, nanotechnology promises better delivery of drugs and nucleic acid-based therapeutics to disease sites. Administration of therapeutics via inhalation provides the opportunity for direct delivery to the lung epithelium, the lining of the respiratory tract. By appropriate selection of particle size, deep lung delivery can be obtained with control of phagocytic uptake, the removal of particles by resident macrophages. Nanotechnology can also help in pulmonary therapies administered by intravenous and oral routes through targeting specific cell types and controlling bioavailability and release kinetics. In the hematology field, nanotechnology can counter multiple drug resistance in leukemia by blocking drug efflux from cancer cells, and provide effective delivery of siRNA into lymphocytes to block apoptosis in sepsis. Controlling the surface properties of materials on devices such as valves and stents promises improved biocompatibility by inhibition of thrombosis, the formation of blood clots, and regulating cell adhesion and activation. Nanoparticle-based thrombolytic agents have the potential to improve the effectiveness of clot removal. Treatment of both lung and blood diseases is also likely to benefit from nano-scaffold-based methods for controlling the differentiation and proliferation of stem and progenitor cells.

CD4+lymphocytes control gut epithelial apoptosis and mediate survival in sepsis

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1(-/-) and wild-type (WT) mice. However, Rag-1(-/-) animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1(-/-) mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4(+) but not CD8(+), gammadelta, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1(-/-) mice. Further, adoptively transferring lymphocytes to Rag-1(-/-) mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4(+) lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.

Death to sepsis: targeting apoptosis pathways in sepsis

Sepsis is a significant public health problem and is one of the leading causes of death in critically ill patients admitted to the intensive care unit. The cost, both in terms of lives lost and annual healthcare expenditures, from sepsis is staggering. Unfortunately, despite an increasing understanding of the unique molecular pathobiology of sepsis, mortality has remained more or less stable over the past decade. Moreover, promising therapies in preclinical models of sepsis have universally failed to live up to initial expectations in subsequent clinical trials. Multiple studies have demonstrated that apoptosis plays a major role in the pathobiology of sepsis and acute lung injury, making the apoptosis pathway an attractive target for therapy. Herein, the role of apoptosis in sepsis is briefly discussed, highlighting studies with one potential therapeutic agent targeting the apoptosis pathway.

Lymphocytes, apoptosis and sepsis: making the jump from mice to humans

Sepsis is an important clinical problem with a mortality rate of 20% to 30%. Lymphocyte apoptosis has been recognized as an important step in the pathogenesis of experimental sepsis, by inducing a state of 'immune paralysis' that renders the host vulnerable to invading pathogens. The importance of lymphocyte apoptosis in human disease is now confirmed by Weber and colleagues, who demonstrate extensive apoptosis in circulating lymphocytes from patients with severe sepsis. Weber and colleagues' data set the basis for further studies aimed at modulating lymphocyte apoptosis in sepsis.

Apoptosis in Sepsis: Mechanisms, Clinical Impact and Potential Therapeutic Targets

The inability of present therapies to mitigate the devastating effects of sepsis and multiple organ failure in the critically ill patient indicates that more knowledge of the pathophysiology of sepsis is needed if we are to develop better, more effective interventions. This review will examine the concept that a portion of the immune and organ dysfunctions encountered in the septic rodent/ patient is a reflection of not only the types of cells stimulating/ mediating the apoptotic response, but also the varying capacity of the target cell in a given tissue/ organ to perceive these death receptor stimuli as either an apoptotic, inflammatory and/or necrotic signal. We hope the discussion of such studies provides not only new insight into the pathobiology of sepsis, but also suggests possible therapeutic targets for the management of this devastating condition.

The Endoplasmic Reticulum Stress-Mediated Apoptosis Signal Pathway Is Involved in Sepsis-Induced Abnormal Lymphocyte Apoptosis

Background/Aim: The mechanisms of abnormal lymphocyte apoptosis in sepsis are only partially defined. The present study was designed to investigate whether the endoplasmic reticulum (ER) is implicated in the extensive apoptosis of lymphocytes in sepsis. Methods: C57BL/6 mice were randomized into cecal ligation and puncture (CLP) and sham operation groups. Apoptosis was detected by the TUNEL method and flow cytometry. The expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was detected by RT-PCR and Western blot. The splicing of X box-binding protein-1 (XBP1) mRNA was detected by RT-PCR. Results: A high degree of lymphocyte apoptosis was observed in the CLP group. Marked induction of GRP78 and accumulation of spliced XBP1 mRNA were observed in the splenocytes from septic mice, indicating activation of unfolded protein responses. Furthermore, both CHOP and its mRNA were markedly upregulated in the CLP group, suggesting that the ER stress response switched to a proapoptotic response. Conclusion: These data demonstrate activation of the unfolded protein response in lymphocytes and that ER stress may contribute to abnormal lymphocyte apoptosis during sepsis. Accordingly, the ER stress-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of sepsis-induced lymphocyte apoptosis.

Pro-apoptotic gene regulation of the bcl-2 family during apoptosis in sepsis

Pro-apoptotic gene regulation of the bcl-2 family during apoptosis in sepsis

APOPTOSIS CONTRIBUTES TO SEPTIC CARDIOMYOPATHY AND IS IMPROVED BY SIMVASTATIN THERAPY

Bacterial toxins cause cardiac dysfunction and death through an inflammatory process, but the mechanism remains unclear. Simvastatin is recognized as having anti-inflammatory properties beyond its lipid-lowering effects. We examined Staphylococcus aureus alpha-toxin in isolated heart and in vivo models and tested simvastatin's effects in sepsis. Isolated Langendorff-perfused rat hearts were exposed to a recirculating perfusate containing alpha-toxin (0.5 microg mL(-1)). Compared with controls, there was a significant increase in coronary perfusion pressure and fall in myocardial performance. Significant increases in p53 expression and apoptosis (1.3 +/- 0.5 to 7.1 +/- 1.4 terminal deoxynucleaotidyl transferase nick end labeling-positive cells; P < 0.05) compared with controls were observed, but markers of necrosis were similar. In parallel experiments, anaesthetized rats receiving alpha-toxin (40 microg kg(-1), i.v.) had in vivo hemodynamic parameters and serum markers of necrosis monitored for 4 h before the hearts were analyzed for histological change, p53 expression, and apoptosis. Over 4 h, alpha-toxin exposure produced substantial hemodynamic effects. In addition, p53 expression (0.2 +/- 0.2 to 7.1 +/- 0.5 p53-positive myocytes; P < 0.05), TNF-alpha levels, the degree of apoptosis, and markers of necrosis were all significantly increased compared with control animals. Pretreatment with simvastatin protected against alpha-toxin-induced sepsis associated with reduced p53, TNF-alpha, apoptosis, and necrosis. We found significant changes in systemic hemodynamics, coronary perfusion pressure, myocardial function, and increased p53 expression with apoptosis due to bacterial exotoxin. In vivo changes were significantly inhibited by pretreatment with simvastatin. We provide novel evidence for the mechanisms by which septicemia causes myocardial depression and hint at a potential role for simvastatin as an inhibitor of apoptosis in sepsis.

Low serum α‐tocopherol and selenium are associated with accelerated apoptosis in severe sepsis

During sepsis, a severe systemic disorder, micronutrients often are decreased. Apoptosis is regarded as an important mechanism in the development of often significant immunosuppression in the course of the disease. This study aimed to investigate alpha-tocopherol and selenium in reference to apoptosis in patients with sepsis. 16 patients were enrolled as soon as they fulfilled the criteria of severe sepsis. 10 intensive care patients without sepsis and 11 healthy volunteers served as controls. alpha-Tocopherol, selenium and nucleosomes were measured in serum. Phosphatidylserine externalization and Bcl-2 expression were analyzed in T-cells by flow cytometry. Serum alpha-tocopherol and selenium were decreased in severe sepsis but not in non-septic critically ill patients (p < 0.05). Conversely, markers of apoptosis were increased in sepsis but not in critically ill control patients: Nucleosomes were found to be elevated 3 fold in serum (p < 0.05) and phosphatidylserine was externalized on an expanded subpopulation of T-cells (p < 0.05) while Bcl-2 was expressed at lower levels (p < 0.05). The decrease of micronutrients correlated with markers of accelerated apoptosis. Accelerated apoptosis in sepsis is associated with low alpha-tocopherol and selenium. The results support the investigation of micronutrient supplementation strategies in severe sepsis.

Inhibitor of apoptosis proteins clAP1, clAP2 and xlAP regulate apoptosis in sepsis

Inhibitor of apoptosis proteins clAP1, clAP2 and xlAP regulate apoptosis in sepsis

Platelet Granzyme B expression is increased in sepsis: a potential mechanism for sepsis‐associated endothelial apoptosis

Platelets induce endothelial cell apoptosis via TGFβ1. We hypothesized that platelets are capable of inducing endothelial cell apoptosis in sepsis via up-regulation of other platelet cell death mediators. BALB/c mice were bled 0 and 24h after cecal-ligation and puncture. All mice developed signs of sepsis. PLT cDNA microarrays showed up-regulation of 13 cell death mediators (GO:0008219), particularly a 10-fold up-regulation of pro-apoptotic, Granzyme (Gzm) B (p=0.06), validated by RT-PCR [24h:0h relative expression (mean±SE) = 10.12±0.07, p=0.04]. This correlated well with megakaryocytic mRNA sampled at the same time points (r=0.996). Flow cytometry of whole blood, gated on platelets by forward/side-scatter properties, showed 0h intracellular GzmB expression of 4.4±1.3%. At 24h, 19.6±6.3% of platelets expressed intracellular GzmB(p=0.04). These data show platelets up-regulate GzmB expression in a mouse model of severe sepsis. This supports the hypothesis that platelets are capable of inducing endothelial apoptosis in sepsis. If GzmB-mediated apoptosis is supported by our ongoing functional studies, this would provide a novel target for sepsis therapy.

The Role of TCR Engagement and Activation-Induced Cell Death in Sepsis-Induced T Cell Apoptosis

Sepsis induces extensive apoptosis in T and B cells suggesting that the loss of immune effector cells could be one explanation for the profound immunosuppression observed in this disorder. Unfortunately, the mechanisms responsible for lymphocyte apoptosis in sepsis remain unknown. In T cells, apoptosis can occur through activation-induced cell death (AICD) in which engagement of the Ag receptors by cognate Ag or polyclonal activators such as bacteria-derived superantigens induces activation, proliferation, and apoptosis. We examined whether proliferation and AICD are necessary for apoptotic cell death in sepsis using normal and TCR transgenic mice. Results show that although sepsis resulted in activation of a small percentage of T cells, no proliferation was detected during the first 48 h following onset, a time when extensive apoptosis is observed. We also observed that T cells do not enter the cell cycle, and stimulation via the TCR in TCR transgenic animals does not enhance or decrease cell death in sepsis. Interestingly, T cells recovered from septic mice retained their ability to proliferate and synthesize cytokines albeit at reduced levels. With the exception of IL-10, which was increased in lymphocytes from mice with sepsis, sepsis caused a decrease in the production of both proinflammatory and anti-inflammatory cytokines. We conclude that lymphocyte apoptosis in sepsis does not require proliferation, TCR engagement, or AICD. Thus the immunosuppression observed in sepsis cannot be the result of T cell deletion via the TCR.

TAT-BH4 and TAT-Bcl-xL Peptides Protect against Sepsis-Induced Lymphocyte Apoptosis In Vivo

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells, thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently, novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study, we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis, and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival.

Pulse High‐Volume Hemofiltration in Critically Ill Patients: A New Approach for Patients with Septic Shock

Mortality rates in septic shock remain unacceptably high despite advances in our understanding of the syndrome and its treatment. Humoral factors are increasingly recognized to participate in the pathogenesis of septic shock, giving a biological rationale to therapies that might remove varied and potentially dangerous humoral mediators. While plasma water exchange in the form of hemofiltration can remove circulating cytokines in septic patients, the procedure, as routinely performed, does not have a substantial impact on their plasma levels. More intensive plasma water exchange, as high-volume hemofiltration (HVHF)can reduce levels of these mediators and potentially improve clinical outcomes. However, there are concerns about the feasibility and costs of HVHF as a continuous modality--very high volumes are difficult to maintain over 24 hours and solute kinetics are not optimized by this regimen. We propose pulse HVHF (PHVHF)-HVHF of 85 ml/kg/hr for 6-8 hours followed by continuous venovenous hemofiltration (CVVH) of 35 ml/kg/hr for 16-18 hours-as a new method to combine the advantages of HVHFimprove solute kinetics, and minimize logistic problems. We treated 15 critically ill patients with severe sepsis and septic shock using daily PHVHF in order to evaluate the feasibility of the technique, its effects on hemodynamics, and the impact of the treatment on pathologic apoptosis in sepsis. Hemodynamic improvements were obtained after 6 hours of PHVHF and were maintained subsequently by standard CVVHas demonstrated by the reduction in norepinephrine dose. PHVHFbut not CVVHsignificantly reduces apoptotic plasma activity within 1 hour and the pattern was maintained in the following hours. PHVHF appears to be a feasible modality that may provide the same or greater benefits as HVHFwhile reducing the workload and cost.

B LYMPHOCYTES UNDERGO APOPTOSIS BECAUSE OF FcγRIIb stress response to infection: A novel mechanism of cell death in sepsis

Sepsis is predominantly characterized by proinflammatory signs in its initial phase, but can be also associated with immune suppression that can be a consequence of apoptotic cell death. The role of Fc receptors (FcRs) is poorly understood in this disease, and it was recently shown that, in addition to the promotion of opposite inflammatory responses, they are implicated in apoptosis. Using a model of peritonitis in mice that do not express activating FcRs, we tested the hypothesis that FcgammaRIIb, the only known immunoglobulin G receptor capable of inducing apoptosis, would participate in the induction of this kind of cell death during serious infection. The blocking of this receptor by a monoclonal antibody significantly decreased the number of apoptotic splenic B cells, demonstrating its involvement in apoptosis. FcgammaRIIb-mediated apoptosis was neither the result of increased TNFalpha levels nor was it associated with IL-10 production. Finally, the decreased apoptosis after mice treatment with FcgammaRIIb-blocking antibody was not sufficient to increase its survival. Thus, we conclude that although apoptosis is a multifactorial phenomenon in sepsis, one of these factors is the inhibitory immunoglobulin G receptor FcgammaRIIb. FcgammaRIIb stress response to infection is a novel mechanism that contributes to the comprehension of apoptosis in sepsis.

The role and regulation of apoptosis in sepsis

Today, sepsis continues to be a growing problem in the critically ill patient population. A number of laboratories have been interested in understanding how changes in immune cell apoptosis during sepsis appear to contribute to septic morbidity. Consistently, it has been found that immune cell apoptosis is altered in a variety of tissue sites and cell populations both in experimental animals and humans. While divergent mediators, such as steroids and TNF, contribute to some of these apoptotic changes, their effects are tissue and cell population selective. Inhibition of FasL—Fas signaling (by either FasL gene deficiency, in vivo gene silencing [siRNA] or with FasL binding protein) protects septic mice from the onset of marked apoptosis and the morbidity/mortality seen in sepsis. Further, this extrinsic apoptosis response appears to utilize aspects of the Bid-induced mitochondrial pathway. This is in keeping with the findings that pan-specific caspase inhibition or the overexpression of Bcl-2 also protect these animals from the sequellae of sepsis.

The role and regulation of apoptosis in sepsis

Today, sepsis continues to be a growing problem in the critically ill patient population. A number of laboratories have been interested in understanding how changes in immune cell apoptosis during sepsis appear to contribute to septic morbidity. Consistently, it has been found that immune cell apoptosis is altered in a variety of tissue sites and cell populations both in experimental animals and humans. While divergent mediators, such as steroids and TNF, contribute to some of these apoptotic changes, their effects are tissue and cell population selective. Inhibition of FasL-Fas signaling (by either FasL gene deficiency, in vivo gene silencing [siRNA] or with FasL binding protein) protects septic mice from the onset of marked apoptosis and the morbidity/mortality seen in sepsis. Further, this extrinsic apoptosis response appears to utilize aspects of the Bid-induced mitochondrial pathway. This is in keeping with the findings that pan-specific caspase inhibition or the overexpression of Bcl-2 also protect these animals from the sequellae of sepsis.

Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity.

The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers. Prospective observational cohort study. Tertiary level intensive care unit and associated research laboratory. Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls. Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential. Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol. Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.

Endothelial cell apoptosis in sepsis.

To discuss a potential role for endothelial cell apoptosis in the pathogenesis of sepsis. Studies published in biomedical journals and studies from the authors' laboratory. In vitro and in vivo studies of endothelial cell apoptosis in endotoxin and sepsis models. Relevant studies that investigate the role of apoptosis in endotoxemia and sepsis are presented. The divergent results of the different studies and the potential reasons for the discrepant findings are presented. The importance of apoptosis in sepsis and the potential impact on endothelial cells and organ function are highlighted. Apoptosis is an important mechanism of lymphocyte and gastrointestinal epithelial cell death in sepsis. Although abundant in vitro studies indicate that endothelial cell apoptosis can occur in response to certain pathogenic organisms (e.g., Rickettsia rickettsii), data documenting endothelial cell apoptosis in in vivo models of sepsis are lacking. Because endothelial cells that undergo apoptosis detach from the vessel basement membrane, enter the circulation, and are rapidly cleared, it may be difficult to detect endothelial cell apoptosis in in vivo models of sepsis. The impact of endothelial cell apoptosis in sepsis may either be detrimental or beneficial to host survival, depending on the particular pathogen.