Agonistic monoclonal antibody against CD40 receptor decreases lymphocyte apoptosis and improves survival in sepsis

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8- T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-x(L) was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-x(L), provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.     

Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis

In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were marked increases in TNF-alpha, IL-1beta, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-alpha and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1-/- mice, which are totally deficient in mature T and B cells, were also studied. Rag-1-/- mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.     

Enhancement of NF-kappaB activation in lymphocytes prevents T cell apoptosis and improves survival in murine sepsis

Sepsis induces extensive lymphocyte apoptosis that contributes to immunosuppression and mortality. Activation of the canonical NF-kappaB pathway, however, prevents TNF-alpha-induced lymphocyte apoptosis. In this study the function of canonical NF-kappaB in T cells was studied in the context of murine sepsis. Upon cecal ligation and puncture (CLP), NF-kappaB DNA binding activity in thymocytes declines relative to sham-operated mice. This decline in NF-kappaB activity is most likely due to posttranslational modifications such as deacetylation of p65. In parallel, cleavage of procaspase-3 is increased, whereas expression of NF-kappaB-dependent antiapoptotic genes Bcl-xL and c-IAP2 is suppressed upon sepsis induction. Interestingly, adoptive transfer of IkappaBalpha-deficient fetal liver stem cells into sublethally irradiated lymphopenic host mice reduced the decline in thymocyte survival, increased peripheral T cell numbers, and improved the mortality rate relative to wild-type reconstituted hosts after cecal ligation and puncture. In conclusion, lymphocyte-directed augmentation of canonical NF-kappaB ameliorates immunosuppression during murine sepsis. These data provide evidence for a new approach in sepsis therapy.     

Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis

Background

Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.

Methodology/Principal Findings

We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.

Conclusions/Significance

Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.     

Akt regulates cell survival and apoptosis at a postmitochondrial level

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.     

Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival

Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis.     

Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine

Previous studies have shown that the liver is the first organ to display signs of injury during hemorrhagic shock. We examined the mechanism by which pyruvate can prevent liver damage during hemorrhagic shock in swine anesthetized with halothane. Thirty minutes after the induction of a 240-min controlled arterial hemorrhage targeted at 40 mmHg, hypertonic sodium pyruvate (0.5 g. kg(-1). h(-1)) was infused to achieve an arterial concentration of 5 mM. The volume and osmolality effects of pyruvate were matched with 10% saline (HTS) and 0.9% saline (NS). Although the peak hemorrhage volume increased significantly in both the pyruvate and HTS group, only the pyruvate treatment was effective in delaying cardiovascular decompensation. In addition, pyruvate effectively maintained the NADH/NAD redox state, as evidenced by increased microdialysate pyruvate levels and a significantly lower lactate-to-pyruvate ratio. Pyruvate also prevented the loss of intracellular antioxidants (GSH) and a reduction in the GSH-to-GSSG ratio. These beneficial effects on the redox environment decreased hepatic cellular death by apoptosis. Pyruvate significantly increased the ratio of Bcl-Xl (antiapoptotic molecule)/Bax (proapoptotic molecule), prevented the release of cytochrome c from mitochondria, and decreased the fragmentation of caspase 3 and poly(ADP ribose) polymerase (DNA repair enzyme). These beneficial findings indicate that pyruvate infused 30 min after the onset of severe hemorrhagic shock is effective in maintaining the redox environment, preventing the loss of the key antioxidant GSH, and decreasing early apoptosis indicators.     

The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway

A number of viral gene products are capable of triggering apoptotic cell death through interfering with cellular signaling cascades, including the Akt kinase pathway. In this study, the pro-apoptotic role of the SARS-CoV Membrane (M) structural protein is described. We found that the SARS-CoV M protein induced apoptosis in both HEK293T cells and transgenic Drosophila. We further showed that M protein-induced apoptosis involved mitochondrial release of cytochrome c protein, and could be suppressed by caspase inhibitors. Over-expression of M caused a dominant rough-eye phenotype in adult Drosophila. By performing a forward genetic modifier screen, we identified phosphoinositide-dependent kinase-1 (PDK-1) as a dominant suppressor of M-induced apoptotic cell death. Both PDK-1 and Akt kinases play essential roles in the cell survival signaling pathway. Altogether, our data show that SARS-CoV M protein induces apoptosis through the modulation of the cellular Akt pro-survival pathway and mitochondrial cytochrome c release.     

Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing

The pleiotropic cytokine IL-6 has favorable and harmful effects on survival from bacterial infections. Although many innate immune cells produce IL-6, little is known about relevant sources in vivo and the nature of its contributions to host responses to severe bacterial infections. To examine these roles, we subjected mast cell-specific IL-6-deficient mice to the cecal ligation and puncture model of septic peritonitis, finding that survival in these mice is markedly worse than in controls. Following intranasal or i.p. inoculation with Klebsiella pneumoniae, IL-6 (-/-) mice are less likely to survive than wild-type controls and at the time of death have higher numbers of bacteria but not inflammatory cells in lungs and peritoneum. Similarly, mast cell-specific IL-6-deficient mice have diminished survival and higher numbers of K. pneumoniae following i.p. infection. Neutrophils lacking IL-6 have greater numbers of live intracellular K. pneumonia, suggesting impaired intracellular killing contributes to reduced clearance in IL-6(-/-) mice. These results establish that mast cell IL-6 is a critical mediator of survival following K. pneumoniae infection and sepsis and suggest that IL-6 protects from death by augmenting neutrophil killing of bacteria.     

Autophagy and Akt promote survival in glioma

Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.     

Autophagy and Akt promote survival in glioma

Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.     

Alpha-chemokine receptor blockade reduces high mobility group box 1 protein-induced lung inflammation and injury and improves survival in sepsis

High mobility group box 1 (HMGB1) protein, a late mediator of lethality in sepsis, can induce acute inflammatory lung injury. Here, we identify the critical role of alpha-chemokine receptors in the HMGB1-induced inflammatory injury and show that alpha-chemokine receptor inhibition increases survival in sepsis, in a clinically relevant time frame. Intratracheal instillation of recombinant HMGB1 induces a neutrophilic leukocytosis, preceded by alveolar accumulation of the alpha-chemokine macrophage inflammatory protein-2 and accompanied by injury and increased inflammatory potential within the air spaces. To investigate the role of alpha-chemokine receptors in the injury, we instilled recombinant HMGB1 (0.5 microg) directly into the lungs and administered a subcutaneous alpha-chemokine receptor inhibitor, Antileukinate (200 microg). alpha-Chemokine receptor blockade reduced HMGB1-induced inflammatory injury (neutrophils: 2.9 +/- 3.2 vs. 8.1 +/- 2.4 x 10(4) cells; total protein: 120 +/- 48 vs. 311 +/- 129 microg/ml; reactive nitrogen species: 2.3 +/- 0.3 vs. 3.5 +/- 1.3 microM; and macrophage migration inhibitory factor: 6.4 +/- 4.2 vs. 37.4 +/- 15.9 ng/ml) within the bronchoalveolar lavage fluid, indicating that HMGB1-induced inflammation and injury are alpha-chemokine mediated. Because HMGB1 can mediate late septic lethality, we administered Antileukinate to septic mice and observed increased survival (from 58% in controls to 89%) even when the inhibitor treatment was initiated 24 h after the induction of sepsis. These data demonstrate that alpha-chemokine receptor inhibition can reduce HMGB1-induced lung injury and lethality in established sepsis and may provide a novel treatment in this devastating disease.     

SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis

Suppressor of Cytokine Signaling (SOCS) proteins are recently identified inhibitors/regulators of cytokine/growth factor signaling pathways. We have previously shown that SOCS-3 is upregulated in mice after sepsis induced by cecal ligation and puncture; however, the contribution of SOCS-1 to septic morbidity and mortality is unclear. In the present study, we characterized SOCS-1 expression in different tissues and delineated putative mechanisms effecting SOCS-1 expression in thymus from septic mice. We observed no difference in SOCS-1 expression in blood, peritoneal leukocytes, lung, and spleen taken from sham or septic animals at 24 h after surgery. In contrast, SOCS-1 expression in thymus declined significantly after sepsis and this down-regulation of SOCS-1 was associated with increased thymocyte apoptosis as well as augmented Bax recruitment to the mitochondria. Administration of RU-38486, a steroid receptor antagonist, reversed the above effects in the septic thymus. Furthermore, SOCS-1+/− mice showed a significant higher mortality when compared to SOCS-1+/+ mice after sepsis. Together, these results show that sepsis increases steroid-induced thymic lymphoid cell apoptosis, which is associated with reduced SOCS-1 expression and increased Bax translocation to mitochondria. Survival data suggests that SOCS-1 protein may play an important role in sepsis.     

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.     

Inhibition of hydrogen peroxide signaling by 4-hydroxynonenal due to differential regulation of Akt1 and Akt2 contributes to decreases in cell survival and proliferation in hepatocellular carcinoma cells

Dysregulation of cell signaling by electrophiles such as 4-hydroxynonenal (4-HNE) is a key component in the pathogenesis of chronic inflammatory liver disease. Another consequence of inflammation is the perpetuation of oxidative damage by the production of reactive oxidative species such as hydrogen peroxide. Previously, we have demonstrated Akt2 as a direct target of 4-HNE in hepatocellular carcinoma cells. In the present study, we used the hepatocellular carcinoma cell line HepG2 as model to understand the combinatorial effects of 4-HNE and hydrogen peroxide. We demonstrate that 4-HNE inhibits hydrogen peroxide-mediated phosphorylation of Akt1 but not Akt2. Pretreatment of HepG2 cells with 4-HNE prevented hydrogen peroxide stimulation of Akt-dependent phosphorylation of downstream targets and intracellular Akt activity compared with untreated control cells. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment resulted in carbonylation of Akt1, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt1 (rAkt1) with 20 or 40 μΜ 4-HNE inhibited rAkt1 activity by 29 and 60%, respectively. We further demonstrate that 4-HNE activates Erk via a PI3 kinase and PP2A-dependent mechanism leading to increased Jnk phosphorylation. At higher concentrations, 4-HNE decreased both cell survival and proliferation as evidenced by MTT assays and EdU incorporation as well as decreased expression of cyclin D1 and β-catenin, an effect only moderately increased by the addition of hydrogen peroxide. The ability of 4-HNE to exert combinatorial effects on Erk, Jnk, and Akt-dependent cell survival pathways provides additional insight into the mechanisms of cellular damage associated with chronic inflammation.