RESUMEN
BACKGROUND: Sepsis remains a leading cause of death in most intensive care units. Many deaths in sepsis are due to nosocomial infections in patients who have entered the immunosuppressive phase of the disorder. One cause of immunosuppression in sepsis is T-cell exhaustion mediated by programmed cell death-1 (PD-1) interaction with its ligand (PD-L1). Studies demonstrated that blocking the interaction of PD-1 with PD-L1 with knockout mice or inhibitory antibodies reversed T-cell dysfunction and improved sepsis survival. This study assessed the efficacy of a novel short-acting peptide (compound 8) that inhibits PD-1:PD-L1 signaling in a clinically relevant second-hit fungal sepsis model. METHODS: Mice underwent cecal ligation and puncture to induce peritonitis. Three days later, mice received intravenous injection of Candida albicans. Forty-eight hours after Candida infection, mice were treated with compound 8 or inactive peptide. The effect of Candida infection on expression of coinhibitory molecules, PD-1, and PD-L1 were quantitated by flow cytometry on CD4+ cells, CD8+ cells, natural killer (NK) cells, and natural killer T-cells (NKT). The effect of compound 8 on survival was also examined. RESULTS: Four days after fungal infection, PD-1 and PD-L1 expressions were markedly increased on CD4+, NK, and NKT cells in septic versus sham-operated mice (%PD-1 on CD4+, 11.9% versus 2.8%; and %PD-L1 on NKT, 14.8% versus 0.5%). Compared with control, compound 8 caused a 2-fold increase in survival from 30% to 60%, P < 0.05. CONCLUSIONS: Compound 8 significantly improved survival in a clinically relevant immunosuppressive model of sepsis. These results support immunoadjuvant therapy targeting T-cell exhaustion in this lethal disease.
Asunto(s)
Antígeno B7-H1/metabolismo , Candidemia/tratamiento farmacológico , Péptidos/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Candidemia/metabolismo , Evaluación Preclínica de Medicamentos , Masculino , Ratones , Péptidos/farmacología , Bazo/metabolismoRESUMEN
Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later, after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was associated with increases in Th1 cytokines TNF-α and IFN-γ in bronchoalveolar lavage fluid and decreased production of the Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed in Bim(-/-) mice. Septic Bim(-/-) mice with cancer also had increased mortality compared with septic WT mice with cancer. These data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia.
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Adenocarcinoma/inmunología , Apoptosis/inmunología , Linfocitos/inmunología , Neoplasias Pancreáticas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Sepsis/inmunología , Adenocarcinoma/genética , Adenocarcinoma/microbiología , Animales , Apoptosis/genética , Citocinas/biosíntesis , Citocinas/inmunología , Humanos , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/microbiología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Infecciones por Pseudomonas/microbiología , Sepsis/genética , Sepsis/microbiologíaRESUMEN
INTRODUCTION: Fungal sepsis is an increasingly common problem in intensive care unit patients.Mortality from fungal sepsis remains high despite antimicrobial therapy that is highly active against most fungal pathogens, a finding consistent with defective host immunity that is present in many patients with disseminated fungemia.One recently recognized immunologic defect that occurs in patients with sepsis is T cell "exhaustion" due to increased expression of programmed cell death -1 (PD-1).This study tested the ability of anti-PD-1 and anti-programmed cell death ligand -1 (anti-PD-L1) antagonistic antibodies to improve survival and reverse sepsis-induced immunosuppression in two mouse models of fungal sepsis. METHODS: Fungal sepsis was induced in mice using two different models of infection, that is, primary fungal sepsis and secondary fungal sepsis occurring after sub-lethal cecal ligation and puncture (CLP).Anti-PD-1 and anti-PD-L1 were administered 24 to 48 h after fungal infection and effects on survival, interferon gamma production, and MHC II expression were examined. RESULTS: Anti-PD-1 and anti-PD-L1 antibodies were highly effective at improving survival in primary and secondary fungal sepsis.Both antibodies reversed sepsis-induced suppression of interferon gamma and increased expression of MHC II on antigen presenting cells.Blockade of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a second negative co-stimulatory molecule that is up-regulated in sepsis and acts like PD-1 to suppress T cell function, also improved survival in fungal sepsis. CONCLUSIONS: Immuno-adjuvant therapy with anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies reverse sepsis-induced immunosuppression and improve survival in fungal sepsis.The present results are consistent with previous studies showing that blockade of PD-1 and CTLA-4 improves survival in bacterial sepsis.Thus, immuno-adjuvant therapy represents a novel approach to sepsis and may have broad applicability in the disorder.Given the relative safety of anti-PD-1 antibody in cancer clinical trials to date, therapy with anti-PD-1 in patients with life-threatening sepsis who have demonstrable immunosuppression should be strongly considered.
Asunto(s)
Anticuerpos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Fungemia/inmunología , Fungemia/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Antígeno B7-H1/inmunología , Antígeno CTLA-4/inmunología , Candidiasis/inmunología , Candidiasis/terapia , Modelos Animales de Enfermedad , Genes MHC Clase I , Antígenos HLA-DR/biosíntesis , Huésped Inmunocomprometido , Interferón gamma/biosíntesis , Masculino , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Análisis de SupervivenciaRESUMEN
We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.
Asunto(s)
Rastreo Celular/métodos , Colorantes Fluorescentes/química , Técnicas de Sonda Molecular , Receptores sigma/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Colorantes Fluorescentes/farmacocinética , Humanos , Leucocitos Mononucleares/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía de Fluorescencia por Excitación Multifotónica , Neoplasias Experimentales/metabolismoRESUMEN
OBJECTIVE: Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals. DESIGN: Prospective, randomized controlled study. SETTING: Animal laboratory in a university medical center. SUBJECTS: C57Bl/6 mice. INTERVENTIONS: Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival. MEASUREMENTS AND MAIN RESULTS: Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice. CONCLUSIONS: When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.
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Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Apoptosis/fisiología , Bacteriemia/mortalidad , Bacteriemia/patología , Modelos Animales de Enfermedad , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa , Sepsis/mortalidad , Sepsis/patología , Animales , Linfocitos B/patología , Línea Celular Tumoral , Comorbilidad , Mucosa Intestinal/patología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Infecciones por Pseudomonas/mortalidad , Linfocitos T/patologíaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Linfocitos T CD4-Positivos/inmunología , Supervivencia Celular , Células Epiteliales/fisiología , Mucosa Intestinal , Sepsis/inmunología , Traslado Adoptivo , Animales , Células Epiteliales/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Bazo/citologíaRESUMEN
Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.
Asunto(s)
Apoptosis/efectos de los fármacos , Péptidos/farmacología , Irradiación Corporal Total , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Transformadores de Poliomavirus/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Señales de Localización Nuclear/farmacología , Estructura Terciaria de Proteína , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteína bcl-X/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
Lymphocyte apoptosis is a hallmark of sepsis and contributes to disease mortality. In other acute injuries, such as myocardial and cerebral ischemia/reperfusion, apoptosis plays a significant role in disease-associated morbidity and mortality. We previously showed that constitutive activation of the potent antiapoptotic Akt/protein kinase B signaling pathway in lymphocytes both reduces sepsis-induced lymphocyte apoptosis and confers a significant survival advantage compared to wild-type littermates. Here, we demonstrate a therapeutic approach to acutely augment Akt activity in a wild-type animal. A cell-permeable peptide conjugated to the Akt-binding domain of the endogenous Akt coactivator, Tcl-1, prolongs Akt activity, activates extracellular regulated kinase (ERK) signaling and protects lymphocytes from numerous apoptotic stimuli both in vitro and in vivo. Molecular approaches to activate the antiapoptotic Akt and ERK signaling pathways may provide a novel tool to study these signaling pathways, as well as a new antiapoptotic strategy for the treatment of sepsis and other acute injuries.
Asunto(s)
Apoptosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Linfocitos/citología , Linfocitos/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/químicaRESUMEN
Sepsis induces widespread lymphocyte apoptosis, resulting in impaired immune defenses and increased morbidity and mortality. There are multiple potential triggers or signaling molecules involved in mediating death signals. Elucidating the specific signaling pathways that are involved in mediating lymphocyte apoptosis may lead to improved therapies of this lethal disorder. We investigated a number of key cellular receptors and intracellular signaling pathways that may be responsible for apoptotic cell death. Specifically, we investigated the role of pathogen-associated molecular patterns (TLR2, TLR4, and IL-1R), intracellular signaling proteins (MyD88 and TRIF), cytoplasmic transcription factors (STAT1 and STAT4), and the MAPK pathway (JNK1) in sepsis-induced lymphocyte apoptosis. Studies were performed in the cecal ligation and puncture (CLP) model of sepsis using specific gene-targeted deletions. CLP-induced lymphocyte apoptosis was evaluated 20 h post-operation by active caspase-3 and TUNEL staining. Surprisingly, the only genetic construct that ameliorated T and B lymphocyte sepsis-induced apoptosis ( approximately 80% and 85%, respectively) occurred in MyD88(-/-) mice. Despite the marked decrease in sepsis-induced apoptosis, MyD88(-/-) mice had a worsened survival. In conclusion, lymphocyte death in sepsis likely involves multiple pathogen-sensing receptors and redundant signaling pathways. MyD88 was effective in blocking apoptosis, as it is essential in mediating most pathogen recognition pathways; however, MyD88 is also critical for host survival in a model of severe peritonitis.
Asunto(s)
Linfocitos B/fisiología , Factor 88 de Diferenciación Mieloide/deficiencia , Sepsis/inmunología , Linfocitos T/fisiología , Animales , Apoptosis , Linfocitos B/inmunología , Caspasa 3/metabolismo , Citocinas/análisis , Muerte , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Linfocitos T/inmunologíaRESUMEN
Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced approximately 40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores sigma/biosíntesis , Arsenicales/química , Caspasas/metabolismo , Endocitosis , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/farmacología , Humanos , Cinética , Ligandos , Microscopía Confocal , Modelos Químicos , Fotones , Receptores sigma/químicaRESUMEN
To assess the degree of lymphocyte apoptosis and survival in mice treated with small interfering RNA (siRNA) targeted to Bim, a proapoptotic molecule from the Bcl-2 family, within a clinically relevant model of sepsis. C57BL/6 mice were treated with a single dose of Bim siRNA complexed in cationic liposomes via tail vein injection. Approximately 24 h later, mice were subjected to either cecal ligation and puncture (CLP) or sham surgery. Animals were killed at 20 h postsurgery, and spleens were harvested for fluorescence-activated cell sorting analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling as a marker for apoptosis. A second cohort of mice was followed for survival for 7 days. The degree of lymphocyte apoptosis in Bim siRNA-treated mice was markedly decreased compared with controls. Fluorescent activated cell sorter analysis demonstrated 13.1% +/- 1.2% B-cell apoptosis and 11.5% +/- 1.5% T-cell apoptosis in control mice compared with 2.7% +/- 0.4% B-cell apoptosis and 3.9% +/- 0.3% T-cell apoptosis in Bim siRNA-treated mice after CLP (P < 0.001 and P < 0.01, respectively). This striking difference in lymphocyte apoptosis correlated with a significant survival advantage in Bim siRNA-treated mice. At 7 days, there was 90% overall survival in Bim siRNA-treated septic mice compared with 50% overall survival in control septic mice (P < 0.05). Treatment with Bim siRNA in vivo has the potential to be an effective therapy in the treatment of sepsis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/genética , Linfocitos/patología , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/fisiología , Sepsis/mortalidad , Sepsis/terapia , Animales , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteína 11 Similar a Bcl2 , Células Cultivadas , Modelos Animales de Enfermedad , Rayos gamma , Humanos , Linfocitos/metabolismo , Linfocitos/efectos de la radiación , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Peritonitis/metabolismo , Peritonitis/mortalidad , Peritonitis/terapia , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/deficiencia , Sepsis/metabolismo , Análisis de SupervivenciaRESUMEN
Lymphocyte apoptosis plays a central role in the pathophysiology of sepsis. Lymphocyte apoptosis was examined in mice with defective death receptor pathways due to transgenic expression of a dominant negative mutant of Fas-associated death domain (FADD-DN) or Bid-/- and in mice with defective mitochondrial-mediated pathways due to loss of Bim-/-, Puma-/-, or Noxa-/-. FADD-DN transgenic and Bid-/- mice had significant albeit incomplete protection, and this protection was associated with increased survival. Surprisingly, splenic B cells were also protected in FADD-DN mice although transgene expression was confined to T cells, providing evidence for an indirect protective mechanism. Bim-/- provided virtually complete protection against lymphocyte apoptosis whereas Puma-/- and Noxa-/- mice had modest or no protection, respectively. Bim-/- mice had improved survival, and adoptive transfer of splenocytes from Bim-/- mice into Rag 1-/- mice demonstrated that this was a lymphocyte intrinsic effect. The improved survival was associated with decreased interleukin (IL) -10 and IL-6 cytokines. Collectively, these data indicate that numerous death stimuli are generated during sepsis, and it therefore appears unlikely that blocking a single "trigger" can inhibit apoptosis. If siRNA becomes practical therapeutically, proapoptotic proteins would be potential targets.
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Apoptosis/fisiología , Linfocitos/citología , Mitocondrias/fisiología , Receptores de Muerte Celular/fisiología , Sepsis/patología , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Citocinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
To determine whether and by which pathway (via the death receptor or mitochondrial mediated pathway) lymphocyte apoptosis occurs in pneumonia and to determine if increased bronchial epithelial cell apoptosis occurs in pneumonia. Prospective randomized study in a university research laboratory. Male C57BL/6 mice (n = 30). Animals received an intratracheal injection of Streptococcus pneumoniae or Pseudomonas aeruginosa to induce gram-positive or gram-negative pneumonia, respectively and were killed 24, 30, or 48 h later. Presence of pneumonia was confirmed via gross visual examination of lungs and by histology. Lymphocyte apoptosis in spleen and thymus was analyzed by flow cytometry for active caspases 3, 8, and 9 and by immunohistochemical (IHC) staining for active caspase 3 and DNA strand breaks. Respiratory epithelial cell apoptosis was assessed by IHC. Histologically, pneumonia was present in all bacteria-treated animals but none in sham-treated mice. Extensive lymphocyte apoptosis in spleen and thymus was documented by characteristic morphological changes on hematoxylin and eosin staining and by IHC staining in both S. pneumonia and P. aeruginosa infection. Flow cytometry confirmed IHC and showed apoptotic lymphocytes positive for active caspases 3, 8, and 9 in both thymi and spleens in both infections. In contrast to the extensive lymphocyte apoptosis, only rare scattered apoptotic changes were seen in respiratory epithelial or endothelial cells in pneumonia due to either organism. Increased lymphocyte but not bronchial cell apoptosis occurs in both gram-positive and gram-negative pneumonia and probably involves both the extrinsic and intrinsic pathway.
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Apoptosis , Infecciones por Bacterias Gramnegativas/patología , Infecciones por Bacterias Grampositivas/patología , Linfocitos/patología , Neumonía Bacteriana/patología , Mucosa Respiratoria/patología , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Daño del ADN , Citometría de Flujo , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Grampositivas/microbiología , Pulmón/microbiología , Pulmón/patología , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Pseudomonas aeruginosa/crecimiento & desarrollo , Bazo/enzimología , Bazo/patología , Streptococcus pneumoniae/crecimiento & desarrollo , Linfocitos T/enzimología , Linfocitos T/metabolismo , Linfocitos T/patología , Timo/enzimología , Timo/patologíaRESUMEN
The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumour cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor-binding site. WC-21, a sigma-2 ligand containing both a photoactive azide moiety and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was identified as PGRMC1 (progesterone receptor membrane component 1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalize with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor-binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.
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Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Receptores de Progesterona/metabolismo , Receptores sigma/metabolismo , Animales , Fluorescencia , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Inmunohistoquímica , Ligandos , Proteínas de la Membrana/genética , Estructura Molecular , Etiquetas de Fotoafinidad/metabolismo , Ratas , Receptores de Progesterona/genéticaRESUMEN
There is increasing recognition that a major pathophysiologic event in sepsis is the progression to an immunosuppressive state in which the host is unable to eradicate invading pathogens. Although there are likely numerous causes for the immunosuppression, expression of negative costimulatory molecules on immune effector cells is a likely contributing factor. PD-1 is a recently described, negative costimulatory molecule that has potent effects to inhibit T cell activation, cytokine production, and cytotoxic functions. PD-1 plays a critical role in the host response to specific pathogens, but relatively little work has been done on the possible effects of PD-1 in sepsis. We hypothesized that the anti-PD-1 antibody would improve survival in sepsis. Mice underwent CLP, and PD-1 expression was quantitated. Additionally, the effects of anti-PD-1 antibody on lymphocyte apoptosis, cytokine production, host immunity, and survival were determined. PD-1 expression increased beginning 48 h after sepsis, and >20% of CD4 and CD8 T cells were positive by 7 days. Anti-PD-1 antibody administered 24 h after sepsis prevented sepsis-induced depletion of lymphocytes and DCs, increased Bcl-xL, blocked apoptosis, and improved survival. Anti-PD-1 also prevented the loss in DTH, a key indicator of immunocompetence in sepsis. Thus, delayed administration of anti-PD-1 antibody, an important therapeutic advantage, was effective in sepsis. Furthermore, these results add to the growing body of evidence that modulation of the positive and negative costimulatory pathways on immune cells represents a viable therapeutic approach in reversing immunosuppression and improving sepsis survival.
Asunto(s)
Anticuerpos/administración & dosificación , Antígenos CD/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Inmunidad/efectos de los fármacos , Sepsis/terapia , Animales , Anticuerpos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Citocinas/biosíntesis , Sistemas de Liberación de Medicamentos/métodos , Interacciones Huésped-Patógeno/inmunología , Ratones , Receptor de Muerte Celular Programada 1 , Sepsis/mortalidad , Tasa de Supervivencia , Linfocitos T , Factores de TiempoRESUMEN
This study examined the fate of dendritic cells (DCs) and macrophages (M Phi) in vivo in a murine model of sepsis. Wild-type, knockout, and transgenic mice were used to examine the role of Bcl-2 family members on the regulation of splenic DCs and M Phi survival. Bim knockout (Bim) mice and mice overexpressing Bcl-2 in selected hematopoietic cells were used: (a) overexpression of Bcl-2 in all hematopoietic cells using a vav promoter (Vav-Bcl-2) and (b) overexpression of Bcl-2 in all Major histocompatibility complex (MHC) class I cells (H-2K-Bcl-2). Mice underwent sham surgery or cecal ligation and puncture, and absolute numbers of splenic DCs and M Phi were determined. Importantly, two distinct M Phi populations, that is, well-differentiated "mature" M Phi population and a less differentiated "immature," "monocyte-like" (IM Phi) population were identified that demonstrated differential susceptibility to apoptosis. In wild-type mice, sepsis induced a 64% +/- 7% and a 77% +/- 3% decrease in absolute cell numbers of splenic DCs and IM Phi, respectively (n = 7, P < 0.05). Mature M Phi were not depleted in sepsis. No significant cell depletion was evident in Vav-Bcl-2, H-2K-Bcl-2, or Bim mice. We conclude that sepsis induces a major depletion of developing M Phi as well as DCs, and this depletion may be an important mechanism of immune suppression in sepsis.
Asunto(s)
Células Dendríticas/inmunología , Macrófagos/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Sepsis/inmunología , Animales , Células Dendríticas/patología , Citometría de Flujo , Regulación de la Expresión Génica , Depleción Linfocítica , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sepsis/patología , Bazo/inmunología , Bazo/patologíaRESUMEN
Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , ARN Interferente Pequeño/inmunología , Sepsis/inmunología , Transfección/métodos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Linfocitos B/inmunología , Proteína 11 Similar a Bcl2 , Celulosa/farmacología , Ciclodextrinas/farmacología , Humanos , Depleción Linfocítica , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Receptores de Transferrina/agonistas , Receptores de Transferrina/genética , Receptores de Transferrina/inmunología , Sepsis/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
BACKGROUND: Clinical signs and symptoms of sepsis are nonspecific and often indistinguishable from those of nonseptic critical illness. This ambiguity frequently delays the diagnosis of sepsis until culture results can confirm the presence or absence of an infectious organism. Lymphocyte phenotyping can be conducted rapidly and may provide information on the presence of infection before culture results are available. In this study, we hypothesized that lymphocyte phenotype can distinguish between septic and nonseptic critical illness. STUDY DESIGN: C57Bl/6 mice were subjected to either P aeruginosa pneumonia or lipopolysaccharide-induced acute lung injury (ALI). Animals were sacrificed 24 hours postinjury and splenic lymphocytes were harvested. Additionally, 13 patients in a surgical ICU were enrolled in the study. Whole blood was obtained and lymphocytes were isolated by density gradient centrifugation. Lymphocyte phenotype was identified through flow cytometry after labeling lymphocytes for CD3, CD4, CD8, CD20, CD40, CD69, and CD86 with fluorochrome-conjugated antibodies. RESULTS: CD69 expression on B cells and CD8+ splenocytes from septic mice was significantly increased compared with acute lung injury mice (p < 0.001 and p < 0.05, respectively). Similarly, CD4+ and CD8+ lymphocytes from septic patients had a two- to threefold increase in the expression of CD69 compared with nonseptic critically ill patients (p < 0.05). CONCLUSIONS: These data indicated that CD69 expression on lymphocytes may be useful in distinguishing between septic and nonseptic critical illness. Continued investigation into the expression of CD69 during sepsis is warranted.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Subgrupos Linfocitarios/fisiología , Sepsis/diagnóstico , Animales , Enfermedad Crítica , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Lectinas Tipo C , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Sepsis/metabolismoRESUMEN
We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the effect of bcl-2 on improving survival during sepsis. Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2. Whole spleens were obtained 6 h after septic injury. DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements. Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene. A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied. At 6 h, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., interleukin-6). These sepsis-induced alterations were blocked by expression of the bcl-2 transgene. Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1. Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1. These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks. In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses.
Asunto(s)
Expresión Génica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sepsis/genética , Bazo/metabolismo , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Heterocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Bazo/citología , Tasa de Supervivencia , Factores de Tiempo , TransgenesRESUMEN
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.