RESUMEN
RIPK3 (receptor-interacting protein kinase 3) activity triggers cell death via necroptosis, whereas scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or killed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality after Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.
Asunto(s)
Neumonía , Pseudomonas aeruginosa , Animales , Humanos , Ratones , Pseudomonas aeruginosa/metabolismo , Apoptosis , Inflamación/metabolismo , ARN Interferente Pequeño , Citocinas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Cell death proteins play a central role in host immune signaling during sepsis. These interconnected mechanisms trigger cell demise via apoptosis, necroptosis, and pyroptosis while also driving inflammatory signaling. Targeting cell death mediators with novel therapies may correct the dysregulated inflammation seen during sepsis and improve outcomes for septic patients.
Asunto(s)
Apoptosis , Sepsis , Humanos , Muerte Celular , Piroptosis , Inflamación , Sepsis/metabolismoRESUMEN
Expression of the tight junction-associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A -/- mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1ß expression. Survival is improved in JAM-A-/- mice. However, intestine-specific JAM-A-/- deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A-/- mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A-/- mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A-/- × RAG-/- mice have improved survival compared with RAG-/- mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A-/- mice, while septic JAM-A-/- mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A-/- mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Molécula A de Adhesión de Unión , Receptores de Superficie Celular/metabolismo , Sepsis , Animales , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Inmunoglobulina A , Ratones , Ratones Endogámicos C57BL , Fagocitosis , Receptores de Superficie Celular/genética , Sepsis/genéticaRESUMEN
During infectious disease, pathogen load drives inflammation and immune response that together contribute to tissue injury often resulting in organ dysfunction. Pulmonary failure in SARS-CoV2-infected hospitalized COVID-19 patients is one such prominent example. Intervention strategies require characterization of the host-pathogen interaction by accurately assessing all of the above-mentioned disease parameters. To study infection in intact mammals, mice are often used as essential genetic models. Due to humane concerns, there is a constant unmet demand to develop studies that reduce the number of mice utilized while generating objective data. Here, we describe an integrated method of evaluating lung inflammation in mice infected with Pseudomonas aeruginosa or murine gammaherpesvirus (MHV)-68. This method conserves animal resources while permitting evaluation of disease mechanisms in both infection settings. Lungs from a single euthanized mouse were used for two purposes-biological assays to determine inflammation and infection load, as well as histology to evaluate tissue architecture. For this concurrent assessment of multiple parameters from a single euthanized mouse, we limit in-situ formalin fixation to the right lung of the cadaver. The unfixed left lung is collected immediately and divided into several segments for biological assays including determination of pathogen titer, assessment of infection-driven cytokine levels and appearance of cell death markers. In situ fixed right lung was then processed for histological determination of tissue injury and confirmation of infection-driven cell death patterns. This method reduces overall animal use and minimizes inter-animal variability that results from sacrificing different animals for different types of assays. The technique can be applied to any lung disease study in mice or other mammals.
Asunto(s)
Infecciones por Herpesviridae/patología , Enfermedades Pulmonares/patología , Pulmón/patología , Infecciones por Pseudomonas/patología , Animales , Gammaherpesvirinae , Ratones , Pseudomonas aeruginosaRESUMEN
Mortality is higher in septic patients with a history of alcohol use disorder than in septic patients without a history of chronic alcohol usage. We have previously described a model of chronic alcohol ingestion followed by sepsis from cecal ligation and puncture in which alcohol-fed septic mice have higher mortality than water-fed septic mice, associated with altered gut integrity and increased production of TNF and IFNγ by splenic CD4 T cells without alterations in CD8 T cell function. The purpose of this study was to determine whether this represents a common host response to the combination of alcohol and sepsis by creating a new model in which mice with chronic alcohol ingestion were subjected to a different model of sepsis. C57Bl/6 mice were randomized to receive either alcohol or water for 12 weeks and then subjected to Pseudomonas aeruginosa pneumonia. Mice were sacrificed either 24âhours after the onset of sepsis or followed for survival. Alcohol-fed septic mice had significantly higher 7-day mortality than water-fed septic mice (96% vs 58%). This was associated with a 5-fold increase in intestinal apoptosis in alcohol-fed septic animals, accompanied by an increase in the pro-apoptotic protein Bax. Serum IL-6 levels were higher and IL-2 levels were lower in alcohol-fed septic mice. In contrast, CD8 T cell frequency was lower in alcohol-fed mice than water-fed septic mice, associated with increased production of IFNγ and TNF in stimulated splenocytes. No significant differences were noted in CD4 T cells, lung injury or bacteremia. Mice with chronic alcohol ingestion thus have increased mortality regardless of their septic insult, associated with changes in both the gut and the immune system.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Linfocitos T CD8-positivos/metabolismo , Mucosa Intestinal/microbiología , Neumonía/metabolismo , Neumonía/microbiología , Neumonía/patología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Sepsis/metabolismo , Sepsis/patología , Alanina Transaminasa/sangre , Animales , Apoptosis/fisiología , Aspartato Aminotransferasas/sangre , Western Blotting , Citocinas/sangre , Femenino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía/sangre , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/complicaciones , Sepsis/etiologíaRESUMEN
Patients with cancer who develop sepsis have a markedly higher mortality than patients who were healthy prior to the onset of sepsis. Potential mechanisms underlying this difference have previously been examined in two preclinical models of cancer followed by sepsis. Both pancreatic cancer/pneumonia and lung cancer/cecal ligation and puncture (CLP) increase murine mortality, associated with alterations in lymphocyte apoptosis and intestinal integrity. However, pancreatic cancer/pneumonia decreases lymphocyte apoptosis and increases gut apoptosis while lung cancer/CLP increases lymphocyte apoptosis and decreases intestinal proliferation. These results cannot distinguish the individual roles of cancer versus sepsis since different models of each were used. We therefore created a new cancer/sepsis model to standardize each variable. Mice were injected with a pancreatic cancer cell line and 3 weeks later cancer mice and healthy mice were subjected to CLP. Cancer septic mice had a significantly higher 10-day mortality than previously healthy septic mice. Cancer septic mice had increased CD4 T cells and CD8 T cells, associated with decreased CD4 T cell apoptosis 24âh after CLP. Further, splenic CD8+ T cell activation was decreased in cancer septic mice. In contrast, no differences were noted in intestinal apoptosis, proliferation, or permeability, nor were changes noted in local bacterial burden, renal, liver, or pulmonary injury. Cancer septic mice thus have consistently reduced survival compared with previously healthy septic mice, independent of the cancer or sepsis model utilized. Changes in lymphocyte apoptosis are common to cancer model and independent of sepsis model, whereas gut apoptosis is common to sepsis model and independent of cancer model. The host response to the combination of cancer and sepsis is dependent, at least in part, on both chronic comorbidity and acute illness.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Neoplasias Experimentales/inmunología , Neoplasias Pancreáticas/inmunología , Sepsis/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Masculino , Ratones , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/patología , Sepsis/patologíaRESUMEN
The gut is a continuously renewing organ, with cell proliferation, migration, and death occurring rapidly under basal conditions. As the impact of critical illness on cell movement from crypt base to villus tip is poorly understood, the purpose of this study was to determine how sepsis alters enterocyte migration. Wild-type, transgenic, and knockout mice were injected with 5-bromo-2'deoxyuridine (BrdU) to label cells in S-phase before and after the onset of cecal ligation and puncture and were sacrificed at predetermined endpoints to determine distance proliferating cells migrated up the crypt-villus unit. Enterocyte migration rate was decreased from 24 to 96âh after sepsis. BrdU was not detectable on villi 6 days after sham laparotomy, meaning all cells had migrated the length of the gut and been exfoliated into its lumen. However, BrdU positive cells were detectable on villi 10 days after sepsis. Multiple components of gut integrity altered enterocyte migration. Sepsis decreased crypt proliferation, which further slowed enterocyte transit as mice injected with BrdU after the onset of sepsis (decreased proliferation) had slower migration than mice injected with BrdU before the onset of sepsis (normal proliferation). Decreasing intestinal apoptosis via gut-specific overexpression of Bcl-2 prevented sepsis-induced slowing of enterocyte migration. In contrast, worsened intestinal hyperpermeability by genetic deletion of JAM-A increased enterocyte migration. Sepsis therefore significantly slows enterocyte migration, and intestinal proliferation, apoptosis and permeability all affect migration time, which can potentially be targeted both genetically and pharmacologically.
Asunto(s)
Apoptosis , Movimiento Celular , Proliferación Celular , Enterocitos/metabolismo , Sepsis/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Enterocitos/patología , Femenino , Masculino , Ratones , Ratones Noqueados , Permeabilidad , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Sepsis/genética , Sepsis/patologíaRESUMEN
CD43 is a large transmembrane protein involved in T cell activation. Previous studies of CD43-/- mice in viral models have demonstrated a role for CD43 in Th1/Th2 skewing, activation of Foxp3+ Treg, and T cell apoptosis. However, the role of CD43 during sepsis has never been tested. Thus, we interrogated the role of CD43 during sepsis using a murine cecal ligation and puncture (CLP) model, and found that CD43-/- mice demonstrated significantly worsened mortality compared to B6 mice following CLP. Phenotypic analysis of splenocytes isolated 24 h after septic insult revealed significantly increased apoptosis of central memory cells in both CD4+ and CD8+ T cell compartments in CD43-/- septic mice compared to WT septic mice. Furthermore, CD43-/-septic mice exhibited a prominent Th2 skewing following sepsis relative to WT septic mice, as evidenced by a significant decrease in the frequency of IL-2+ CXCR3+ TH1 cells as a significant increase in the frequency of IL-4+ CCR4+ TH2 cells. Finally, septic CD43-/- animals contained significantly fewer CD25+ Foxp3+ TReg cells as compared to WT septic animals. Importantly, depleting CD25+ Treg eliminated the increased mortality observed in CD43-/- mice. Taken together, these data demonstrate an important role of CD43 in modulating immune dysregulation and mortality following sepsis.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Leucosialina/genética , Sepsis/mortalidad , Bazo/inmunología , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Interleucinas/metabolismo , Masculino , Ratones , Receptores CXCR/metabolismo , Sepsis/genética , Sepsis/inmunología , Bazo/citología , Células TH1/citología , Células TH1/inmunología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
Asunto(s)
Caspasa 8/metabolismo , Caspasas/metabolismo , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/fisiopatología , Choque Séptico/enzimología , Choque Séptico/fisiopatología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 8/genética , Caspasas/genética , Caspasas Iniciadoras , Células Cultivadas , Femenino , Inflamación/metabolismo , Inflamación/patología , Factor 3 Regulador del Interferón/genética , Interferón beta/sangre , Interferón beta/metabolismo , Intestino Delgado/patología , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/toxicidad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Fosfato , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Bazo/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Receptores CXCR4/antagonistas & inhibidores , Sepsis/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Femenino , Citometría de Flujo , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Sepsis/microbiología , Análisis de SupervivenciaRESUMEN
Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild-type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi-TAg mice expressing large T-antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic fabpi-TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic fabpi-TAg mice. To examine the relationship between apoptosis and proliferation in a compartment-specific manner, fabpi-TAg mice were crossed with fabpl-Bcl-2 mice, resulting in expression of both genes in the villus but Bcl-2 alone in the crypt. Septic bi-transgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi-TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment-specific proliferative and apoptotic regulation that is not present under homeostatic conditions.-Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.
Asunto(s)
Apoptosis/fisiología , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Enterocitos/citología , Intestinos/citología , Sepsis/metabolismo , Sepsis/patología , Animales , Western Blotting , Enterocitos/fisiología , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Masculino , Ratones , Ratones TransgénicosRESUMEN
OBJECTIVE: To describe and summarize the data supporting the gut as the motor driving critical illness and multiple organ dysfunction syndrome presented at the National Institute of Child Health and Human Development MODS Workshop (March 26-27, 2015). DATA SOURCES: Summary of workshop keynote presentation. STUDY SELECTION: Not applicable. DATA EXTRACTION: Presented by an expert in the field, the data assessing the role of gastrointestinal dysfunction driving critical illness were described with a focus on identifying knowledge gaps and research priorities. DATA SYNTHESIS: Summary of presentation and discussion supported and supplemented by relevant literature. CONCLUSIONS: The understanding of gut dysfunction in critical illness has evolved greatly over time, and the gut is now often considered as the "motor" of critical illness. The association of the gut with critical illness is supported by both animal models and clinical studies. Initially, the association between gut dysfunction and critical illness focused primarily on bacterial translocation into the bloodstream. However, that work has evolved to include other gut-derived products causing distant injury via other routes (e.g., lymphatics). Additionally, alterations in the gut epithelium may be associated with critical illness and influence outcomes. Gut epithelial apoptosis, intestinal hyperpermeability, and perturbations in the intestinal mucus layer have all been associated with critical illness. Finally, there is growing evidence that the intestinal microbiome plays a crucial role in mediating pathology in critical illness. Further research is needed to better understand the role of each of these mechanisms and their contribution to multiple organ dysfunction syndrome in children.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Mucosa Intestinal/microbiología , Insuficiencia Multiorgánica/microbiología , Insuficiencia Multiorgánica/fisiopatología , Traslocación Bacteriana , Niño , Enfermedad Crítica , Humanos , Mucosa Intestinal/fisiopatología , PediatríaRESUMEN
Epidermal growth factor (EGF) is a cytoprotective protein that improves survival in preclinical models of sepsis through its beneficial effects on intestinal integrity. Alcohol use disorder worsens intestinal integrity and is associated with increased morbidity and mortality in critical illness. We sought to determine whether chronic alcohol ingestion alters the host response to systemic administration of EGF in sepsis. Six-week-old FVB/N mice were randomized to receive 20% alcohol or water for 12 weeks. All mice then underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive either intraperitoneal injection of EGF (150âµg/kg/day) or normal saline. Water-fed mice given EGF had decreased 7-day mortality compared with water-fed mice (18% vs. 55%). Alcohol-fed mice given EGF also had decreased 7-day mortality compared with alcohol-fed mice (48% vs. 79%). Notably, while systemic EGF improved absolute survival to a similar degree in both water-fed and alcohol-fed mice, mortality was significantly higher in alcohol+EGF mice compared with water+EGF mice. Compared with water-fed septic mice, alcohol-fed septic mice had worsened intestinal integrity with intestinal hyperpermeability, increased intestinal epithelial apoptosis, decreased proliferation and shorter villus length. Systemic administration of EGF to septic alcohol-fed mice decreased intestinal permeability compared with septic alcohol-fed mice given vehicle, with increased levels of the tight junction mediators claudin-5 and JAM-A. Systemic administration of EGF to septic alcohol-fed mice also decreased intestinal apoptosis with an improvement in the Bax/Bcl-2 ratio. EGF also improved both crypt proliferation and villus length in septic alcohol-fed mice. EGF administration resulted in lower levels of both pro- and anti-inflammatory cytokines monocyte chemoattractant protein-1, tumor necrosis factor, and interleukin 10 in alcohol-fed mice. EGF is therefore effective at improving both intestinal integrity and mortality following sepsis in mice with chronic alcohol ingestion. However, the efficacy of EGF in sepsis is blunted in the setting of chronic alcohol ingestion, as intestinal integrity and mortality in alcohol-fed mice given EGF improves animals to levels seen in water-fed mice given vehicle but does not approach levels seen in water-fed mice given EGF.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Epidérmico/uso terapéutico , Intestinos/citología , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Claudina-5/metabolismo , Interleucina-10/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Receptores de Superficie Celular/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Sepsis is the leading cause of death in intensive care units in the US, and it is known that chronic alcohol use is associated with higher incidence of sepsis, longer ICU stays, and higher mortality from sepsis. Both sepsis and chronic alcohol use are associated with immune deficits such as decreased lymphocyte numbers, impaired innate immunity, delayed-type hypersensitivity reactions, and susceptibility to infections; however, understanding of specific pathways of interaction or synergy between these two states of immune dysregulation is lacking. This study therefore sought to elucidate mechanisms underlying the immune dysregulation observed during sepsis in the setting of chronic alcohol exposure. Using a murine model of chronic ethanol ingestion followed by sepsis induction via cecal ligation and puncture, we determined that while CD4+ and CD8+ T cells isolated from alcohol fed mice eventually expressed the same cellular activation markers (CD44, CD69, and CD43) and effector molecules (IFN-γ, TNF) as their water fed counterparts, there was an overall delay in the acquisition of these phenotypes. This early lag in T cell activation was associated with significantly reduced IL-2 production at a later timepoint in both the CD4+ and CD8+ T cell compartments in alcohol sepsis, as well as with a reduced accumulation of CD8dim activated effectors. Taken together, these data suggest that delayed T cell activation may result in qualitative differences in the immune response to sepsis in the setting of chronic alcohol ingestion.
Asunto(s)
Alcoholes/administración & dosificación , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Sepsis/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Animales , Biomarcadores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Glicosilación/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Recuento de Linfocitos , Masculino , Ratones , Sepsis/metabolismo , Subgrupos de Linfocitos T/metabolismo , Factores de TiempoRESUMEN
CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69+CD25intCD62LHI) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain "unactivated" (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Antígeno-1 Asociado a Función de Linfocito/inmunología , Sepsis/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis , Efecto Espectador , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Citocinas/sangre , Progresión de la Enfermedad , Genes RAG-1 , Inmunoterapia Adoptiva , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/trasplanteRESUMEN
BACKGROUND: Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered. METHODS: C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival. RESULTS: Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury. CONCLUSIONS: Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/citología , Sepsis/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Hígado/enzimología , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Receptores CXCR3/metabolismo , Sepsis/metabolismo , Sepsis/mortalidad , Tasa de Supervivencia , Trasplante HomólogoAsunto(s)
Técnicas Bacteriológicas , Enfermedad Crítica , Carga Bacteriana , Medios de Cultivo , Humanos , MicrobiotaRESUMEN
BACKGROUND: Cancer is known to modulate tumor-specific immune responses by establishing a microenvironment that leads to the upregulation of T-cell inhibitory receptors, resulting in the progressive loss of function and eventual death of tumor-specific T-cells. However, the ability of cancer to impact the functionality of the immune system on a systemic level is much less well characterized. Because cancer is known to predispose patients to infectious complications including sepsis, we hypothesized that the presence of cancer alters pathogen-directed immune responses on a systemic level. MATERIALS AND METHODS: We assessed systemic T-cell coinhibitory receptor expression, cytokine production, and apoptosis in mice with established subcutaneous lung cancer tumors and in unmanipulated mice without cancer. RESULTS: Results indicated that the frequencies of programmed death-1-positive, B and T lymphocyte attenuator-positive, and 2B4(+) cells in both the CD4(+) and CD8(+) T-cell compartments were increased in mice with localized cancer relative to non-cancer controls, and the frequencies of both CD4(+) and CD8(+) T-cells expressing multiple different inhibitory receptors were increased in cancer animals relative to non-cancer controls. Additionally, 2B4(+)CD8(+) T-cells in cancer mice exhibited reduced interleukin-2 and interferon-γ, whereas B and T lymphocyte attenuator-positive CD8(+) T-cells in cancer mice exhibited reduced interleukin-2 and tumor necrosis factor. Conversely, CD4(+) T-cells in cancer animals demonstrated an increase in the frequency of annexin V(+) apoptotic cells. CONCLUSIONS: Taken together, these data suggest that the presence of cancer induces systemic T-cell exhaustion and generalized immune suppression.
Asunto(s)
Neoplasias Pulmonares/inmunología , Linfocitos T/inmunología , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/fisiología , Receptores Inmunológicos/fisiologíaRESUMEN
Complications after tracheal repair in the past have included wound infections, tracheal stenosis, "spitting" of sutures, and tracheoesophageal fistulas. Modern operative approaches have significantly decreased the incidence of these complications. We conducted retrospective data collection using the TRACS database. Changes that preceded the time interval of the study included the following: 1) an emphasis on clinical (rather than endoscopic) recognition of injury; 2) minimal peritracheal dissection and repair with absorbable sutures; 3) limited use of "protective" tracheostomies; and 4) use of muscle buttresses to cover tracheal repairs, especially in patients with combined injuries. From 1997 to 2010, 22 patients were treated for wounds to the trachea (cervical 20, thoracic 2). The mechanism of injury was a gunshot wound in 15 patients and a stab wound in seven. A clinical diagnosis of the need for cervical operation or of a tracheal injury was made in 19 patients (86%), whereas three patients had positive diagnostic studies. Direct tracheal repair (No. 19) or evaluation of a superficial injury (No. 1) was performed in 20 patients, and three (15%) had a tracheostomy performed. Combined injuries were present in 12 patients (55%), most commonly to the esophagus (10 of 12 [83%]), and 10 of these 12 patients had vascularized buttresses applied to the tracheal repair. There were seven significant complications in patients with combined injuries to the esophagus or carotid artery. One patient (4.5%) died. Patients with penetrating tracheal injuries most commonly present with overt findings. Modern techniques of repair have eliminated many of the complications noted in the past.
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Procedimientos de Cirugía Plástica , Complicaciones Posoperatorias/prevención & control , Tráquea/lesiones , Traqueostomía , Heridas por Arma de Fuego/cirugía , Heridas Punzantes/cirugía , Adolescente , Adulto , Niño , Preescolar , Esófago/lesiones , Esófago/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/cirugía , Complicaciones Posoperatorias/epidemiología , Procedimientos de Cirugía Plástica/métodos , Procedimientos de Cirugía Plástica/estadística & datos numéricos , Estudios Retrospectivos , Tráquea/cirugía , Traqueostomía/estadística & datos numéricos , Resultado del Tratamiento , Heridas por Arma de Fuego/mortalidad , Heridas Punzantes/mortalidad , Adulto JovenRESUMEN
BACKGROUND: Thyroidectomy continues to be the least common form of therapy for Graves' disease in the US, presumably based upon concerns about injuries to the recurrent laryngeal nerves and/or parathyroid glands. STUDY DESIGN: Concurrent and retrospective data were collected on 65 consecutive patients undergoing subtotal or total thyroidectomy for Graves' disease at one community-type hospital by one surgeon from 1992 to 2010. RESULTS: A total of 65 patients (50 women, 15 men) with a mean age of 38 years underwent subtotal (52.3%) or near total or total thyroidectomy (47.7%). Six patients (9.2%) had high lying or ischemic parathyroid glands reimplanted into the ipsilateral sternocleidomastoid muscle at the completion of the thyroidectomy, and none developed hypocalcemia. No injuries to the recurrent laryngeal nerve or external branch of the superior laryngeal nerve occurred. Seven patients (10.8%) had 1 parathyroid gland removed inadvertently during thyroidectomy, but none developed hypocalcemia. Nine patients (13.8%) were discharged from the hospital on supplemental calcium tablets for symptomatic or asymptomatic hypocalcemia (calcium < 7.5 mg/dL). Four patients were weaned off calcium tablets, and 5, all of whom had normocalcemia on supplementation, were lost to long-term follow-up. One patient had reopening of the cervical incision under the same anesthetic for stridor that was subsequently found to have been caused by bacterial tonsillitis, and 2 other patients had reoperations for a cervical hematoma (same day) and a reaction to hydrocodone with facial and cervical edema (tracheostomy, 11th day). No patient is known to have developed a recurrence of Graves' disease. CONCLUSIONS: Total thyroidectomy offers a rapid and complete cure for Graves' disease. Extensive thyroidectomy for Graves' goiters can be safely performed in a community-type hospital.