RESUMEN
Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.
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Leucemia , Monocitos , Diferenciación Celular , Línea Celular Tumoral , Humanos , Macrófagos , Fenotipo , Células THP-1 , Microambiente TumoralRESUMEN
Immune dysfunction can occur during sepsis or following major trauma. Decreased monocyte HLA-DR expression and cytokine responses are associated with mortality. Recent studies have shown that adaptive immune system defects can also occur in such patients, characterised by increased PD-L1 expression and associated T-cell anergy. The aim of this study was to determine the effects of an immune adjuvant, interferon-gamma, on monocyte PD-L1 expression and T-cell activation in an ex-vivo human whole blood model of infection. We found that with interferon-gamma treatment, monocytes had increased HLA-DR expression and augmented TNF-α production in response to LPS stimulation, with a decrease in IL-10 levels. Both LPS and interferon-gamma increased the level of monocyte PD-L1 expression, and that a combination of both agents synergistically stimulated a further increase in PD-L1 levels as measured by flow cytometry. However, despite elevated PD-L1 expression, both CD4 and CD8 T-cell activation was not diminished by the addition of interferon-gamma treatment. These findings suggest that PD-L1 may not be a reliable marker for T-cell anergy, and that interferon-gamma remains an adjuvant of interest that can improve the monocyte inflammatory response while preserving T-cell activation.
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Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Interferón gamma/farmacología , Monocitos/inmunología , Adulto , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Femenino , Citometría de Flujo , Expresión Génica , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Following major trauma, sepsis or surgery, some patients exhibit an impaired monocyte inflammatory response that is characterized by a decreased response to a subsequent bacterial challenge. To investigate this poorly understood phenomenon, we adopted an in-vitro model of endotoxin tolerance utilising primary human CD14 + monocytes to focus on the effect of impairment on IκKα/ß, a critical part of the NFκB pathway. Impaired monocytes had decreased IκKα mRNA and protein expression and decreased phosphorylation of the IκKα/ß complex. The impaired monocyte secretome demonstrated a distinct cytokine/chemokine footprint from the naïve monocyte, and that TNF-α was the most sensitive cytokine or chemokine in this setting of impairment. Inhibition of IκKα/ß with a novel selective inhibitor reproduced the impaired monocyte phenotype with decreased production of TNF-α, IL-6, IL-12p70, IL-10, GM-CSF, VEGF, MIP-1ß, TNF-ß, IFN-α2 and IL-7 in response to an LPS challenge. Surgical patients with infection also exhibited an impaired monocyte phenotype and had decreased SITPEC, TAK1 and MEKK gene expression, which are important for IκKα/ß activation. Our results emphasize that impaired monocyte function is, at least in part, related to dysregulated IκKα/ß activation, and that IκKα/ß is likely involved in mounting a sufficient monocyte inflammatory response. Future studies may wish to focus on adjuvant therapies that augment IκKα/ß function to restore monocyte function in this clinically important problem.
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Quinasa I-kappa B/metabolismo , Monocitos/metabolismo , Adulto , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
Predation threat impacts prey behavior, physiology, and fitness. Stress-mediated alterations to the paternal epigenome can be transmitted to offspring via the germline, conferring a potential advantage to offspring in predator-rich environments. While intergenerational epigenetic transmission of paternal experience has been demonstrated in mammals, how paternal predator exposure might alter offspring phenotypes across development is unstudied. We exposed male mice to a predator odor (2,4,5-trimethylthiazoline, TMT) or a neutral odor (banana extract) prior to mating and measured offspring behavioral phenotypes throughout development, together with adult stress reactivity and candidate gene expression in the prefrontal cortex, hippocampus, amygdala, and hypothalamus. We predicted that offspring of TMT-exposed males would be less active, would display elevated anxiety-like behaviors, and would have a more efficient stress response relative to controls, phenotypes that should enhance predator avoidance in a high predation risk environment. Unexpectedly, we found that offspring of TMT-exposed males are more active, exhibit less anxiety-like behavior, and have decreased baseline plasma corticosterone relative to controls. Effects of paternal treatment on neural gene expression were limited to the prefrontal cortex, with increased mineralocorticoid receptor expression and a trend towards increased Bdnf expression in offspring of TMT-exposed males. These results suggest that fathers exposed to predation threat produce offspring that are buffered against non-acute stressors and, potentially, better adapted to a predator-dense environment because they avoid trade-offs between predator avoidance and foraging and reproduction. This study provides evidence that ecologically relevant paternal experience can be transmitted through the germline, and can impact offspring phenotypes throughout development.
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Ansiedad , Encéfalo/metabolismo , Exposición Paterna , Conducta Predatoria/fisiología , Estrés Fisiológico/genética , Animales , Ansiedad/genética , Ansiedad/metabolismo , Ansiedad/fisiopatología , Ansiedad/psicología , Conducta Animal/fisiología , Encéfalo/patología , Corticosterona/sangre , Señales (Psicología) , Padre , Femenino , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Exposición Paterna/efectos adversos , Fenotipo , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Efectos Tardíos de la Exposición Prenatal/psicologíaRESUMEN
BACKGROUND: Severe injury can lead to immune dysfunction and predispose patients to infection and death. Micro-RNAs regulate gene expression and may act as biomarkers for susceptibility to infection. The aim of this study was to examine the temporal and differential expression of previously identified dysregulated micro-RNAs in patients with severe injury. METHODS: Fourteen severely injured patients requiring transfusion were enrolled prospectively in this study approved by our institutional review board. Inclusion criteria consisted of adult patients deemed clinically to be in hemorrhagic shock necessitating transfusion in the acute phase of their injury care. Peripheral blood samples were obtained after admission to the surgical intensive care unit and again at 6, 12, 24, and 48 hours after admission. The samples obtained at arrival to the intensive care unit and 24 and 48 hours later were analyzed in this data set. Fourteen healthy volunteers served as controls. The 10 dysregulated micro-RNAs identified in a prior study at the 12-hour time point and important genes in innate immunity were measured using quantitative reverse transcription-polymerase chain reaction. RESULTS: The participants were 21-77 years old (median, 42), 78% were male, and their Injury Severity Score ranged from 11 to 43 (median, 27); 11 had blunt and 3 had penetrating injuries. Three were intubated and 5 had received blood products before arrival at the hospital. Base deficit on hospital admission was 3-20 (median, 9). All patients required blood transfusion secondary to blood loss sustained during injury. Eleven of the 14 patients went directly to the operating room from the emergency department for control of the source of hemorrhage. Survival to discharge was 93%. Seven patients developed infection. Compared with healthy controls, miR-106a was downregulated at all time points compared with controls (P < .05). miR-618 was upregulated in initial blood draws (P < .05) and at 24 and 48 hours (P < .06). Tumor necrosis factor α and human leukocyte antigen-DR (HLA-DR) were downregulated, and interleukin-10 and PD-L1 were upregulated (P < .05). In patients who developed infection, miR-106a levels appeared more downregulated than those who did not develop infection. CONCLUSION: miR-106a was downregulated in trauma patients after major injury for up to 48 hours after intensive care unit admission. Tumor necrosis factor α and interleukin-10 are targeted by miR-106a, which are regulators of the immune response. Manipulation of micro-RNA expression may be a therapeutic target for immune dysfunction.
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MicroARNs/sangre , Choque Hemorrágico/sangre , Choque Hemorrágico/etiología , Heridas no Penetrantes/sangre , Heridas Penetrantes/sangre , Adulto , Anciano , Transfusión Sanguínea , Estudios de Casos y Controles , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Choque Hemorrágico/terapia , Factores de Tiempo , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/terapia , Heridas Penetrantes/complicaciones , Heridas Penetrantes/terapia , Adulto JovenRESUMEN
This study focuses on impaired monocyte function, which occurs in some patients after trauma, major elective surgery, or sepsis. This monocyte impairment increases the risk of secondary infection and death. We aimed to determine the influence IκK-16 had on monocytes using an ex-vivo model of human monocyte impairment. We included the effects of the well-studied comparators interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on impaired monocytes. Primary human monocytes were stimulated with 10ng/mL of lipopolysaccharide (LPS) for 16h and then challenged with 100ng/mL LPS to assess the monocyte inflammatory response. Treatment regimens, consisting of either IκK-16, IFN-γ, or GM-CSF, were administered to impaired monocytes near the time of initial LPS stimulation. Stimulation with 10ng/mL LPS initially promoted a pro-inflammatory response but subsequently impaired production of both tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) and decreased HLA-DR expression. IκK-16 treatment attenuated TNF-α production and programmed death-ligand 1 (PD-L1) expression and increased IL-10 and CD14 expression. IFN-γ treatment increased TNF-α production as well as PD-L1 and HLA-DR expression. In conclusion, limiting early inflammation with IκK-16 suppresses TNF-α production and PD-L1 expression but enhances IL-10 production and preserves CD14 expression for potential future exposure to infective stimuli.
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Trastornos de Traumas Acumulados/inmunología , Cirugía General , Quinasa I-kappa B/antagonistas & inhibidores , Inflamación/inmunología , Monocitos/inmunología , Piperidinas/farmacología , Complicaciones Posoperatorias/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/farmacología , Sepsis/inmunología , Adulto , Células Cultivadas , Procedimientos Quirúrgicos Electivos , Femenino , Humanos , Separación Inmunomagnética , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Masculino , Adulto JovenRESUMEN
We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.
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Adenosina Trifosfato/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Adenosina Trifosfato/administración & dosificación , Animales , Quimiocina CCL2/metabolismo , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Liposomas , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Células RAW 264.7/efectos de los fármacos , Células RAW 264.7/fisiología , Factores de TiempoRESUMEN
OBJECTIVE: To investigate whether warming to normal body temperature or to febrile range temperature (39°C) is able to reverse the detrimental effects of hypothermia. BACKGROUND: Unintentional intraoperative hypothermia is a well-described risk factor for surgical site infections but also sepsis. We have previously shown that hypothermia prolongs the proinflammatory response whereas normothermia and especially febrile range temperature enhance the anti-inflammatory response. METHODS: Primary human monocytes were isolated from healthy volunteers. After stimulation with LPS (Lipopolysaccharide), the monocytes were exposed to 32°C for 3â hours or 6â hours and then warmed at either 37°C or 39°C for the remaining 33â hours or 36 âhours, respectively. Tumor necrosis factor α, interleukin 10, and the expression of miR-155 and miR-101 were assessed at 24â hours and 36â hours. RESULTS: Warming to 37°C does not normalize monocyte cytokine secretion within 36â hours, whereas warming to 39°C partially reverses the effects of hypothermia on monocyte function. Both miR-155 and miR-101 were suppressed after the warming episode. However, 39°C had a stronger suppressive effect than 37°C. The duration of hypothermia and the warming temperature seem to be critical for a full reversibility of the effects of hypothermia. CONCLUSION: Warming to normal body temperature (37°C) does not restore normal monocyte function in vitro. These data suggest that hypothermic patients should be warmed to febrile range temperatures. Furthermore, febrile range temperatures should be investigated as a means to modulate the inflammatory response in patients with systemic infections.
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Citocinas/metabolismo , Hipotermia/metabolismo , Hipotermia/terapia , Monocitos/metabolismo , Recalentamiento/métodos , Biomarcadores/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , MicroARNs/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
INTRODUCTION: Hypothermia is a well-known risk factor for postoperative complications because it prolongs the monocyte inflammatory response. The purpose of this study was to investigate whether temperature-activated ion channels (transient receptor protein channels [TRP] A1 and V1) mediate the effects of temperature on monocytes. METHODS: Primary human monocytes were isolated and stimulated with lipopolysaccharide at 32°C or 39°C. RNA was isolated for analysis of microRNA (miR)-155 expression, and cytokines in the supernatant were measured with an enzyme-linked immunosorbent assay. Specific inhibitors of TRPA1 (HC- 030031) and a specific activator of TRPV1 (capsaicin) were used to block or activate TRPA1 and TRPV1, respectively. Statistical analysis was performed using the Wilcoxon signed-rank test. RESULTS: TRPM8 mRNA was not expressed in primary human monocytes, whereas TRPA1 and TRPV1 were expressed. TRPV1 mRNA expression was suppressed at 32°C but not at 39°C. TRPA1 was induced strongly at 32°C and 39°C. Immunofluorescence microscopy confirmed that monocytes express TRPA1 and TRPV1 on their cell surface. Interleukin-10 secretion was increased by blocking TRPA1 (77.8 ± 3 2.8 pg/mL) and activating TRPA1 (79.4 ± 16.1 pg/mL) after 24 hours at 32°C (control 37.4 ± 17.1 pg/mL, P < .05). At 36 hours, tumor necrosis factor secretion was decreased after TRPA1 blockade (2,321 ± 439 pg/mL) and TRPV1 activation (2,137 ± 411 pg/mL) compared with control (2,567 ± 495 pg/mL, P < .05). Furthermore, miR-155 expression also was suppressed at 24 hours by TRPA1 blockade and TRPV1 activation (both P < .05). Silencing of TRPA1 normalized monocyte IL-10 secretion at 32°C. CONCLUSION: These results demonstrate that hypothermia mediates its effects on monocytes through TRPA1. Blockade of TRPA1 or activation of TRPV1 may be used to modify the effects of hypothermia on the monocyte inflammatory response.
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Canales de Calcio/metabolismo , Frío/efectos adversos , Hipotermia/inmunología , Monocitos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Hipotermia/metabolismo , Microscopía Fluorescente , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidoresRESUMEN
Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS-induced production of TNF and IL-6, while blunting IL-10 production. This dysregulation was associated with increased expression of microRNA-155 (miR-155), which potentiates Toll-like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR-155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold-exposed monocytes. In contrast, miR-155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL-10 production. In a murine model of LPS-induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR-155, suppression of Ship1 and Socs1, and alterations in TNF and IL-10. Importantly, miR-155-deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL-10. These results establish a causal role of miR-155 in the dysregulation of the inflammatory response to hypothermia.
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Hipotermia/complicaciones , Inflamación/fisiopatología , Interleucina-10/antagonistas & inhibidores , MicroARNs/fisiología , Animales , Células Cultivadas , Citocinas/biosíntesis , Humanos , Inflamación/etiología , Interleucina-10/biosíntesis , Ratones , Monocitos/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
Unintentional hypothermia is a well-described risk factor for death and complications after elective and emergency surgery. The molecular mechanisms by which hypothermia exerts its detrimental effects are not well understood. Differences in cytokine production and the overall cell function have been reported under hypothermic conditions. We investigated the effect of a range of clinically relevant temperatures on cytokine production and microRNA (miRNA) expression in a whole-blood model. We found that there was a wide variation in tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 production among different subjects, ranging from low to high TNF-α producers. The intersubject variation can also be found on the transcriptional level: high producers had higher upregulation of TNF-α messenger RNA than intermediate and low producers. This variation in TNF-α was reproducible in each individual. Temperature seems to modulate TNF-α production among these different groups. miRNA expression was modulated by temperature. miRNA-181a might control, or be a part of the mechanism which controls, TNF-α production. However, an analysis of whole-leukocyte RNA does not allow the investigation of mechanisms in a specific leukocyte subpopulation such as monocytes, because these changes may be concealed by miRNA expression changes in the other leukocyte subsets. In conclusion, TNF-α, IL-6, and IL-10 production is highly variable among different persons, but temperature affects the expression of miRNAs, which may consequently alter the production of TNF-α.
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Frío/efectos adversos , Citocinas/inmunología , Hipotermia/inmunología , Leucocitos/inmunología , MicroARNs/biosíntesis , Adolescente , Adulto , Circulación Sanguínea , Células Cultivadas , Citocinas/genética , Regulación de la Expresión Génica/inmunología , Humanos , Recuento de Leucocitos , MicroARNs/genética , Persona de Mediana Edad , Biosíntesis de Proteínas/inmunología , Adulto JovenRESUMEN
OBJECTIVE: To investigate liver damage and abscess formation in murine, secondary peritonitis. SUBJECTS: Male C57BL/6 mice. TREATMENT: Intraperitoneal injection with 10(3) CFU Klebsiella pneumoniae and treatment with gentamicin 5 mg/kg/day (BID), subcutaneously. METHODS: Animals were killed at 12, 24, 48 and 72 h after infection. Bacterial burden was determined in the blood and the liver. Liver abscess formation was assessed macroscopically and microscopically. Plasma levels of alkaline phosphatase (ALP) and alanine transaminase (ALT) were measured. Polymorphonuclear leukocyte (PMN) accumulation was assessed via tissue myeloperoxidase (MPO) concentrations. Liver interleukin-10 (IL-10) levels were determined by ELISA. RESULTS: K. pneumoniae was detectable in the blood and the liver at 12 h after infection. Liver abscess formation was visible earliest at 24 h after infection and most pronounced within the right liver lobes. ALP and ALT levels peaked at 12 and 24 h after infection, respectively. MPO was elevated in the right and left liver lobes at 12 h but only in the right lobes at 48 h after infection, compared to tissue levels in naïve mice. Liver IL-10 concentrations were not significantly increased. CONCLUSION: Peritonitis led to liver injury and abscess formation but did not significantly affect tissue concentrations of anti-inflammatory IL-10.
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Absceso Hepático/etiología , Hígado/lesiones , Peritonitis/complicaciones , Animales , Infecciones Bacterianas/sangre , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/patología , Humanos , Interleucina-10/inmunología , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/patogenicidad , Hígado/enzimología , Hígado/inmunología , Hígado/patología , Absceso Hepático/inmunología , Absceso Hepático/microbiología , Absceso Hepático/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Peritonitis/inmunología , Peritonitis/microbiología , Peroxidasa/metabolismoRESUMEN
PURPOSE AND METHODS: Microbial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model. C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2×10(8) CFU (hkKlebsiella), LPS 10mg/kg (LPS 10), or BLP 10mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24h later using 10(3) intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss. RESULTS: Following in vitro stimulation with Klebsiella 10(5) and 10(3) CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups. CONCLUSION: Pretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.
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Tolerancia Inmunológica/inmunología , Klebsiella pneumoniae/inmunología , Peritonitis/inmunología , Peritonitis/microbiología , Animales , Carga Bacteriana/efectos de los fármacos , Tampones (Química) , Recuento de Células , Citocinas/metabolismo , Tolerancia Inmunológica/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Ligandos , Lipopolisacáridos/farmacología , Hígado/microbiología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Cavidad Peritoneal/microbiología , Cavidad Peritoneal/patología , Peritonitis/patología , Análisis de Supervivencia , Receptores Toll-Like/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
BACKGROUND: Recent clinical trials investigating the role of hyperoxia in decreasing surgical site infection have reported conflicting results. Immunologic mechanisms through which supplemental oxygen could act have not been elucidated fully. The authors sought to investigate the effects of hyperoxia on previously tested and prognostically significant innate immune parameters to uncover the potential effects of hyperoxia at the cellular level. METHODS: After formal approval and informed consent, venous blood samples were collected from young healthy volunteers. Corresponding samples were incubated at 21 or 80% O2 following a 1 ng/ml lipopolysaccharide challenge and analyzed to determine human leukocyte antigen-DR surface receptor expression, cytokine release, phagocytic capacity, and formation of reactive oxygen species. Data are presented as mean +/- SD. RESULTS: After the 2 h of incubation at 21% O2 (room air) and in 80% O2 chambers, the change in human leukocyte antigen-DR mean channel fluorescence in lipopolysaccharide-stimulated monocytes was 2,177 +/- 383 and 2,179 +/- 338 (P = 0.96), respectively. Tumor necrosis factor-alpha concentrations were significantly lower for samples incubated at 80% O2 when compared with 21% O2 (P < 0.05). The phagocytic capacity of the innate immune system was not significantly enhanced by supplemental oxygen. However, the formation of reactive oxygen species increased by 87% (P < 0.05). CONCLUSION: Hyperoxia exerts significant effects on multiple cellular and immunologic parameters, providing a potential mechanism for benefits from the use of supplemental oxygen. However, the ability to translate positive basic scientific findings to the operating suite or bedside require the existence of similar innate immune processes in vivo and the efficient transfer of oxygen to the sites where it may be used.
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Quimiotaxis de Leucocito/inmunología , Hiperoxia/inmunología , Inmunidad Innata , Oxígeno/administración & dosificación , Infección de la Herida Quirúrgica/inmunología , Adolescente , Adulto , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Citocinas/biosíntesis , Citocinas/sangre , Femenino , Antígenos HLA-DR/biosíntesis , Antígenos HLA-DR/sangre , Humanos , Hiperoxia/prevención & control , Inmunidad Innata/efectos de los fármacos , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Oxígeno/sangre , Fagocitosis/inmunología , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/inmunología , Complicaciones Posoperatorias/prevención & control , Especies Reactivas de Oxígeno/sangre , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Infección de la Herida Quirúrgica/sangre , Infección de la Herida Quirúrgica/prevención & control , Adulto JovenRESUMEN
BACKGROUND: Early clinical trials investigating the role of tightly controlled glucose levels showed marked benefit in survival of critically ill patients. However, a recent meta-analysis and large randomized controlled trial have failed to reproduce the benefit, showing instead substantially increased risk of dangerous hypoglycemia. We sought to investigate the effects of varying glucose concentrations on previously tested, prognostically significant, innate immune parameters, to define any potential effects of glucose at the cellular level. STUDY DESIGN: After formal approval and informed consent, venous blood samples were collected from young healthy volunteers. Up to 11 corresponding (same-subject) samples were incubated at 100, 350, or 600 mg/dL glucose concentrations and analyzed to determine human leukocyte antigen-DR surface receptor expression, cytokine release, phagocytic capacity, and formation of reactive oxygen species. Data are presented as mean +/- SEM. RESULTS: After incubation, the change in human leukocyte antigen-DR mean channel fluorescence from resting baseline values in lipopolysaccharide-stimulated monocytes was not significantly different between 100, 350, and 600 mg/dL (1,749 +/- 110; 1,748 +/- 120; and 1,725 +/- 96, respectively; p = 0.89). Tumor necrosis factor-alpha concentrations were significantly lower for samples incubated at higher glucose concentrations (179 +/- 50 pg/mL, 125 +/- 30 pg/mL, and 107 +/- 29 pg/mL; p < 0.05). The phagocytic capacity of the innate immune system was marginally enhanced by glucose. However, the formation of reactive oxygen species was markedly impaired by rising glucose (55% to 66% impairment; p < 0.05). CONCLUSIONS: Increasing glucose concentrations exert considerable opposing effects on several well-established innate immunologic processes. The opposing findings might contribute to recent clinical controversies. Physician judgment and experience are essential to imminent treatment of critically ill and perioperative surgical patients.
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Glucemia/análisis , Sepsis/inmunología , Adolescente , Adulto , Análisis de Varianza , Enfermedad Crítica , Citocinas/sangre , Femenino , Citometría de Flujo , Antígenos HLA-DR/sangre , Humanos , Hipoglucemia/sangre , Hipoglucemia/inmunología , Inmunidad Celular , Receptores de Lipopolisacáridos/sangre , Masculino , Fagocitosis , Especies Reactivas de Oxígeno/sangre , Sepsis/sangre , Procedimientos Quirúrgicos OperativosRESUMEN
OBJECTIVE: To examine cellular and immunologic mechanisms by which intraoperative hypothermia affects surgical patients. SUMMARY BACKGROUND DATA: Avoidance of perioperative hypothermia has recently become a focus of attention as an important quality performance measure, aimed at optimizing the care of surgical patients. Anesthetized surgical patients are particularly at risk for hypothermia, which has been directly linked to the development of sequelae, such as coagulopathy, infection, morbid myocardial events, and death after surgery. However, many of the underlying immunologic mechanisms remain unclear. METHODS: Venous blood samples from healthy volunteers were exposed for up to 4 hours to various temperatures following the addition of a 1 ng/mL lipopolysaccharide challenge. Innate immune function, assessed by the ability of monocytes to present antigen and coordinate cytokine release, was determined by qualitative and quantitative measurements of HLA-DR surface expression 2 hours following incubation, and proinflammatory tumor necrosis factor-alpha (TNF-alpha) and anti-inflammatory (IL-10) cytokine release in the first 4 hours. RESULTS: Monocyte incubation at hypothermic temperatures (34 degrees C) reduced HLA-DR surface expression, delayed TNF-alpha clearance, and increased IL-10 release. Conversely, hyperthermia (40 degrees C) increased monocyte antigen presentation and resulted in rapid decay of TNF-alpha. However, IL-10 release was also increased. Normothermia (37 degrees C) attenuated IL-10 release following the initial proinflammatory surge. CONCLUSION: Hypothermia exerts multiple effects at the cellular level, which impair innate immune function, and are associated with increased septic complications and mortality. These findings provide a physiological basis for perioperative temperature monitoring, which is a valid surgical performance measure that can be used to reduce surgical complications associated with avoidable hypothermia.
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Sangre/inmunología , Hipotermia/inmunología , Inmunidad Innata , Procedimientos Quirúrgicos Operativos/efectos adversos , Humanos , Hipotermia/etiología , Periodo Intraoperatorio , Lipopolisacáridos/inmunología , TemperaturaRESUMEN
Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24h (median: 5.04 log CFU) and 48h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10(5) CFU and 2 x 10(8) CFU Klebsiella. Cells harvested from animals 48h after infection, were unresponsive to an ex vivo stimulation with 10(5) CFU Klebsiella, but fully responded to 10(8) CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.
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Citocinas/metabolismo , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/inmunología , Macrófagos Peritoneales/inmunología , Peritonitis/inmunología , Animales , Antibacterianos/uso terapéutico , Citocinas/inmunología , Modelos Animales de Enfermedad , Gentamicinas/uso terapéutico , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peritonitis/tratamiento farmacológico , Peritonitis/microbiologíaRESUMEN
We previously observed insufficient neutrophil accumulation and a lack of TNF-alpha response at the site of infection until bacteria numbers >10(5) colony forming units in our model of chronic murine peritonitis, suggesting a defective host response after bacterial challenge with Klebsiella pneumoniae (Klebsiella). The aim of this study was to determine a potentially immunosuppressive effect of IL-10 in this model of chronic peritonitis. Balb/c animals were injected with 10(3) colony forming units Klebsiella intraperitoneally. Gentamicin (5 mg/kg/day BID) was given subcutaneously (s.c.) for two days and then withdrawn. Animals were treated with anti-IL-10 antibody or IgG isotype control (s.c.) before or after Klebsiella administration. Survival was determined over 14 days. Similarly treated animals were harvested after 48 h to obtain liver tissue, peritoneal fluid and blood. Bacteria and neutrophil counts were determined. TNF-alpha and IL-10 were measured by ELISA. Anti-IL-10 antibody significantly increased survival and bacterial clearance in the observed compartments. Anti-IL-10 administration did not lead to an increase in TNF-alpha concentrations or neutrophil accumulation at the site of infection at lower levels of Klebsiella. We conclude that endogenous IL-10 is detrimental for survival and bacterial clearance in this model of chronic peritonitis.
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Interleucina-10/inmunología , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/inmunología , Peritonitis/inmunología , Animales , Antibacterianos/farmacología , Anticuerpos/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Gentamicinas/farmacología , Interleucina-10/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos BALB C , Peritonitis/microbiología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.
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Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Peritonitis/genética , Peritonitis/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Citocinas/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-18/inmunología , Interleucina-18/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Neutralización , Factor de Transcripción STAT4 , Bazo/inmunología , Bazo/metabolismo , Tasa de Supervivencia , Transactivadores/deficiencia , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Natural killer (NK) cells are major cytokine producers during bacterial sepsis, but their precise role is undefined. This study investigates the effect of NK cell depletion with and without prior activation on macrophage function and bacterial clearance during cecal ligation and puncture. Two different NK cell-depleting antibodies were used: anti-asialo-GM1 (GM1), a nonactivating antibody, and anti-NK1.1 (NK1.1), an NK cell-activating antibody. C57BL/6 mice were NK depleted with either GM1 or NK1.1 by intraperitoneal injection 7 and 3 days before experimentation. Control animals received isotype immunoglobulin G. Depletion was confirmed by flow cytometry. Bacterial levels in peritoneal washout, blood, and liver were determined 4 hours after cecal ligation and puncture. Macrophage activation was measured by phagocytosis ability and by production of nitric oxide and interleukin-6. Depletion with GM1 resulted in significantly higher bacterial levels at 4 hours, whereas depletion with NK1.1 had the opposite effect of significantly decreasing bacterial levels. Macrophage phagocytosis ability was significantly increased in mice depleted with NK1.1 compared with those mice depleted with GM1. We conclude that activation of NK cells improves bacterial clearance by priming macrophages to help clear a subsequent bacterial challenge. Macrophages are less able to clear bacteria when NK cells are depleted without activation. NK cells are therefore important in bacterial clearance through interactions with macrophages.