RESUMEN
Hydrogen sulfide (H2S) is now recognized as an endogenous signaling gasotransmitter in mammals. It is produced by mammalian cells and tissues by various enzymes - predominantly cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - but part of the H2S is produced by the intestinal microbiota (colonic H2S-producing bacteria). Here we summarize the available information on the production and functional role of H2S in the various cell types typically associated with innate immunity (neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, basophils, eosinophils) and adaptive immunity (T and B lymphocytes) under normal conditions and as it relates to the development of various inflammatory and immune diseases. Special attention is paid to the physiological and the pathophysiological aspects of the oral cavity and the colon, where the immune cells and the parenchymal cells are exposed to a special "H2S environment" due to bacterial H2S production. H2S has many cellular and molecular targets. Immune cells are "surrounded" by a "cloud" of H2S, as a result of endogenous H2S production and exogenous production from the surrounding parenchymal cells, which, in turn, importantly regulates their viability and function. Downregulation of endogenous H2S producing enzymes in various diseases, or genetic defects in H2S biosynthetic enzyme systems either lead to the development of spontaneous autoimmune disease or accelerate the onset and worsen the severity of various immune-mediated diseases (e.g. autoimmune rheumatoid arthritis or asthma). Low, regulated amounts of H2S, when therapeutically delivered by small molecule donors, improve the function of various immune cells, and protect them against dysfunction induced by various noxious stimuli (e.g. reactive oxygen species or oxidized LDL). These effects of H2S contribute to the maintenance of immune functions, can stimulate antimicrobial defenses and can exert anti-inflammatory therapeutic effects in various diseases.
Asunto(s)
Inmunidad Adaptativa , Gasotransmisores/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sistema Inmunológico/metabolismo , Inmunidad Innata , Animales , Antiinflamatorios/farmacología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Autoinmunidad , Bacterias/inmunología , Bacterias/metabolismo , Gasotransmisores/inmunología , Gasotransmisores/farmacología , Microbioma Gastrointestinal/inmunología , Interacciones Huésped-Patógeno , Humanos , Sulfuro de Hidrógeno/inmunología , Sulfuro de Hidrógeno/farmacología , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Transducción de SeñalRESUMEN
The biological mediator hydrogen sulfide (H2S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H2S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus Elevations in H2S levels increased the resistance of both species to immune-mediated killing. Clearances of infections with wild-type and genetically H2S-deficient E. coli and S. aureus were compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H2S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H2S production can increase the susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H2S as a bacterial defense mechanism against the host response and implicate bacterial H2S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.
Asunto(s)
Infecciones por Escherichia coli/patología , Escherichia coli/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Sulfuro de Hidrógeno/metabolismo , Infecciones Estafilocócicas/patología , Staphylococcus aureus/crecimiento & desarrollo , Animales , Escherichia coli/inmunología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Evasión Inmune , Leucocitos/inmunología , Masculino , Ratones Endogámicos BALB C , Viabilidad Microbiana , Sepsis/microbiología , Sepsis/patología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Infección de Heridas/microbiología , Infección de Heridas/patologíaRESUMEN
Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 µM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.
Asunto(s)
Antiinflamatorios/uso terapéutico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Ciego , Citocinas/sangre , ADN/efectos de los fármacos , Reposicionamiento de Medicamentos , Femenino , Humanos , Ligadura , Hígado/efectos de los fármacos , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Recuento de Linfocitos , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Punciones , Sepsis/sangre , Sepsis/inmunología , Sepsis/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Células U937RESUMEN
BACKGROUND: Patients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset. METHODS: Mice were subjected to full-thickness scald burns under deep anesthesia and were provided analgesia. pDCs were depleted by injection of anti-plasmacytoid dendritic cell antigen-1 antibodies. Survival, bacterial clearance, and neutrophil responses in vivo and in vitro were measured. RESULTS: Depletion of preexisting pDCs, but not FL-expanded pDCs, abrogated the beneficial effects of FL on survival, bacterial clearance, and neutrophil migration in response to burn wound infection. This requisite role of pDCs for FL-mediated enhancement of neutrophil migratory capacity is not due to direct effects of pDCs on neutrophils. cDCs, but not pDCs, directly increased neutrophil migratory capacity after co-culture. CONCLUSIONS: The protective effects of FL treatment after burn injury are mediated by both pDCs and cDCs. Pharmacological enhancement of both DC subtypes by FL is a potential therapeutic intervention to enhance immune responses to infection and improve outcome after burn injury.
Asunto(s)
Quemaduras/inmunología , Células Dendríticas/inmunología , Proteínas de la Membrana/metabolismo , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/fisiología , Sepsis/inmunología , Animales , Quemaduras/microbiología , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Activación NeutrófilaRESUMEN
Adequate wound healing is vital for burn patients to reduce the risk of infections and prolonged hospitalization. Dendritic cells (DCs) are antigen presenting cells that release cytokines and are central for the activation of innate and acquired immune responses. Studies have showed their presence in human burn wounds; however, their role in burn wound healing remains to be determined. This study investigated the role of DCs in modulating healing responses within the burn wound. A murine model of full-thickness contact burns was used to study wound healing in the absence of DCs (CD11c promoter-driven diphtheria toxin receptor transgenic mice) and in a DC-rich environment (using fms-like tyrosine kinase-3 ligand, FL- a DC growth factor). Wound closure was significantly delayed in DC-deficient mice and was associated with significant suppression of early cellular proliferation, granulation tissue formation, wound levels of TGFß1 and formation of CD31+ vessels in healing wounds. In contrast, DC enhancement significantly accelerated early wound closure, associated with increased and accelerated cellular proliferation, granulation tissue formation, and increased TGFß1 levels and CD31+ vessels in healing wounds. We conclude that DCs play an important role in the acceleration of early wound healing events, likely by secreting factors that trigger the proliferation of cells that mediate wound healing. Therefore, pharmacological enhancement of DCs may provide a therapeutic intervention to facilitate healing of burn wounds.
Asunto(s)
Quemaduras , Proliferación Celular/fisiología , Células Dendríticas/fisiología , Tejido de Granulación , Neovascularización Fisiológica/fisiología , Cicatrización de Heridas/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Antígeno CD11c , Proliferación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Masculino , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neovascularización Fisiológica/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
INTRODUCTION: The chemokine CXCL10 is produced during infection and inflammation to activate the chemokine receptor CXCR3, an important regulator of lymphocyte trafficking and activation. The goal of this study was to assess the contributions of CXCL10 to the pathogenesis of experimental septic shock in mice. METHODS: Septic shock was induced by cecal ligation and puncture (CLP) in mice resuscitated with lactated Ringer's solution and, in some cases, the broad spectrum antibiotic Primaxin. Studies were performed in CXCL10 knockout mice and mice treated with anti-CXCL10 immunoglobulin G (IgG). Endpoints included leukocyte trafficking and activation, core body temperature, plasma cytokine concentrations, bacterial clearance and survival. RESULTS: CXCL10 was present at high concentrations in plasma and peritoneal cavity during CLP-induced septic shock. Survival was significantly improved in CXCL10 knockout (CXCL10KO) mice and mice treated with anti-CXCL10 IgG compared to controls. CXCL10KO mice and mice treated with anti-CXCL10 IgG showed attenuated hypothermia, lower concentrations of interleukin-6 (IL-6) and macrophage inhibitory protein-2 (MIP-2) in plasma and lessened natural killer (NK) cell activation compared to control mice. Compared to control mice, bacterial burden in blood and lungs was lower in CXCL10-deficient mice but not in mice treated with anti-CXCL10 IgG. Treatment of mice with anti-CXCL10 IgG plus fluids and Primaxin at 2 or 6 hours after CLP significantly improved survival compared to mice treated with non-specific IgG under the same conditions. CONCLUSIONS: CXCL10 plays a role in the pathogenesis of CLP-induced septic shock and could serve as a therapeutic target during the acute phase of septic shock.
Asunto(s)
Quimiocina CXCL10/metabolismo , Choque Séptico/inmunología , Animales , Temperatura Corporal , Quimiocina CXCL10/antagonistas & inhibidores , Quimiocina CXCL10/sangre , Citocinas/sangre , Femenino , Inmunoglobulina G/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/fisiología , Choque Séptico/microbiologíaRESUMEN
RATIONALE: Lymphocytes have been shown to facilitate systemic inflammation and physiologic dysfunction in experimental models of severe sepsis. Our previous studies show that natural killer (NK) cells migrate into the peritoneal cavity during intraabdominal sepsis, but the trafficking of NKT and T lymphocytes has not been determined. The factors that regulate lymphocyte trafficking during sepsis are currently unknown. OBJECTIVES: To ascertain the importance of CXC chemokine receptor 3 (CXCR3) as a regulator of lymphocyte trafficking during sepsis and determine the contribution of CXCR3-mediated lymphocyte trafficking to the pathogenesis of septic shock. METHODS: Lymphocyte trafficking was evaluated in control and CXCR3-deficient mice using flow cytometry during sepsis caused by cecal ligation and puncture (CLP). Survival, core temperature, cytokine production, and bacterial clearance were measured as pathobiological endpoints. MEASUREMENTS AND MAIN RESULTS: This study shows that concentrations of the CXCR3 ligands CXCL9 (monokine induced by interferon γ, MIG) and CXCL10 (interferon γ-induced protein 10, IP-10) increase in plasma and the peritoneal cavity after CLP, peak at 8 hours after infection, and are higher in the peritoneal cavity than in plasma. The numbers of CXCR3(+) NK cells progressively decreased in spleen after CLP with a concomitant increase within the peritoneal cavity, a pattern that was ablated in CXCR3-deficient mice. CXCR3-dependent recruitment of T cells was also evident at 16 hours after CLP. Treatment of mice with anti-CXCR3 significantly attenuated CLP-induced hypothermia, decreased systemic cytokine production, and improved survival. CONCLUSIONS: CXCR3 regulates NK- and T-cell trafficking during sepsis and blockade of CXCR3 attenuates the pathogenesis of septic shock.
Asunto(s)
Infecciones Intraabdominales/inmunología , Células Asesinas Naturales/metabolismo , Receptores CXCR3/metabolismo , Choque Séptico/inmunología , Linfocitos T/metabolismo , Animales , Movimiento Celular/fisiología , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Infecciones Intraabdominales/mortalidad , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/metabolismo , Receptores CXCR3/antagonistas & inhibidores , Choque Séptico/mortalidadRESUMEN
Burn patients are highly susceptible to infections due to increased exposure through wounds and impairments in a number of immune functions. Dendritic cells (DCs) are important in activation of numerous immune responses that are essential for the clearance of infections. We have found that prophylactic treatment of burn-injured mice with the DC growth factor FLT3 ligand (FL) significantly increases resistance to burn wound infections in a DC-dependent manner that is correlated closely with enhanced bacterial clearance. However, as DCs are not typically microbicidal, the mechanisms by which DC modulation enhances bacterial clearance are not known. Due to the rapid response of neutrophils to cutaneous wounds, and the reported interactions between DCs and neutrophils, we investigated the role of neutrophils in FL-mediated resistance to burn wound infection. This was examined both in vivo and in vitro through neutrophil depletion, supplementation of neutrophils, and assessment of neutrophil chemotaxis following FL treatment. To test the involvement of DCs, CD11c-diphtheria toxin receptor transgenic mice were used to deplete DCs during FL treatment. Studies revealed that neutrophils do play a critical role in FL-mediated resistance to a burn wound infection. Additionally, treatment with FL after a burn injury enhances neutrophil-mediated control of bacterial spread, neutrophil migratory capacity, and myeloperoxidase production in a DC-dependent manner. The results of this study provide new insight into immunological mechanisms that can offer protection against infection after burn injury.
Asunto(s)
Quemaduras/inmunología , Quemaduras/terapia , Células Dendríticas/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/terapia , Infección de Heridas/inmunología , Infección de Heridas/terapia , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Traslado Adoptivo , Animales , Quemaduras/microbiología , Comunicación Celular/inmunología , Células Dendríticas/microbiología , Células Dendríticas/trasplante , Modelos Animales de Enfermedad , Calor , Inmunidad Innata , Masculino , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Infiltración Neutrófila/inmunología , Neutrófilos/microbiología , Neutrófilos/trasplante , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/inmunología , Infección de Heridas/microbiologíaRESUMEN
INTRODUCTION: In our previous studies we demonstrated that CXC chemokine receptor 3 (CXCR3) participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 immunoglobulin (IgG) and antibiotics on outcome during septic shock caused by CLP. METHODS: C57BL/6J mice were treated with neutralizing IgG against CXCR3 plus Primaxin either 24 hours prior to, 2 hours after or 6 hours after CLP. Control mice received nonspecific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated. RESULTS: Our results show that treatment with anti-CXCR3 IgG plus Primaxin significantly improved survival when administered 24 hours prior to CLP (50% vs. 10%), 2 hours after CLP (55% vs. 10%) or 6 hours after CLP (55% vs. 25%) compared with mice receiving nonspecific IgG plus Primaxin. Treatment with anti-CXCR3 plus Primaxin 24 hours prior to CLP attenuated hypothermia and IL-6 and macrophage inflammatory protein 2 (MIP-2) production but did not alter bacterial clearance. Treatment with anti-CXCR3 IgG and Primaxin 2 hours after CLP did not improve bacterial clearance and systemic cytokine production compared with mice treated with IgG and Primaxin, whereas 6 hours after CLP the bacterial clearance and IL-6 and MIP-2 concentrations, both in plasma and peritoneal lavage fluid, were significantly improved in mice receiving anti-CXCR3 IgG and Primaxin compared with mice that only received nonspecific IgG and Primaxin. CONCLUSION: The results from this study indicate that neutralization of CXCR3 prior to, 2 hours after or 6 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.
Asunto(s)
Anticuerpos Antiidiotipos/uso terapéutico , Modelos Animales de Enfermedad , Inmunoglobulina G , Receptores CXCR3/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Animales , Anticuerpos Antiidiotipos/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores CXCR3/metabolismo , Sepsis/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: This study was designed to determine the ability of insulin to improve outcome following a Pseudomonas aeruginosa wound infection in a rodent model of severe burn injury. BACKGROUND: Severe burn injury predisposes patients to burn wound infections that can disseminate, lead to uncontrolled inflammation, and induce septic shock. Whereas insulin administration has been extensively discussed to improve morbidity and mortality in critically ill patients, the ability of insulin to improve outcomes of severely burned patients with infected burn wounds is not known. DESIGN: Sprague-Dawley rats. SETTING: University setting. INTERVENTION: Burn-injured Sprague Dawley rats were randomized into treatment groups that received either saline or insulin. Burn wounds were topically inoculated with a lethal dose of Pseudomonas aeruginosa 6 days after injury. MEASUREMENTS AND MAIN RESULTS: Survival, systemic dissemination of bacteria, systemic inflammation, and immune activation were examined. Insulin decreased the early inflammatory response to a severe burn injury. Treatment with low doses of insulin following burn injury improved the outcome of rats in response to a lethal burn wound infection. Specifically, survival was improved and systemic dissemination of bacteria from the wound was decreased. Systemic inflammation, indicated by serum interleukin-6 levels, was significantly decreased by insulin treatments after injury. Additionally, insulin treatments were associated with alterations in B and T lymphocyte responses to wound infection. CONCLUSIONS: Although the mechanisms by which insulin improves outcome following a lethal burn wound infection are not known, the data suggest that immunologic responses to infection may be altered by postburn insulin treatments.
Asunto(s)
Bacteriemia/tratamiento farmacológico , Quemaduras/tratamiento farmacológico , Insulina/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Infección de Heridas/tratamiento farmacológico , Animales , Bacteriemia/mortalidad , Bacteriemia/prevención & control , Quemaduras/complicaciones , Quemaduras/mortalidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Citometría de Flujo , Inflamación/prevención & control , Activación de Linfocitos/efectos de los fármacos , Análisis Multivariante , Probabilidad , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/mortalidad , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Estadísticas no Paramétricas , Tasa de Supervivencia , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiología , Infección de Heridas/complicaciones , Infección de Heridas/mortalidadRESUMEN
BACKGROUND: Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine and dendritic cell (DC) growth factor that promotes the proliferation and differentiation of progenitor cells into DCs. We have previously found that treatment of severely burned mice with recombinant Flt3L significantly enhances DC production and bacterial clearance from infected burn wounds, and increases global immune cell activation and survival in response to a burn wound infection. These significant benefits of Flt3L treatment after burn injury have prompted the question of whether or not severe burn injury induces deficits in endogenous Flt3L levels that could affect DCs and subsequent responses to infection. RESULTS: To address this, male BALB/c mice received a 30% total body surface area scald burn. Blood, spleens, and wound-draining lymph nodes were harvested at various time-points after injury. Some mice received a wound inoculation with P. aeruginosa. Murine Flt3L and G-CSF levels were measured by ELISA. Burn injury had no significant effect on Flt3L levels at any post-burn time-point examined compared to normal Flt3L levels in the sera, spleen, or lymph nodes. Additionally, Flt3L levels in the sera, spleen, and lymph nodes were not significantly altered when wounds were inoculated on the day of burn injury or at post-burn time points examined. Alternatively, levels of G-CSF were increased in response to burn injury and burn wound infection. Additionally, DC numbers and functions were not altered following burn injury alone. There was no significant difference between the number of DCs in the spleens of sham-injured mice and mice at 5 days after burn injury. When naïve T cells from sham-injured mice were co-cultured with DCs from either sham- or burn-injured mice, IFN-gamma production was similar, however, IFN-gamma levels produced by T cells harvested from burn-injured mice were significantly lower than those produced by T cells from sham mice, regardless of which DC group, sham or burn, was used in the coculture. CONCLUSION: These data suggest that the beneficial effects of Flt3L treatments after burn injury are not due to correction of a burn-associated Flt3L deficiency but rather, are likely due to supplementary stimulation of DC production and immune responses to infection.
Asunto(s)
Quemaduras/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Proteínas de la Membrana/metabolismo , Infecciones por Pseudomonas/inmunología , Linfocitos T/inmunología , Animales , Quemaduras/metabolismo , Quemaduras/microbiología , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Factor Estimulante de Colonias de Granulocitos/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Pseudomonas/microbiología , Linfocitos T/metabolismo , Linfocitos T/microbiologíaRESUMEN
BACKGROUND: Burn injury induces immunosuppression and promotes infection with opportunistic pathogens. Pneumonia and sepsis are leading causes of post-burn morbidity and mortality. Fms-like tyrosine kinase-3 ligand (Flt3L) improves local and systemic resistance to P aeruginosa-associated burn wound infection. This study evaluates the effects of post-burn prophylactic Flt3L treatment on local and systemic infection and inflammation in a murine model of pneumonia and sepsis. METHODS: Mice received a severe scald burn, were treated with Flt3L or vehicle (CTR) for 5 days, and inoculated trans-nasally with P aeruginosa. Lung, blood, and spleen were harvested at 24 and 48âh postinoculation (p.i.) to assess infection (bacterial burden, bacteremia, distant organ manifestation) and inflammation (interleukin-6 (IL-6) and myeloperoxidase (MPO) levels). Histology correlated infection and inflammation parameters with morphology. Survival at various bacterial concentrations was monitored for 14 days p.i. RESULTS: Bacterial burden was significantly reduced in lung and spleen of Flt3L-treated mice. Flt3L treatment was associated with decreased signs of pulmonary inflammation (reduced wet weight and IL-6 levels), lower incidences of bacteremia and septic distant organ manifestation, and reduced systemic inflammation (IL-6 and MPO). Histologically, reduced alveolar and peribronchiolar neutrophil and lymphocyte infiltration indicated attenuated pulmonary inflammation after Flt3L treatment. Overall survival was comparable between groups for all doses of P aeruginosa, but mortality delayed in the Flt3L-treated group. CONCLUSION: Prophylactic treatment with Flt3L could augment antimicrobial therapy of post-burn pneumonia through improvement of the initial host response to challenge with P aeruginosa, attenuate local, and systemic inflammation as well as septic pathogen dissemination.
Asunto(s)
Bacteriemia/metabolismo , Quemaduras/metabolismo , Proteínas de la Membrana/metabolismo , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Animales , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Bacteriemia/patología , Quemaduras/tratamiento farmacológico , Quemaduras/microbiología , Quemaduras/patología , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/patologíaRESUMEN
BACKGROUND: A complete understanding of the role of the liver in burn-induced hypermetabolism is lacking. We investigated the acute effect of severe burn trauma on liver mitochondrial respiratory capacity and coupling control as well as the signaling events underlying these alterations. METHODS: Male BALB/c mice (8-12 weeks) received full-thickness scald burns on â¼30% of the body surface. Liver tissue was harvested 24âh postinjury. Mitochondrial respiration was determined by high-resolution respirometry. Citrate synthase activity was determined as a proxy of mitochondrial density. Male Sprague-Dawley rats received full-thickness scald burns to â¼60% of the body surface. Serum was collected 24âh postinjury. HepG2 cells were cultured with serum-enriched media from either sham- or burn-treated rats. Protein levels were analyzed via western blot. RESULTS: Mass-specific (Pâ=â0.01) and mitochondrial-specific (Pâ=â0.01) respiration coupled to ATP production significantly increased in the liver after burn. The respiratory control ratio for ADP (Pâ=â0.04) and the mitochondrial flux control ratio (Pâ=â0.03) were elevated in the liver of burned animals. Complex III and Complex IV protein abundance in the liver increased after burn by 17% and 14%, respectively. Exposure of HepG2 cells to serum from burned rats increased the pAMPKα:AMPKα ratio (Pâ<â0.001) and levels of SIRT1 (Pâ=â0.01), Nrf2 (Pâ<â0.001), and PGC1α (Pâ=â0.02). CONCLUSIONS: Severe burn trauma augments respiratory capacity and function of liver mitochondria, adaptations that augment ATP production. This response may be mediated by systemic factors that activate signaling proteins responsible for regulating cellular energy metabolism and mitochondrial biogenesis.
Asunto(s)
Quemaduras/metabolismo , Mitocondrias Hepáticas/metabolismo , Mitocondrias/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Transporte de Electrón/fisiología , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-DawleyRESUMEN
The present study was undertaken to determine whether the mice depleted of alphabeta or gammadelta T cells show resistance to acute polymicrobial sepsis caused by cecal ligation and puncture (CLP). T-cell receptor beta knockout (betaTCRKO) and T-cell receptor delta knockout (deltaTCRKO) mice were used. An additional group of mice was treated with an antibody against the alphabeta T-cell receptor to induce alphabeta T-cell depletion; a subset of alphabeta T cell-deficient mice was also treated with anti-asialoGM1 to deplete natural killer (NK) cells. The mice underwent CLP and were monitored for survival, temperature, acid-base balance, bacterial counts, and cytokine production. The betaTCRKO mice and the wild-type mice treated with anti-beta T-cell receptor (anti-TCRbeta) antibody showed improved survival after CLP compared with wild-type mice. The treatment of alphabeta T cell-deficient mice with anti-asialoGM1further improved survival after CLP, especially when the mice were treated with imipenem. The improved survival observed in alphabeta T cell-deficient mice was associated with less hypothermia, improved acid-base balance, and decreased production of the proinflammatory cytokines interleukin (IL) 6 and macrophage inflammatory protein (MIP) 2. Compared with wild-type controls, the overall survival was not improved in deltaTCRKO mice. The concentrations of IL-6 and MIP-2 in plasma and cytokine mRNA expression in tissues were not significantly different between wild-type and deltaTCRKO mice. These studies indicate that mice depleted of alphabeta but not of gammadelta T cells are resistant to mortality in an acutely lethal model of CLP. The depletion of NK cells caused further survival benefit in alphabeta T cell-deficient mice. These findings suggest that alphabeta T and NK cells mediate or facilitate CLP-induced inflammatory injury.
Asunto(s)
Ciego/lesiones , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Linfocitos T/inmunología , Animales , Antibacterianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Bacteriemia/inmunología , Bacteriemia/mortalidad , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Quimiocina CXCL2 , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Imipenem/uso terapéutico , Interleucina-6/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ligadura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocinas/metabolismo , Punciones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Sepsis/mortalidad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Temperatura , Factores de TiempoRESUMEN
Severe burn injury produces a plethora of metabolic abnormalities which contribute to the prolonged morbidity of burn survivors. The authors have recently demonstrated trans-differentiation of white adipose tissue (WAT) after burn trauma, toward a more thermogenic phenotype. However, the impact of burn injury on subcutaneous WAT (sWAT) morphology in humans is unknown. Here, the authors studied the effect of severe burn injury on the architecture of sWAT. sWAT was collected from 11 severely burned children (11 ± 3 years; 55 ± 16% total BSA burned) and 12 nonburned healthy children (9 ± 3 years). Histology, electron microscopy, immunohistochemistry, and immunofluorescence were performed on fixed adipose tissue sections. sWAT cytokine and collagen concentrations were measured by multiplex assay and sirius/fast green staining method, respectively. sWAT histology demonstrated multiple fat droplets, significantly (P < .05) reduced mean cell size (104 ± 6 vs 68 ± 3 µm) and higher collagen content (7 ± 0.8 vs 4 ± 0.4) in burn patients. sWAT from burn victims stained positive for CD68 suggesting infiltration of macrophages. Furthermore, electron microscopic analysis showed multiple fat droplets and greater mitochondrial abundance in sWAT of burn survivors. In agreement with this, mitochondrial respiratory capacity in the leak and coupled state increased by 100% in sWAT of burned children from 1 to 3 weeks postinjury. The cytokines IL-6, IL-8, IL-13, IL-1a, IL-1b, MCP-1, and TNF-α were all significantly greater in the sWAT of burned children versus healthy children (P < .05). Furthermore, IL-6, IL-8, IL1-a, IL-1b, and TNF-α significantly increased after injury in sWAT of burned children (P < .05). This study provides detailed evidence of morphological and functional changes in sWAT of burn survivors which was associated with tissue inflammation. A better understanding of morphological and functional changes in sWAT will help discern the mechanisms underlying hypermetabolism in burned patients.
Asunto(s)
Tejido Adiposo Blanco/lesiones , Tejido Adiposo Blanco/metabolismo , Quemaduras/metabolismo , Grasa Subcutánea/lesiones , Grasa Subcutánea/metabolismo , Estudios de Casos y Controles , Niño , Colágeno/metabolismo , Citocinas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica , FenotipoRESUMEN
Altered skeletal muscle mitochondrial function contributes to the pathophysiological stress response to burns. However, the acute and chronic impact of burn trauma on skeletal muscle bioenergetics remains poorly understood. Here, we determined the temporal relationship between burn trauma and mitochondrial function in murine skeletal muscle local to and distal from burn wounds. Male BALB/c mice (8-10 weeks old) were burned by submersion of the dorsum in water (â¼ 95 °C) to create a full thickness burn on â¼ 30% of the body. Skeletal muscle was harvested spinotrapezius underneath burn wounds (local) and the quadriceps (distal) of sham and burn treated mice at 3h, 24h, 4d and 10d post-injury. Mitochondrial respiration was determined in permeabilized myofiber bundles by high-resolution respirometry. Caspase 9 and caspase 3 protein concentration were determined by western blot. In muscle local to burn wounds, respiration coupled to ATP production was significantly diminished at 3h and 24h post-injury (P<0.001), as was mitochondrial coupling control (P<0.001). There was a 5- (P<0.05) and 8-fold (P<0.001) increase in respiration in response to cytochrome at 3h and 24h post burn, respectively, indicating damage to the outer mitochondrial membranes. Moreover, we also observed greater active caspase 9 and caspase 3 in muscle local to burn wounds, indicating the induction of apoptosis. Distal muscle mitochondrial function was unaltered by burn trauma until 10d post burn, where both respiratory capacity (P<0.05) and coupling control (P<0.05) were significantly lower than sham. These data highlight a differential response in muscle mitochondrial function to burn trauma, where the timing, degree and mode of dysfunction are dependent on whether the muscle is local or distal to the burn wound.
Asunto(s)
Quemaduras/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Enfermedad Aguda , Animales , Apoptosis , Músculos de la Espalda/metabolismo , Músculos de la Espalda/patología , Western Blotting , Respiración de la Célula , Enfermedad Crónica , Masculino , Ratones , Ratones Endogámicos BALB C , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Músculo Cuádriceps/metabolismo , Músculo Cuádriceps/patologíaRESUMEN
Brown adipose tissue (BAT) plays an important role in mammalian thermoregulation. The component of BAT mitochondria that permits this function is the inner membrane carrier protein uncoupling protein 1 (UCP1). To the best of our knowledge, no studies have directly quantified UCP1 function in human BAT. Further, whether human and rodent BAT have comparable thermogenic function remains unknown. We employed high-resolution respirometry to determine the respiratory capacity, coupling control, and, most importantly, UCP1 function of human supraclavicular BAT and rodent interscapular BAT. Human BAT was sensitive to the purine nucleotide GDP, providing the first direct evidence that human BAT mitochondria have thermogenically functional UCP1. Further, our data demonstrate that human and rodent BAT have similar UCP1 function per mitochondrion. These data indicate that human and rodent BAT are qualitatively similar in terms of UCP1 function.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Mitocondrias/metabolismo , Proteína Desacopladora 1/metabolismo , Tejido Adiposo Pardo/ultraestructura , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/ultraestructura , Animales , Respiración de la Célula , Humanos , Masculino , Ratones Endogámicos BALB C , Mitocondrias/ultraestructura , Músculo Esquelético/metabolismo , CuelloRESUMEN
These studies evaluated the effects treatment with glucan phosphate, a soluble polysaccharide immunomodulator, on the inflammatory response induced by burn injury and on resistance to Pseudomonas aeruginosa burn wound infection. Mice were exposed to 35% total body surface area burns and were resuscitated with lactated Ringer's (LR) solution alone or LR supplemented with glucan phosphate (40 mg/kg). Glucan phosphate treatment attenuated burn-induced expression of interleukin (IL)-1beta, IL-6, and IL-10 mRNAs in spleen, lung, and heart. Plasma concentrations of IL-1beta, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10 were also decreased in burned mice treated with glucan phosphate compared with vehicle-treated controls. Early postburn mortality was not significantly different between control (20%) and glucan phosphate-treated (10%) mice, but there was a small improvement in acid-base balance in the glucan phosphate-treated group. Mice received a second injection of glucan phosphate or LR on day 4 postburn and were infected by topical application of P. aeruginosa to the burn wound on day 5. Glucan phosphate treatment significantly improved survival in mice exposed to P. aeruginosa burn wound infection. The improved survival correlated with lower bacterial burden in the burn wound, attenuated production of proinflammatory cytokines, and enhanced production of Th1 cytokines. These studies show that glucan phosphate treatment attenuates burn-induced inflammation and increases resistance to P. aeruginosa burn wound infection in an experimental model of burn injury.
Asunto(s)
Quemaduras/tratamiento farmacológico , Glucanos/uso terapéutico , Inflamación/prevención & control , Infecciones por Pseudomonas/prevención & control , Infección de Heridas/prevención & control , Equilibrio Ácido-Base/efectos de los fármacos , Animales , Quemaduras/complicaciones , Quemaduras/genética , Quemaduras/inmunología , Quimiocina CXCL2 , Quimiocinas/sangre , Femenino , Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Interferón gamma/biosíntesis , Interleucina-1/biosíntesis , Interleucina-1/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Interleucina-6/genética , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Chronic cold exposure induces functionally thermogenic mitochondria in the inguinal white adipose tissue (iWAT) of mice. Whether this response occurs in pathophysiological states remains unclear. The purpose of this study was to determine the impact of severe burn trauma on iWAT mitochondrial function in mice. Male BALB/c mice (10-12 weeks) received full-thickness scald burns to â¼30% of the body surface area. Inguinal white adipose tissue was harvested from mice at 1, 4, 10, 20, and 40 days postinjury. Total and uncoupling protein 1 (UCP1)-dependent mitochondrial thermogenesis were determined in iWAT. Citrate synthase activity was determined as a proxy of mitochondrial abundance. Immunohistochemistry was performed to assess iWAT morphology and UCP1 expression. Uncoupling protein 1-dependent respiration was significantly greater at 4 and 10 days after burn compared with sham, peaking at 20 days after burn (P < 0.001). Citrate synthase activity was threefold greater at 4, 10, 20, and 40 days after burn versus sham (P < 0.05). Per mitochondrion, UCP1 function increased after burn trauma (P < 0.05). After burn trauma, iWAT exhibited numerous multilocular lipid droplets that stained positive for UCP1. The current findings demonstrate the induction of thermogenically competent mitochondria within rodent iWAT in a model of severe burn trauma. These data identify a specific pathology that induces the browning of white adipose tissue in vivo and may offer a mechanistic explanation for the chronic hypermetabolism observed in burn victims.