Delayed Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Inhibition by Trametinib Attenuates Systemic Inflammatory Responses and Multiple Organ Injury in Murine Sepsis.
ABSTRACT: The mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway is an essential component of innate immunity necessary for mediating proinflammatory responses in the setting of sepsis. We previously demonstrated that the mitogen-activated protein kinase 1/2 inhibitor trametinib prevents endotoxin-induced renal injury in mice. We therefore assessed efficacy of trametinib in a more clinically relevant experimental model of sepsis.Controlled in vivo laboratory study.University animal research laboratory.Male C57BL/6 mice.Mice were subjected to cecal ligation and puncture to induce sepsis or underwent sham operation as controls. Six hours after cecal ligation and puncture, mice were randomized to four experimental groups as follows: 1) sham control; 2) sham control + trametinib (1?mg/kg, IP); 3) cecal ligation and puncture; and 4) cecal ligation and puncture + trametinib. All animals received buprenorphine (0.05?mg/kg, SC) and imipenem/cilastatin (14?mg/kg, SC) in 1.5?mL of warm saline (40?mL/kg) at the 6-hour time point. Mice were euthanized at 18 hours after induction of cecal ligation and puncture.Trametinib inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase signaling 6 hours after cecal ligation and puncture attenuated increases in circulating proinflammatory cytokines (tumor necrosis factor-?, interleukin-1?, interleukin-6, and granulocyte macrophage colony-stimulating factor) and hypothermia at 18 hours. Trametinib also attenuated multiple organ injury as determined by serum creatinine, alanine aminotransferase, lactate dehydrogenase, and creatine kinase. At the organ level, trametinib completely restored peritubular capillary perfusion in the kidney. Restoration of microvascular perfusion was associated with reduced messenger RNA expression of well-characterized markers of proximal tubule injury. mitogen-activated protein kinase/extracellular signal-regulated kinase blockade attenuated cecal ligation and puncture-mediated up-regulation of cytokines (tumor necrosis factor-?, interleukin-1?) and restored interleukin-6 to control levels in the renal cortex, indicating the protective effects on the proximal tubule occur primarily through modulation of the proinflammatory response in sepsis.These data reveal that the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor trametinib attenuates systemic inflammation and multiple organ damage in a clinically relevant model of sepsis. Because trametinib has been safely used in humans, we propose that this drug might represent a translatable approach to limit organ injury in septic patients.
Project description:Peroxisome proliferator-activated receptor-? is significantly down-regulated in circulating leukocytes from children with sepsis. Peroxisome proliferator-activated receptor-? null (Ppara) mice have greater mortality than wild-type mice when subjected to sepsis by cecal ligation and puncture. We sought to characterize the role of peroxisome proliferator-activated receptor-? in sepsis and to identify the mechanism whereby peroxisome proliferator-activated receptor-? confers a survival advantage.Prospective randomized preclinical study.Laboratory investigation.Male C57Bl/6J and Ppara mice (B6.129S4-Ppara/J), aged 12-16 weeks.Bone marrow chimeric mice were generated and subjected to cecal ligation and puncture. Survival was measured for 7 days. Separate groups of nontransplanted mice underwent cecal ligation and puncture and were euthanized 24 hours later for plasma and tissue analyses.Ppara mice had dramatically reduced survival compared with wild-type mice irrespective of the peroxisome proliferator-activated receptor-? status of the bone marrow they received (3% vs 63%; p < 0.0001). No difference in survival was observed between Ppara mice that received wild-type versus Ppara marrow or in wild-type mice receiving wild-type versus Ppara marrow. In septic, nontransplanted mice at 24 hours, Ppara mice had elevated cardiac troponin levels compared with wild-type mice. Cardiac histologic injury scores were greater in Ppara versus wild-type mice. Expression of transcription factors and enzymes related to fatty acid oxidation in the heart were profoundly down-regulated in both wild-type and Ppara mice, but more so in the Ppara mice.Peroxisome proliferator-activated receptor-? expression in nonhematopoietic tissues plays a critical role in determining clinical outcome in experimental polymicrobial sepsis and is more important to survival in sepsis than hematopoietic peroxisome proliferator-activated receptor-? expression. Cardiac injury due to inadequate energy production from fatty acid substrate is a probable mechanism of decreased survival in Ppara mice. These results suggest that altered peroxisome proliferator-activated receptor-?-mediated cellular metabolism may play an important role in sepsis-related end-organ injury and dysfunction, especially in the heart.
Project description:Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release.We investigated the contribution of calpain in eliciting tissue damage during sepsis.To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor.In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy.These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.
Project description:OBJECTIVE:Sepsis-induced multi-organ failure continues to have a high mortality. The liver is an organ central to the disease pathogenesis. The objective of this study was to identify the liver proteins that change in abundance with sepsis and subsequently identify new drug targets. DESIGN:Proteomic discovery study and drug target validation. For the proteomics study, three biological replicate mice were used per group. SETTING:Research institute laboratory. SUBJECTS:Three-month-old C57BL/6 mice. INTERVENTIONS:We used a mouse model of sepsis based on cecal ligation and puncture, but with fluid and antibiotic resuscitation. Liver proteins that changed in abundance were identified by difference in gel electrophoresis. We compared liver proteins from 6-hr post-cecal ligation and puncture to sham-operated mice ("early proteins") and 24-hr post-cecal ligation and puncture with 6-hr post-cecal ligation and puncture ("late proteins"). Proteins that changed in abundance were identified by tandem mass spectrometry. We then inhibited the receptor for one protein and determined the effect on sepsis-induced organ dysfunction. RESULTS:The liver proteins that changed in abundance after sepsis had a range of functions such as acute phase response, coagulation, endoplasmic reticulum stress, oxidative stress, apoptosis, mitochondrial electron transfer proteins, and nitric oxide metabolism. We found that cyclophilin increased in abundance after cecal ligation and puncture. When the receptor for this protein, CD147, was inhibited, sepsis-induced renal dysfunction was reduced. There was also a significant reduction in serum cytokine production when CD147 was inhibited. CONCLUSION:By applying proteomics to a clinically relevant mouse model of sepsis, we identified a number of novel proteins that changed in abundance. The inhibition of the receptor for one of these proteins, cyclophilin, attenuated sepsis-induced acute renal failure. The application of proteomics to sepsis research can facilitate the discovery of new therapeutic targets.
Project description:OBJECTIVES:Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN:Experimental study. SETTING:Research laboratory. SUBJECTS:Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS:Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS:Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS:The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.
Project description:There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the ?-common receptor ('tissue-protective receptor'), which is pharmacologically distinct from the 'classical' erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the ?-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the ?-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and ?-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16-18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a ?-common-receptor-dependent fashion. Activation by erythropoietin of the ?-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3?, nuclear factor-?B and interleukin-1?). All the above pleiotropic effects of erythropoietin were lost in ?-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the ?-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation.
Project description:Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. ?-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of ?-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that ?-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of ?-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, ?-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, ?-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of ?-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.
Project description:RATIONALE:BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. OBJECTIVE:We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. METHODS AND RESULTS:The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis. CONCLUSIONS:Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.
Project description:Sepsis is a life-threatening condition that is caused by an abnormal immune response to infection and can lead to tissue damage, organ failure, and death. Erastin is a small molecule capable of initiating ferroptotic cell death in cancer cells. However, the function of erastin in the inflammatory response during sepsis remains unknown. Here, we showed that erastin ameliorates septic shock induced by cecal ligation and puncture or lipopolysaccharides (LPS) in mice, which was associated with a reduced production of inflammatory mediators such as nitric oxide, tumor necrosis factor (TNF)-?, and interleukin (IL)-1?. Pretreatment with erastin in bone marrow-derived macrophages (BMDMs) significantly attenuated the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-?, and IL-1? mRNA in response to LPS treatment. Furthermore, we also showed that erastin suppresses phosphorylation of I?B kinase ?, phosphorylation and degradation of I?B?, and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) in LPS-stimulated BMDMs. Our findings suggest that erastin attenuates the inflammatory response by suppressing the NF-?B signaling pathway, resulting in inhibition of sepsis development. This study provides new insights regarding the potential therapeutic properties of erastin in sepsis.
Project description:We determined the contribution of vascular BK channels to endotoxin (lipopolysaccharide, LPS)-induced hypotension, organ damage, and mortality using smooth muscle BK channel deficiency (BK channel ?1-subunit knockout, BK ?1-KO) mice. BK ?1-KO mice were more sensitive to LPS-induced mortality compared with wild-type mice. After LPS (20 mg/kg, intraperitoneally), BK ?1-KO mice had a more rapid fall in heart rate and blood pressure (measured by radiotelemetry), shorter latency to mortality, and higher mortality rate than wild-type mice. Twenty-two hours after LPS treatment, wild-type and BK ?1-KO mice had reduced norepinephrine reactivity and impaired constrictor responses to the BK channel blocker paxilline in mesenteric arteries in vitro and higher iNOS expression in the heart, but not in mesenteric arteries. Endotoxemic BK ?1-KO mice also showed more severe lung and intestinal injury, higher myeloperoxidase activity and polymorphonuclear neutrophil infiltration in lung and liver. Endotoxemic BK ?1-KO mice had higher plasma tumor necrosis factor ? and interleukin 6 levels at 22 hours, but not 6 hours post-LPS. Exaggerated mortality in BK ?1-KO mice also occurred in the cecal ligation/puncture model of septic shock. Reduced vascular BK channel function does not protect against hypotension in the early stage of septic shock; in the later stage, smooth muscle BK channel deficiency enhances organ damage and mortality.
Project description:Selegiline, an anti-Parkinson drug, has antioxidant and anti-apoptotic effects. To explore the effect of selegiline on sepsis, we used a clinically relevant animal model of polymicrobial sepsis. Cecal ligation and puncture (CLP) or sham operation was performed in male rats under anesthesia. Three hours after surgery, animals were randomized to receive intravenously selegiline (3 mg/kg) or an equivalent volume of saline. The administration of CLP rats with selegiline (i) increased arterial blood pressure and vascular responsiveness to norepinephrine, (ii) reduced plasma liver and kidney dysfunction, (iii) attenuated metabolic acidosis, (iv) decreased neutrophil infiltration in liver and lung, and (v) improved survival rate (from 44% to 65%), compared to those in the CLP alone rats. The CLP-induced increases of plasma interleukin-6, organ superoxide levels, and liver inducible nitric oxide synthase and caspase-3 expressions were ameliorated by selegiline treatment. In addition, the histological changes in liver and lung were significantly attenuated in the selegiline -treated CLP group compared to those in the CLP group. The improvement of organ dysfunction and survival through reducing inflammation, oxidative stress and apoptosis in peritonitis-induced sepsis by selegiline has potential as an adjuvant agent for critical ill.