RESUMO
Extensive studies demonstrate the importance of the STING1 (also known as STING) protein as a signaling hub that coordinates immune and autophagic responses to ectopic DNA in the cytoplasm. Here, we report a nuclear function of STING1 in driving the activation of the transcription factor aryl hydrocarbon receptor (AHR) to control gut microbiota composition and homeostasis. This function was independent of DNA sensing and autophagy and showed competitive inhibition with cytoplasmic cyclic guanosine monophosphate (GMP)-AMP synthase (CGAS)-STING1 signaling. Structurally, the cyclic dinucleotide binding domain of STING1 interacted with the AHR N-terminal domain. Proteomic analyses revealed that STING1-mediated transcriptional activation of AHR required additional nuclear partners, including positive and negative regulatory proteins. Although AHR ligands could rescue colitis pathology and dysbiosis in wild-type mice, this protection was abrogated by mutational inactivation of STING1. These findings establish a key framework for understanding the nuclear molecular crosstalk between the microbiota and the immune system.
Assuntos
Proteômica , Receptores de Hidrocarboneto Arílico , Animais , Camundongos , DNA , Homeostase , Intestinos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
Heparin, a mammalian polysaccharide, is a widely used anticoagulant medicine to treat thrombotic disorders. It is also known to improve outcomes in sepsis, a leading cause of mortality resulted from infection-induced immune dysfunction. Whereas it is relatively clear how heparin exerts its anticoagulant effect, the immunomodulatory mechanisms enabled by heparin remain enigmatic. Here, we show that heparin prevented caspase-11-dependent immune responses and lethality in sepsis independent of its anticoagulant properties. Heparin or a chemically modified form of heparin without anticoagulant function inhibited the alarmin HMGB1-lipopolysaccharide (LPS) interaction and prevented the macrophage glycocalyx degradation by heparanase. These events blocked the cytosolic delivery of LPS in macrophages and the activation of caspase-11, a cytosolic LPS receptor that mediates lethality in sepsis. Survival was higher in septic patients treated with heparin than those without heparin treatment. The identification of this previously unrecognized heparin function establishes a link between innate immune responses and coagulation.
Assuntos
Anticoagulantes/uso terapêutico , Caspases/metabolismo , Heparina/uso terapêutico , Macrófagos/imunologia , Sepse/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Caspases/genética , Linhagem Celular , Feminino , Glucuronidase/genética , Glucuronidase/metabolismo , Glicocálix/metabolismo , Proteína HMGB1/metabolismo , Humanos , Imunomodulação , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Sepse/mortalidade , Análise de Sobrevida , Adulto JovemRESUMO
Excessive activation of the coagulation system leads to life-threatening disseminated intravascular coagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. We found that caspase-11, a cytosolic LPS receptor, activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores and subsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD pores mediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, or neutralization of phosphatidylserine or TF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α and IL-1ß, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.
Assuntos
Caspases Iniciadoras/metabolismo , Coagulação Intravascular Disseminada/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosfatidilserinas/metabolismo , Tromboplastina/metabolismo , Animais , Coagulação Sanguínea/fisiologia , Caspases Iniciadoras/genética , Linhagem Celular Tumoral , Endotoxemia/patologia , Ativação Enzimática , Células HT29 , Células HeLa , Humanos , Interleucina-1alfa/sangue , Interleucina-1beta/sangue , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Piroptose/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.
Assuntos
Caspases/imunologia , Endotoxinas/imunologia , Proteína HMGB1/imunologia , Piroptose/imunologia , Sepse/imunologia , Animais , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotoxinas/metabolismo , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada/imunologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Sepse/genética , Sepse/metabolismo , Células THP-1RESUMO
Sepsis-induced acute kidney injury (S-AKI) is highly lethal, and effective drugs for treatment are scarce. Previously, we reported the robust therapeutic efficacy of fibroblastic reticular cells (FRCs) in sepsis. Here, we demonstrate the ability of FRC-derived exosomes (FRC-Exos) to improve C57BL/6 mouse kidney function following cecal ligation and puncture-induced sepsis. In vivo imaging confirmed that FRC-Exos homed to injured kidneys. RNA-Seq analysis of FRC-Exo-treated primary kidney tubular cells (PKTCs) revealed that FRC-Exos influenced PKTC fate in the presence of lipopolysaccharide (LPS). FRC-Exos promoted kinase PINK1-dependent mitophagy and inhibited NLRP3 inflammasome activation in LPS-stimulated PKTCs. To dissect the mechanism underlying the protective role of Exos in S-AKI, we examined the proteins within Exos by mass spectrometry and found that CD5L was the most upregulated protein in FRC-Exos compared to macrophage-derived Exos. Recombinant CD5L treatment in vitro attenuated kidney cell swelling and surface bubble formation after LPS stimulation. FRCs were infected with a CD5L lentivirus to increase CD5L levels in FRC-Exos, which were then modified in vitro with the kidney tubular cell targeting peptide LTH, a peptide that binds to the biomarker protein kidney injury molecule-1 expressed on injured tubule cells, to enhance binding specificity. Compared with an equivalent dose of recombinant CD5L, the modified CD5L-enriched FRC-Exos selectively bound PKTCs, promoted kinase PINK-ubiquitin ligase Parkin-mediated mitophagy, inhibiting pyroptosis and improved kidney function by hindering NLRP3 inflammasome activation, thereby improving the sepsis survival rate. Thus, strategies to modify FRC-Exos could be a new avenue in developing therapeutics against kidney injury.
Assuntos
Injúria Renal Aguda , Exossomos , Sepse , Camundongos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Exossomos/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Injúria Renal Aguda/metabolismo , Sepse/complicações , Sepse/metabolismoRESUMO
OBJECTIVE: To create a blueprint for surgical department leaders, academic institutions, and funding agencies to optimally support surgeon-scientists. BACKGROUND: Scientific contributions by surgeons have been transformative across many medical disciplines. Surgeon-scientists provide a distinct approach and mindset toward key scientific questions. However, lack of institutional support, pressure for increased clinical productivity, and growing administrative burden are major challenges for the surgeon-scientist, as is the time-consuming nature of surgical training and practice. METHODS: An American Surgical Association Research Sustainability Task Force was created to outline a blueprint for sustainable science in surgery. Leaders from top NIH-sponsored departments of surgery engaged in video and in-person meetings between January and April 2023. A strength, weakness, opportunities, threats analysis was performed, and workgroups focused on the roles of surgeons, the department and institutions, and funding agencies. RESULTS: Taskforce recommendations: (1) SURGEONS: Growth mindset : identifying research focus, long-term planning, patience/tenacity, team science, collaborations with disparate experts; Skill set : align skills and research, fill critical skill gaps, develop team leadership skills; DEPARTMENT OF SURGERY (DOS): (2) MENTORSHIP: Chair : mentor-mentee matching/regular meetings/accountability, review of junior faculty progress, mentorship training requirement, recognition of mentorship (eg, relative value unit equivalent, awards; Mentor: dedicated time, relevant scientific expertise, extramural funding, experience and/or trained as mentor, trusted advisor; Mentee : enthusiastic/eager, proactive, open to feedback, clear about goals; (3) FINANCIAL SUSTAINABILITY: diversification of research portfolio, identification of matching funding sources, departmental resource awards (eg, T-/P-grants), leveraging of institutional resources, negotiation of formalized/formulaic funds flow investment from academic medical center toward science, philanthropy; (4) STRUCTURAL/STRATEGIC SUPPORT: Structural: grants administrative support, biostats/bioinformatics support, clinical trial and research support, regulatory support, shared departmental laboratory space/equipment; Strategic: hiring diverse surgeon-scientist/scientists faculty across DOS, strategic faculty retention/ recruitment, philanthropy, career development support, progress tracking, grant writing support, DOS-wide research meetings, regular DOS strategic research planning; (5) COMMUNITY AND CULTURE: Community: right mix of faculty, connection surgeon with broad scientific community; Culture: building research infrastructure, financial support for research, projecting importance of research (awards, grand rounds, shoutouts); (6) THE ROLE OF INSTITUTIONS: Foundation: research space co-location, flexible start-up packages, courses/mock study section, awards, diverse institutional mentorship teams; Nurture: institutional infrastructure, funding (eg, endowed chairs), promotion friendly toward surgeon-scientists, surgeon-scientists in institutional leadership positions; Expectations: RVU target relief, salary gap funding, competitive starting salaries, longitudinal salary strategy; (7) THE ROLE OF FUNDING AGENCIES: change surgeon research training paradigm, offer alternate awards to K-awards, increasing salary cap to reflect market reality, time extension for surgeon early-stage investigator status, surgeon representation on study section, focused award strategies for professional societies/foundations. CONCLUSIONS: Authentic recommitment from surgeon leaders with intentional and ambitious actions from institutions, corporations, funders, and society is essential in order to reap the essential benefits of surgeon-scientists toward advancements of science.
Assuntos
Pesquisa Biomédica , Cirurgiões , Humanos , Estados Unidos , Mentores , Docentes , Centros Médicos Acadêmicos , Mobilidade Ocupacional , National Institutes of Health (U.S.)RESUMO
Sepsis and septic shock driven by microbial infections are still among the most challenging health problems, causing 11 million deaths worldwide every year. How does the host's response to pathogen infections effectively restore homeostasis instead of precipitating pathogenic and potentially fatal feedforward reactions? Recently, there have been significant new advances in our understanding of the interface between mammalian immunity and coagulation ('immunocoagulation') and its impact on sepsis. In particular, the release and activation of F3 (the main initiator of coagulation) from and on myeloid or epithelial cells is facilitated by activating inflammasomes and consequent gasdermin D (GSDMD)-mediated pyroptosis, coupled to signaling via high mobility group box 1 (HMGB1), stimulator of interferon response CGAMP interactor 1 (STING1), or sequestosome 1 (SQSTM1). Pharmacological modulation of the immunocoagulation pathways emerge as novel and potential therapeutic strategies for sepsis.
Assuntos
Sepse , Choque Séptico , Animais , Caspases Iniciadoras/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Fosfato , PiroptoseRESUMO
Inflammation, the body's primary defensive response system to injury and infection, is triggered by molecular signatures of microbes and tissue injury. These molecules also stimulate specialized sensory neurons, termed nociceptors. Activation of nociceptors mediates inflammation through antidromic release of neuropeptides into infected or injured tissue, producing neurogenic inflammation. Because HMGB1 is an important inflammatory mediator that is synthesized by neurons, we reasoned nociceptor release of HMGB1 might be a component of the neuroinflammatory response. In support of this possibility, we show here that transgenic nociceptors expressing channelrhodopsin-2 (ChR2) directly release HMGB1 in response to light stimulation. Additionally, HMGB1 expression in neurons was silenced by crossing synapsin-Cre (Syn-Cre) mice with floxed HMGB1 mice (HMGB1f/f). When these mice undergo sciatic nerve injury to activate neurogenic inflammation, they are protected from the development of cutaneous inflammation and allodynia as compared to wild-type controls. Syn-Cre/HMGB1fl/fl mice subjected to experimental collagen antibody-induced arthritis, a disease model in which nociceptor-dependent inflammation plays a significant pathological role, are protected from the development of allodynia and joint inflammation. Thus, nociceptor HMGB1 is required to mediate pain and inflammation during sciatic nerve injury and collagen antibody-induced arthritis.
Assuntos
Proteína HMGB1/metabolismo , Neurônios/fisiologia , Nociceptores/metabolismo , Animais , Anticorpos/imunologia , Artrite/induzido quimicamente , Células Cultivadas , Colágeno/imunologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica , Proteína HMGB1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Neuropatia Ciática/metabolismoRESUMO
OBJECTIVE: Evaluate the association of survival with helicopter transport directly to a trauma center compared with ground transport to a non-trauma center (NTC) and subsequent transfer. SUMMARY BACKGROUND DATA: Helicopter transport improves survival after injury. One potential mechanism is direct transport to a trauma center when the patient would otherwise be transported to an NTC for subsequent transfer. METHODS: Scene patients 16 years and above with positive physiological or anatomic triage criteria within PTOS 2000-2017 were included. Patients transported directly to level I/II trauma centers by helicopter were compared with patients initially transported to an NTC by ground with a subsequent helicopter transfer to a level I/II trauma center. Propensity score matching was used to evaluate the association between direct helicopter transport and survival. Individual triage criteria were evaluated to identify patients most likely to benefit from direct helicopter transport. RESULTS: In all, 36,830 patients were included. Direct helicopter transport was associated with a nearly 2-fold increase in odds of survival compared with NTC ground transport and subsequent transfer by helicopter (aOR 2.78; 95% CI 2.24-3.44, P <0.01). Triage criteria identifying patients with a survival benefit from direct helicopter transport included GCS≤13 (1.71; 1.22-2.41, P <0.01), hypotension (2.56; 1.39-4.71, P <0.01), abnormal respiratory rate (2.30; 1.36-3.89, P <0.01), paralysis (8.01; 2.03-31.69, P <0.01), hemothorax/pneumothorax (2.34; 1.36-4.05, P <0.01), and multisystem trauma (2.29; 1.08-4.84, P =0.03). CONCLUSIONS: Direct trauma center access is a mechanism driving the survival benefit of helicopter transport. First responders should consider helicopter transport for patients meeting these criteria who would otherwise be transported to an NTC.
Assuntos
Resgate Aéreo , Serviços Médicos de Emergência , Ferimentos e Lesões , Humanos , Estudos Retrospectivos , Aeronaves , Triagem , Centros de Traumatologia , Escala de Gravidade do Ferimento , Ferimentos e Lesões/terapiaRESUMO
OBJECTIVE: The aim of this study was to assess the survival impact of low-titer group O whole blood (LTOWB) in injured pediatric patients who require massive transfusion. SUMMARY BACKGROUND DATA: Limited data are available regarding the effectiveness of LTOWB in pediatric trauma. METHODS: A prospective observational study of children requiring massive transfusion after injury at UPMC Children's Hospital of Pittsburgh, an urban academic pediatric Level 1 trauma center. Injured children ages 1 to 17 years who received a total of >40 mL/kg of LTOWB and/or conventional components over the 24 hours after admission were included. Patient characteristics, blood product utilization and clinical outcomes were analyzed using Kaplan-Meier survival curves, log rank tests and Cox proportional hazards regression analyses. The primary outcome was 28-day survival. RESULTS: Of patients analyzed, 27 of 80 (33%) received LTOWB as part of their hemostatic resuscitation. The LTOWB group was comparable to the component therapy group on baseline demographic and physiologic parameters except older age, higher body weight, and lower red blood cell and plasma transfusion volumes. After adjusting for age, total blood product volume transfused in 24 hours, admission base deficit, international normalized ratio (INR), and injury severity score (ISS), children who received LTOWB as part of their resuscitation had significantly improved survival at both 72 hours and 28 days post-trauma [adjusted odds ratio (AOR) 0.23, P = 0.009 and AOR 0.41, P = 0.02, respectively]; 6-hour survival was not statistically significant (AOR = 0.51, P = 0.30). Survivors at 28 days in the LTOWB group had reduced hospital LOS, ICU LOS, and ventilator days compared to the CT group. CONCLUSION: Administration of LTOWB during the hemostatic resuscitation of injured children requiring massive transfusion was independently associated with improved 72-hour and 28-day survival.
Assuntos
Transfusão de Componentes Sanguíneos , Ferimentos e Lesões , Humanos , Criança , Lactente , Pré-Escolar , Adolescente , Plasma , Transfusão de Sangue , Ressuscitação , Estudos Prospectivos , Sistema ABO de Grupos Sanguíneos , Ferimentos e Lesões/terapiaRESUMO
BACKGROUND: Sepsis induces group 2 innate lymphoid cell (ILC2) expansion in the lung. However, the origin of these lung-recruited ILC2 and the mechanism of ILC2 expansion are unclear. This study aims to determine the origin of lung-recruited ILC2 and its underlying mechanism in sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP) model in wild-type, IL-33-deficient and ST2-deficient mice. The frequency, cell number and C-X-C chemokine receptor 4 (CXCR4) expression of ILC2 in bone marrow (BM), blood and lung were measured by flow cytometry. In the in vitro studies, purified ILC2 progenitor (ILC2p) were challenged with IL-33 or G protein-coupled receptor kinase 2 (GRK2) inhibitor, the CXCR4 expression and GRK2 activity were detected by confocal microscopy or flow cytometry. RESULTS: We show that IL-33 acts through its receptor, ST2, on BM ILC2p to induce GRK2 expression and subsequent downregulation of cell surface expression of CXCR4, which results in decreasing retention of ILC2p in the BM and promoting expansion of ILC2 in the lung. Importantly, we demonstrate that reduced IL-33 level in aging mice contributes to impaired ILC2 mobilization from BM and accumulation in the lung following sepsis. CONCLUSION: This study identifies a novel pathway in regulating ILC2p mobilization and expansion during sepsis and indicates BM as the main source of ILC2 in the lung following sepsis.
Assuntos
Interleucina-33 , Sepse , Animais , Quinase 2 de Receptor Acoplado a Proteína G , Imunidade Inata , Proteína 1 Semelhante a Receptor de Interleucina-1 , Pulmão/metabolismo , Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Sepse/metabolismoRESUMO
OBJECTIVES: The authors sought to identify causal factors that explain the selective benefit of prehospital administration of thawed plasma (TP) in traumatic brain injury (TBI) patients using mediation analysis of a multiomic database. BACKGROUND: The Prehospital Air Medical Plasma (PAMPer) Trial showed that patients with TBI and a pronounced systemic response to injury [defined as endotype 2 (E2)], have a survival benefit from prehospital administration of TP. An interrogation of high dimensional proteomics, lipidomics and metabolomics previously demonstrated unique patterns in circulating biomarkers in patients receiving prehospital TP, suggesting that a deeper analysis could reveal causal features specific to TBI patients. METHODS: A novel proteomic database (SomaLogic Inc., aptamer-based assay, 7K platform) was generated using admission blood samples from a subset of patients (n=149) from the PAMPer Trial. This proteomic dataset was combined with previously reported metabolomic and lipidomic datasets from these same patients. A 2-step analysis was performed to identify factors that promote survival in E2-TBI patients who had received early TP. First, features were selected using both linear and multivariate-latent-factor regression analyses. Then, the selected features were entered into the causal mediation analysis. RESULTS: Causal mediation analysis of observable features identified 16 proteins and 41 lipids with a high proportion of mediated effect (>50%) to explain the survival benefit of early TP in E2-TBI patients. The multivariate latent-factor regression analyses also uncovered 5 latent clusters of features with a proportion effect >30%, many in common with the observable features. Among the observable and latent features were protease inhibitors known to inhibit activated protein C and block fibrinolysis (SERPINA5 and CPB2), a clotting factor (factor XI), as well as proteins involved in lipid transport and metabolism (APOE3 and sPLA(2)-XIIA). CONCLUSIONS: These findings suggest that severely injured patients with TBI process exogenous plasma differently than those without TBI. The beneficial effects of early TP in E2-TBI patients may be the result of improved blood clotting and the effect of brain protective factors independent of coagulation.
Assuntos
Lesões Encefálicas Traumáticas , Serviços Médicos de Emergência , Traumatismo Múltiplo , Lesões Encefálicas Traumáticas/terapia , Serviços Médicos de Emergência/métodos , Humanos , Traumatismo Múltiplo/terapia , Plasma , ProteômicaRESUMO
Bacterial infection not only stimulates innate immune responses but also activates coagulation cascades. Overactivation of the coagulation system in bacterial sepsis leads to disseminated intravascular coagulation (DIC), a life-threatening condition. However, the mechanisms by which bacterial infection activates the coagulation cascade are not fully understood. Here we show that type 1 interferons (IFNs), a widely expressed family of cytokines that orchestrate innate antiviral and antibacterial immunity, mediate bacterial infection-induced DIC by amplifying the release of high-mobility group box 1 (HMGB1) into the bloodstream. Inhibition of the expression of type 1 IFNs and disruption of their receptor IFN-α/ßR or downstream effector (eg, HMGB1) uniformly decreased gram-negative bacteria-induced DIC. Mechanistically, extracellular HMGB1 markedly increased the procoagulant activity of tissue factor by promoting the externalization of phosphatidylserine to the outer cell surface, where phosphatidylserine assembles a complex of cofactor-proteases of the coagulation cascades. These findings not only provide novel insights into the link between innate immune responses and coagulation, but they also open a new avenue for developing novel therapeutic strategies to prevent DIC in sepsis.
Assuntos
Coagulação Intravascular Disseminada/imunologia , Endotoxemia/imunologia , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Interferon-alfa/imunologia , Interferon beta/imunologia , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Coagulação Sanguínea , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/etiologia , Endotoxemia/sangue , Endotoxemia/complicações , Infecções por Bactérias Gram-Negativas/sangue , Infecções por Bactérias Gram-Negativas/complicações , Proteína HMGB1/sangue , Proteína HMGB1/imunologia , Humanos , Imunidade Inata , Camundongos Endogâmicos C57BLRESUMO
Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.
Assuntos
Modelos Animais de Doenças , Leucócitos/metabolismo , Fenótipo , Sepse/genética , Transcriptoma , Adulto , Fatores Etários , Idoso , Animais , Ceco/cirurgia , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Punções , Sepse/sangue , Fatores SexuaisRESUMO
The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).
Assuntos
Glutarredoxinas/metabolismo , Glutationa/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , S-Nitrosotióis/metabolismo , Óxidos N-Cíclicos/química , Glutarredoxinas/química , Células Hep G2 , Humanos , Ribonucleosídeo Difosfato Redutase/química , S-Nitrosotióis/química , Marcadores de Spin , Detecção de Spin , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMO
BACKGROUND: Bacterial lipopolysaccharide (LPS) induces a multi-organ, Toll-like receptor 4 (TLR4)-dependent acute inflammatory response. METHODS: Using network analysis, we defined the spatiotemporal dynamics of 20, LPS-induced, protein-level inflammatory mediators over 0-48 h in the heart, gut, lung, liver, spleen, kidney, and systemic circulation, in both C57BL/6 (wild-type) and TLR4-null mice. RESULTS: Dynamic Network Analysis suggested that inflammation in the heart is most dependent on TLR4, followed by the liver, kidney, plasma, gut, lung, and spleen, and raises the possibility of non-TLR4 LPS signaling pathways at defined time points in the gut, lung, and spleen. Insights from computational analyses suggest an early role for TLR4-dependent tumor necrosis factor in coordinating multiple signaling pathways in the heart, giving way to later interleukin-17A-possibly derived from pathogenic Th17 cells and effector/memory T cells-in the spleen and blood. CONCLUSIONS: We have derived novel, systems-level insights regarding the spatiotemporal evolution acute inflammation.
Assuntos
Suscetibilidade a Doenças , Endotoxinas/efeitos adversos , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-17/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Biomarcadores , Biologia Computacional/métodos , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-17/genética , Masculino , Camundongos , Camundongos Transgênicos , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown. METHODS: WT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury. RESULTS: MaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt. CONCLUSIONS: MaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Hepatopatias/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Formil Peptídeo/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Sobrevivência Celular/efeitos dos fármacos , Citocinas/sangue , Ácidos Docosa-Hexaenoicos/farmacologia , Glutationa Peroxidase/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias/sangue , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Malondialdeído/metabolismo , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Receptores de Formil Peptídeo/genética , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: The aim of this study was to determine whether prehospital blood products reduce 30-day mortality in patients at risk for hemorrhagic shock compared with crystalloid only resuscitation. SUMMARY OF BACKGROUND DATA: Hemorrhage is the primary cause of preventable death after injury. Large volume crystalloid resuscitation can be deleterious. The benefits of prehospital packed red blood cells (PRBCs), plasma, or transfusion of both products among trauma patients is unknown compared with crystalloid. METHODS: Secondary analysis of the multicenter PAMPer trial was performed on hypotensive injured patients from the scene. The trial randomized 27 helicopter bases to prehospital plasma or standard resuscitation. Standard resuscitation at the sites was equally divided between crystalloid and crystalloid + PRBC. This led to 4 prehospital resuscitation groups: crystalloid only; PRBC; plasma; and PRBC+plasma. Cox regression determined the association between resuscitation groups and risk-adjusted 30-day mortality. The dose effect of resuscitation fluids was also explored. RESULTS: Four hundred seven patients were included. PRBC+plasma had the greatest benefit [hazard ratio (HR) 0.38; 95% confidence interval (95% CI) 0.26-0.55, P < 0.001], followed by plasma (HR 0.57; 95% CI 0.36-0.91, P = 0.017) and PRBC (HR 0.68; 95% CI 0.49-0.95, P = 0.025) versus crystalloid only. Mortality was lower per-unit of PRBC (HR 0.69; 95% CI 0.52-0.92, p = 0.009) and plasma (HR 0.68; 95% CI 0.54-0.88, P = 0.003). Crystalloid volume was associated with increased mortality among patients receiving blood products (HR 1.65; 95% CI 1.17-2.32, P = 0.004). CONCLUSION: Patients receiving prehospital PRBC+plasma had the greatest mortality benefit. Crystalloid only had the worst survival. Patients with hemorrhagic shock should receive prehospital blood products when available, preferably PRBC+plasma. Prehospital whole blood may be ideal in this population.
Assuntos
Transfusão de Sangue , Soluções Cristaloides/uso terapêutico , Serviços Médicos de Emergência , Ressuscitação , Choque Hemorrágico/mortalidade , Choque Hemorrágico/terapia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Choque Hemorrágico/etiologia , Taxa de Sobrevida , Ferimentos e Lesões/complicações , Ferimentos e Lesões/mortalidade , Ferimentos e Lesões/terapiaRESUMO
BACKGROUND: After a person has been injured, prehospital administration of plasma in addition to the initiation of standard resuscitation procedures in the prehospital environment may reduce the risk of downstream complications from hemorrhage and shock. Data from large clinical trials are lacking to show either the efficacy or the risks associated with plasma transfusion in the prehospital setting. METHODS: To determine the efficacy and safety of prehospital administration of thawed plasma in injured patients who are at risk for hemorrhagic shock, we conducted a pragmatic, multicenter, cluster-randomized, phase 3 superiority trial that compared the administration of thawed plasma with standard-care resuscitation during air medical transport. The primary outcome was mortality at 30 days. RESULTS: A total of 501 patients were evaluated: 230 patients received plasma (plasma group) and 271 received standard-care resuscitation (standard-care group). Mortality at 30 days was significantly lower in the plasma group than in the standard-care group (23.2% vs. 33.0%; difference, -9.8 percentage points; 95% confidence interval, -18.6 to -1.0%; P=0.03). A similar treatment effect was observed across nine prespecified subgroups (heterogeneity chi-square test, 12.21; P=0.79). Kaplan-Meier curves showed an early separation of the two treatment groups that began 3 hours after randomization and persisted until 30 days after randomization (log-rank chi-square test, 5.70; P=0.02). The median prothrombin-time ratio was lower in the plasma group than in the standard-care group (1.2 [interquartile range, 1.1 to 1.4] vs. 1.3 [interquartile range, 1.1 to 1.6], P<0.001) after the patients' arrival at the trauma center. No significant differences between the two groups were noted with respect to multiorgan failure, acute lung injury-acute respiratory distress syndrome, nosocomial infections, or allergic or transfusion-related reactions. CONCLUSIONS: In injured patients at risk for hemorrhagic shock, the prehospital administration of thawed plasma was safe and resulted in lower 30-day mortality and a lower median prothrombin-time ratio than standard-care resuscitation. (Funded by the U.S. Army Medical Research and Materiel Command; PAMPer ClinicalTrials.gov number, NCT01818427 .).
Assuntos
Transfusão de Componentes Sanguíneos , Serviços Médicos de Emergência/métodos , Plasma , Ressuscitação/métodos , Choque Hemorrágico/prevenção & controle , Ferimentos e Lesões/terapia , Adulto , Resgate Aéreo , Transfusão de Componentes Sanguíneos/efeitos adversos , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Tempo de Protrombina , Ferimentos e Lesões/complicações , Ferimentos e Lesões/mortalidadeRESUMO
BACKGROUND: Aicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate the IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS-associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double-stranded RNA regions. Whether an AGS-associated mutation in ADAR1 activates the IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined. METHODS: Mutations in the ADAR1 gene found in AGS patients were introduced into the mouse genome via CRISPR/Cas9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays, respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. RESULTS: Mice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in the mutant brain. CONCLUSIONS: We demonstrated that an AGS-associated mutation in ADAR1, specifically the p.K999N mutation, activates the IFN pathway in the mouse brain. The ADAR1 p.K999N mutant mouse replicates aspects of the brain interferonopathy of AGS. Neurons and microglia express different ISGs. Basal ganglia calcification and leukodystrophy seen in AGS patients were not observed in K999N mutant mice, indicating that development of the full clinical phenotype may need an additional stimulus besides AGS mutations. This mutant mouse presents a robust tool for the investigation of AGS and neuroinflammatory diseases including the modeling of potential "second hits" that enable severe phenotypes of clinically variable diseases.