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1.
Curr Protoc ; 2(1): e342, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35038380

RESUMO

The protocols presented here describe steps for cryosectioning tissue samples to be used in light microscopy methodologies including histochemistry, enzyme immunohistochemistry, and immunofluorescence. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cryosectioning.

2.
Curr Protoc ; 2(1): e343, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35030299

RESUMO

Gravity flow whole-body perfusion maintains effective and reproducible preservation of tissue architecture critical to investigate pathobiology of multiple organs from the same specimen. The purpose of the protocols described within this article is to help the researcher optimize tissue harvest for multisystem pathobiology comparison. The protocols presented here describe tissue harvest for processing and cryopreservation to generate optimal samples for microscopy and parallel biochemical and molecular biology analysis. First, this paper outlines a protocol for tissue perfusion and organ harvest that allows the researcher multiple analysis options from the same research subject simultaneously. Second, this paper outlines a model to optimize ex-vivo tissue fixation for precious human sample preparation. Finally, this paper outlines a methodology for freezing tissue samples to optimize their capacity for biochemical and immunohistochemical analysis. Benefits and alternative approaches to retain cellular morphology in tissue harvest and processing are discussed. Also described within each section are common technical issues to assist problem-solving and troubleshooting. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Whole body in vivo tissue perfusion by gravity flow: preparation and surgical procedures Alternate Protocol: Human ex vivo tissue fixation Basic Protocol 2: Freezing of tissue samples.


Assuntos
Manejo de Espécimes , Coleta de Tecidos e Órgãos , Humanos , Perfusão , Fixação de Tecidos
3.
Artigo em Inglês | MEDLINE | ID: mdl-34889809

RESUMO

Acute kidney injury (AKI) is common after trauma, but contributory factors are incompletely understood. Increases in plasma von Willebrand Factor (vWF) with concurrent decreases in ADAMTS13 are associated with renal microvascular thrombosis in other disease states, but similar findings have not been shown in trauma. We hypothesized that molecular changes in circulating vWF and ADAMTS13 promote AKI following traumatic injury. VWF antigen, vWF multimer composition and ADAMTS13 levels were compared in plasma samples from 16 trauma patients with and without trauma-induced AKI, obtained from the Prehospital Air Medical Plasma (PAMPer) biorepository. Renal histopathology and function, vWF and ADAMTS13 levels were assessed in parallel in a murine model of polytrauma and haemorrhage. VWF antigen was higher in trauma patients when compared with healthy controls [314% (253-349) vs. 100% (87-117)] [median (IQR)], while ADAMTS13 activity was lower [36.0% (30.1-44.7) vs. 100.0% (83.1-121.0)]. Patients who developed AKI showed significantly higher levels of high molecular weight multimeric vWF at 72-h when compared with non-AKI counterparts [32.9% (30.4-35.3) vs. 27.8% (24.6-30.8)]. Murine plasma cystatin C and vWF were elevated postpolytrauma model in mice, with associated decreases in ADAMTS13, and immunohistologic analysis demonstrated renal injury with small vessel plugs positive for fibrinogen and vWF. Following traumatic injury, the vWF-ADAMTS13 axis shifted towards a prothrombotic state in both trauma patients and a murine model. We further demonstrated that vWF-containing, microangiopathic deposits were concurrently produced as the prothrombotic changes were sustained during the days following trauma, potentially contributing to AKI development.

4.
Artigo em Inglês | MEDLINE | ID: mdl-34890842

RESUMO

INTRODUCTION: Pyroptosis, gasdermin-mediated programmed cell death, is readily induced in macrophages by activation of the canonical inflammasome (caspase-1), or by intracellular lipopolysaccharide (LPS)-mediated non-canonical inflammasome (caspase-11) activation. However, whether pyroptosis is induced similarly in hepatocytes is still largely controversial but highly relevant to liver pathologies such as alcoholic/non-alcoholic liver disease, drug-induced liver injury, ischemia-reperfusion and liver transplant injury, or organ damage secondary to sepsis. APPROACH AND RESULTS: In this study, we find that hepatocytes activate and cleave gasdermin-D (GSDMD) at low levels after treatment with LPS. Overexpression of caspase-1 or caspase-11 p10/p20 activated domains was able to induce typical GSDMD-dependent pyroptosis in hepatocytes both in vitro and in vivo. However, morphological features of pyroptosis in macrophages (e.g. pyroptotic bodies, cell flattening, loss of cell structure) did not occur in pyroptotic hepatocytes, with cell structure remaining relatively intact despite the cell membrane being breached. Our results suggest that hepatocytes activate pyroptosis pathways and cleave GSDMD, but this does not result in cell rupture and confer the same pyroptotic morphologic changes as previously reported in macrophages. This is even with caspase-1 or caspase-11 artificial overexpression way above levels seen endogenously even after priming or in pathological conditions. CONCLUSION: Our novel findings characterize hepatocyte morphology in pyroptosis, and suggest alternative use for canonical/non-canonical inflammasome activation/signaling, and subsequent GSDMD cleavage, as there is no rapid cell death as in macrophages. Improved understanding and recognition of the role of these pathways in hepatocytes may result in novel therapeutics for a range of liver diseases.

5.
Cancer Med ; 10(20): 7233-7241, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34559451

RESUMO

INTRODUCTION: Preoperative autophagy inhibition with hydroxychloroquine (HCQ) in combination with gemcitabine in pancreatic adenocarcinoma (PDAC) has been shown to be safe and effective in inducing a serum biomarker response and increase resection rates in a previous phase I/II clinical trial. We aimed to analyze the long-term outcomes of preoperative HCQ with gemcitabine for this cohort. METHODS: A review of patients enrolled between July 2010 and February 2013 in the completed phase I/II single arm (two doses of fixed-dose gemcitabine (1500 mg/m2 ) in combination with oral hydroxychloroquine administered for 31 consecutive days until the day of surgery for high-risk pancreatic cancer) was undertaken. Progression-free survival (PFS) and overall survival analysis (OS) using Kaplan-Meier estimates were performed. RESULTS: Of 35 patients initially enrolled, 29 patients underwent surgical resection (median age at diagnosis: 62 years, 45% females). Median duration of follow-up was 7.5 years. There was a median 15% decrease in the serum CA19-9 levels following completion of neoadjuvant therapy and 83% of the cohort underwent a pancreaticoduodenectomy, 7 (24%) patients had a concomitant venous resection. On histopathology, 14 (48%) patients had at least a partial treatment response. The median PFS and OS were 11 months (95% Confidence interval [CI]: 7-28) and 31 months (95% CI: 13-47), respectively, while 9 (31%) patients survived beyond 5 years from diagnosis; a rate that compares very favorably with contemporaneous series. CONCLUSION: Compared to historical data, neoadjuvant autophagy inhibition with HCQ plus gemcitabine is associated with encouraging long-term survival for patients with PDAC.

6.
Cancer Res ; 81(9): 2373-2385, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33687949

RESUMO

Surgical removal of malignant tumors is a mainstay in controlling most solid cancers. However, surgical insult also increases the risk of tumor recurrence and metastasis. Tissue trauma activates the innate immune system locally and systemically, mounting an inflammatory response. Platelets and neutrophils are two crucial players in the early innate immune response that heals tissues, but their actions may also contribute to cancer cell dissemination and distant metastasis. Here we report that surgical stress-activated platelets enhance the formation of platelet-tumor cell aggregates, facilitating their entrapment by neutrophil extracellular traps (NET) and subsequent distant metastasis. A murine hepatic ischemia/reperfusion (I/R) injury model of localized surgical stress showed that I/R promotes capturing of aggregated circulating tumor cells (CTC) by NETs and eventual metastasis to the lungs, which are abrogated when platelets are depleted. Hepatic I/R also increased deposition of NETs within the lung microvasculature, but depletion of platelets had no effect. TLR4 was essential for platelet activation and platelet-tumor cell aggregate formation in an ERK5-GPIIb/IIIa integrin-dependent manner. Such aggregation facilitated NET-mediated capture of CTCs in vitro under static and dynamic conditions. Blocking platelet activation or knocking out TLR4 protected mice from hepatic I/R-induced metastasis with no CTC entrapment by NETs. These results uncover a novel mechanism where platelets and neutrophils contribute to metastasis in the setting of acute inflammation. Targeted disruption of the interaction between platelets and NETs holds therapeutic promise to prevent postoperative distant metastasis. SIGNIFICANCE: Targeting platelet activation via TLR4/ERK5/integrin GPIIb/IIIa signaling shows potential for preventing NET-driven distant metastasis in patients post-resection.


Assuntos
Plaquetas/imunologia , Armadilhas Extracelulares/metabolismo , Fígado/lesões , Neoplasias Pulmonares/secundário , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células Neoplásicas Circulantes/metabolismo , Neutrófilos/imunologia , Traumatismo por Reperfusão/metabolismo , Receptor 4 Toll-Like/deficiência , Animais , Plaquetas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recidiva Local de Neoplasia , Neutrófilos/metabolismo , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética
7.
Mol Med ; 27(1): 18, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33632134

RESUMO

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 fármacos
8.
Hepatology ; 73(6): 2494-2509, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32924145

RESUMO

BACKGROUND AND AIMS: Liver ischemia/reperfusion injury (IRI) induces local and systemic inflammation in which neutrophil extracellular traps (NETs) are major drivers. IRI markedly augments metastatic growth, which is consistent with the notion that the liver IRI can serve as a premetastatic niche. Exercise training (ExT) confers a sustainable protection, reducing IRI in some animal models, and has been associated with improved survival in patients with cancer; however, the impact of ExT on liver IRI or development of hepatic metastases is unknown. APPROACH AND RESULTS: Mice were randomized into exercise (ExT) and sedentary groups before liver IRI and tumor injection. Computerized dynamic network analysis of 20 inflammatory mediators was used to dissect the sequence of mediator interactions after ischemia/reperfusion (I/R) that induce injury. ExT mice showed a significant decrease in hepatic IRI and tissue necrosis. This coincided with disassembly of complex networks among inflammatory mediators seen in sedentary mice. Neutrophil infiltration and NET formation were decreased in the ExT group, which suppressed the expression of liver endothelial cell adhesion molecules. Concurrently, ExT mice revealed a distinct population of infiltrating macrophages expressing M2 phenotypic genes. In a metastatic model, fewer metastases were present 3 weeks after I/R in the ExT mice, a finding that correlated with a marked increase in tumor-suppressing T cells within the tumor microenvironment. CONCLUSIONS: ExT preconditioning mitigates the inflammatory response to liver IRI, protecting the liver from injury and metastases. In light of these findings, potential may exist for the reduction of liver premetastatic niches induced by liver IRI through the use of ExT as a nonpharmacologic therapy before curative surgical approaches.

9.
JCI Insight ; 6(2)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33320841

RESUMO

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.


Assuntos
Ferimentos e Lesões/genética , Ferimentos e Lesões/imunologia , Adulto , Animais , Células da Medula Óssea/imunologia , Queimaduras/sangue , Queimaduras/genética , Queimaduras/imunologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA-Seq , Sepse/sangue , Sepse/genética , Sepse/imunologia , Choque Hemorrágico/sangue , Choque Hemorrágico/genética , Choque Hemorrágico/imunologia , Análise de Célula Única , Fatores de Tempo , Transcriptoma , Ferimentos e Lesões/classificação , Adulto Jovem
10.
PLoS One ; 15(11): e0239119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33137133

RESUMO

Autophagy is an important factor in liver ischemia-reperfusion injury. In the current study we investigate the function of interferon regulatory factor-1 (IRF1) in regulating autophagy to promote hepatic ischemia reperfusion injury (IR). The high expression of IRF1 during hepatic IR exhibited increased liver damage and was associated with activation of autophagy shown by Western blot markers, as well as immunofluorescent staining for autophagosomes. These effects were diminished by IRF1 deficiency in IRF1 knock out (KO) mice. Moreover, the autophagy inhibitor 3-MA decreased IR-induced liver necrosis and markedly abrogated the rise in liver injury tests (AST/ALT). ß-catenin expression decreased during liver IR and was increased in the IRF1 KO mice. Immunoprecipitation assay showed the binding between IRF1 and ß-catenin. Overexpression of IRF1 induced autophagy and also inhibited the expression of ß-catenin. ß-catenin inhibitor increased autophagy while ß-catenin agonist suppressed autophagy in primary mouse hepatocytes. These results indicate that IRF1 induced autophagy aggravates hepatic IR injury in part by inhibiting ß-catenin and suggests that targeting IRF1 may be an effective strategy in reducing hepatic IR injury.


Assuntos
Autofagia/fisiologia , Fator Regulador 1 de Interferon/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Traumatismo por Reperfusão/metabolismo , beta Catenina/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
11.
Mol Med ; 26(1): 115, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33238880

RESUMO

BACKGROUND: Circulating high-mobility group box 1 (HMGB1) plays important roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Intracellular HMGB1 is critical for the biology of hepatocytes. However, the intracellular role of HMGB1 in hepatocellular steatosis is unknown. Therefore, we aimed to investigate the role of hepatocyte-specific HMGB1 (HC-HMGB1) in development of hepatic steatosis. METHODS: Wild type (WT) C57BL/6 and HC-HMGB1-/- mice were fed high-fat diet (HFD) or low-fat diet (LFD) for up to 16 weeks. RESULTS: As expected, HMGB1 translocated from nuclear into cytoplasm and released into circulation after HFD treatment. HC-HMGB1 deficiency significantly reduced circulating HMGB1, suggesting that hepatocyte is a major source of circulating HMGB1 during NAFLD. Unexpectedly, HC-HMGB1 deficiency promoted rapid weight gain with enhanced hepatic fat deposition compared with WT at as early as 4 weeks after HFD treatment. Furthermore, there was no difference between WT and HC-HMGB1-/- mice in glucose tolerance, energy expenditure, liver damage or systemic inflammation. Interestingly, hepatic gene expression related to free fatty acid (FFA) ß-oxidation was significantly down-regulated in HC-HMGB1-/- mice compared with WT, and endoplasmic reticulum (ER) stress markers were significantly higher in livers of HC-HMGB1-/- mice. In vitro experiments using primary mouse hepatocytes showed absence of HMGB1 increased FFA-induced intracellular lipid accumulation, accompanied by increased ER-stress, significant downregulation of FFA ß-oxidation, and reduced oxidative phosphorylation. CONCLUSIONS: Our findings suggest that hepatocyte HMGB1 protects against dysregulated lipid metabolism via maintenance of ß-oxidation and prevention of ER stress. This represents a novel mechanism for HMGB1-regulation of hepatocellular steatosis, and suggests that stabilizing HMGB1 in hepatocytes may be effective strategies for prevention and treatment of NAFLD.


Assuntos
Dieta Hiperlipídica , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Proteína HMGB1/genética , Hepatócitos/metabolismo , Estresse Fisiológico , Animais , Biópsia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Proteína HMGB1/sangue , Proteína HMGB1/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Oxirredução
12.
Sci Adv ; 6(39)2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32967837

RESUMO

Dysregulation of T cell apoptosis contributes to the pathogenesis of acute systemic inflammation-induced immunosuppression, as seen in sepsis and trauma. However, the regulatory mechanisms of T cell apoptosis are unclear. Activation of stimulator of interferon genes (STING) has been shown to induce T cell apoptosis. Notch was previously identified as the top negative regulator of STING in macrophages through a kinase inhibitor library screening. However, how Notch signaling regulates STING activation in T cells is unknown. Here, using a γ-secretase inhibitor to block Notch signaling, we found that Notch protected CD4 T cells from STING-mediated apoptosis during endotoxemia. Mechanistically, Notch intracellular domain (NICD) interacted with STING at the cyclic dinucleotide (CDN) binding domain and competed with CDN to inhibit STING activation. In conclusion, our data reveal a previously unidentified role of Notch in negative regulation of STING-mediated apoptosis in CD4 T cells.

13.
Mol Med ; 26(1): 69, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641037

RESUMO

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.


Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/patologia , Isquemia/patologia , Músculo Esquelético/patologia , Pneumonia Viral/patologia , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Betacoronavirus , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Glicólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Fosforilação Oxidativa , Pandemias , Doença Arterial Periférica/patologia , Pneumonia Viral/tratamento farmacológico , Regeneração , SARS-CoV-2 , Transdução de Sinais
14.
Front Immunol ; 11: 987, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32528475

RESUMO

Innate immunity can initiate platelet activation during the development of thrombosis through a process, termed immunothrombosis. Neutrophils form neutrophil extracellular traps (NETs) that have been shown to interact directly with platelets and play pro-coagulant roles in a variety of infectious and sterile inflammatory settings. Hepatic surgical stress initiated by ischemia/reperfusion (I/R) injury has wide systemic consequences on distant organs. However, the mechanisms of this remote injury phenomenon are not well-understood. Here, we sought to determine the role of NETs in causing systemic immunothrombosis and distant organ injury following a local inflammatory insult with liver I/R. Postoperative thromboelastographic revealed that the speed of clot formation (alpha-angle) was significantly increased whereas time to clot formation (R-time) were decreased by in patients undergoing liver resection, indicating a hypercoagulable state after surgery. In mice subjected to liver I/R, circulating platelet activation and platelet-neutrophil aggregates were significantly increased. Injured distant organs such as the lung and kidney displayed NETs and platelet-rich micro-thrombi in the microvasculature following liver I/R. The immune-thrombi and organ damage were dramatically decreased when NETs were inhibited by DNase treatment. Depletion of Tlr4 on platelets limited NET-induced activation of platelets but had no effect on NET formation. Furthermore, platelet-specific TLR4 KO mice had significantly reduced distant organ injury with decreased circulating platelet activation, platelet-neutrophil aggregates following liver I/R in comparison to their control counterparts. These data establish that after an acute local inflammatory process, NET-activated platelets can lead to a systemic pro-coagulant state with resultant remote organ injury by immunothrombosis.


Assuntos
Coagulação Sanguínea , Plaquetas/imunologia , Armadilhas Extracelulares/imunologia , Hepatectomia/efeitos adversos , Neutrófilos/imunologia , Ativação Plaquetária , Traumatismo por Reperfusão/imunologia , Trombose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Plaquetas/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/deficiência , Proteína-Arginina Desiminase do Tipo 4/genética , Traumatismo por Reperfusão/sangue , Transdução de Sinais , Estresse Fisiológico , Trombose/sangue , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Adulto Jovem
15.
Front Immunol ; 11: 229, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32328059

RESUMO

High-mobility group box-1 (HMGB1), a ubiquitous nuclear protein, acts as a late mediator of lethality when released extracellularly during sepsis. The major source of circulating HMGB1 in sepsis is hepatocytes. However, the mechanism of HMGB1 release of hepatocytes during sepsis is not very clear. We have previously shown that bacterial endotoxin [lipopolysaccharide (LPS)] sensing pathways, including Toll-like receptor (TLR)4 and caspase-11, regulate hepatocyte HMGB1 release in response to LPS. Here, we report the novel function of caspase-11 and gasdermin D (GsdmD) in LPS-induced active HMGB1 released from hepatocytes. HMGB1 release during endotoxemia was caspase-11/GsdmD dependent via an active way in vivo and in vitro. Caspase-11/GsdmD was responsible for HMGB1 translocation from nucleus to the cytoplasm via calcium changing-induced phosphorylation of calcium-calmodulin kinase kinase (camkk)ß during endotoxemia. Cleaved GsdmD accumulated on the endoplasmic reticulum, suggesting this may lead to calcium leak and intracellular calcium increase. Furthermore, we investigated that exosome was an important pathway for HMGB1 release from hepatocytes; this process was dependent on TLR4, independent of caspase-11 and GsdmD in vivo and in vitro. These findings provide a novel mechanism that TLR4 signaling results in an increase in caspase-11 expression, as well as increased exosome release, while caspase-11/GsdmD activation/cleavage leads to accumulation of HMGB1 in the cytoplasm through a process associated with the release of calcium from the endoplasmic reticulum and camkkß activation.


Assuntos
Caspases Iniciadoras/metabolismo , Exossomos/metabolismo , Proteína HMGB1/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Sepse/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Cálcio/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Caspases Iniciadoras/genética , Retículo Endoplasmático/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Transdução de Sinais , Receptor 4 Toll-Like/genética
16.
Antioxid Redox Signal ; 33(1): 1-19, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32103677

RESUMO

Aims: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies. Results: In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria. Innovation: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis. Conclusion: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.


Assuntos
Gastroenterite/etiologia , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mitofagia/genética , Animais , Antioxidantes/farmacologia , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Colite/etiologia , Colite/metabolismo , Colite/patologia , Óxidos N-Cíclicos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Gastroenterite/metabolismo , Gastroenterite/patologia , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta
17.
Hepatology ; 72(4): 1394-1411, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-31997373

RESUMO

BACKGROUND AND AIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. APPROACH AND RESULTS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CONCLUSIONS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.


Assuntos
Anti-Inflamatórios/farmacologia , Carboxiliases/fisiologia , Hepatócitos/metabolismo , Fígado/irrigação sanguínea , Fator 2 Relacionado a NF-E2/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Succinatos/farmacologia , Animais , Humanos , Hidroliases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Transdução de Sinais/fisiologia , Succinatos/uso terapêutico
18.
JCI Insight ; 4(22)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31723054

RESUMO

Thymic stromal lymphopoietin (TSLP) is a cytokine mainly released by epithelial cells that plays important roles in inflammation, autoimmune disease, and cancer. While TSLP is expressed in the liver at high levels, the role of TSLP in liver ischemia/reperfusion (I/R) injury remains unknown. Experiments were carried out to determine the role of TSLP in liver I/R injury. Wild-type (WT) and TSLP receptor-knockout (TSLPR-/-) mice were subjected to liver partial warm I/R injury. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) level, necrotic areas by liver histology, hepatocyte death, and local hepatic inflammatory responses. Signal pathways were explored in vivo and in vitro to identify possible mechanisms for TSLP in I/R injury. TSLP and TSLPR protein expression increased during liver I/R in vivo and following hepatocyte hypoxia/reoxygenation in vitro. Deletion of TSLPR or neutralization of TSLP with anti-TSLP antibody exacerbated liver injury in terms of serum ALT levels as well as necrotic areas in liver histology. Administration of exogenous recombinant mouse TSLP to WT mice significantly reduced liver damage compared with controls, but failed to prevent I/R injury in TSLPR-/- mice. TSLP induced autophagy in hepatocytes during liver I/R injury. Mechanistically, Akt was activated in WT mice during liver I/R injury. The opposite results were observed in TSLPR-/- mice. In addition, TSLP could directly induce Akt activation in hepatocytes independent of nonparenchymal cells in vitro. Furthermore, the Akt agonist, insulin-like growth factor-1 (IGF-1), prevented I/R injury in TSLPR-/- mice and an Akt inhibitor, LY294002, blocked the protective effects of TSLP in WT mice subjected to I/R. Our data indicate that TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. Through this pathway, TSLP induces autophagy in hepatocytes. Thus, TSLP is a potent inhibitor of stress-induced hepatocyte necrosis.


Assuntos
Citocinas , Fígado/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Fígado/citologia , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
19.
Cancer Res ; 79(21): 5626-5639, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31519688

RESUMO

Neutrophil infiltration and neutrophil extracellular traps (NET) in solid cancers are associated with poorer prognosis, but the mechanisms are incompletely understood. We hypothesized that NETs enhance mitochondrial function in tumor cells, providing extra energy for accelerated growth. Metastatic colorectal cancer tissue showed increased intratumoral NETs and supranormal preoperative serum MPO-DNA, a NET marker. Higher MPO-DNA correlated with shorter survival. In mice, subcutaneous tumor implants and hepatic metastases grew slowly in PAD4-KO mice, genetically incapable of NETosis. In parallel experiments, human cancer cell lines grew slower in nu/nu mice treated with DNAse, which disassembles NETs. PAD4-KO tumors manifested decreased proliferation, increased apoptosis, and increased evidence of oxidative stress. PAD4-KO tumors had decreased mitochondrial density, mitochondrial DNA, a lesser degree of ATP production, along with significantly decreased mitochondrial biogenesis proteins PGC1α, TFAM, and NRF-1. In vitro, cancer cells treated with NETs upregulated mitochondrial biogenesis-associated genes, increased mitochondrial density, increased ATP production, enhanced the percentage of cancer cells with reduced mitochondrial membrane potential, and increased the oxygen consumption rate. Furthermore, NETs increased cancer cells' expression of fission and fusion-associated proteins, DRP-1 and MFN-2, and mitophagy-linked proteins, PINK1 and Parkin. All of which were decreased in PAD4-KO tumors. Mechanistically, neutrophil elastase released from NETs activated TLR4 on cancer cells, leading to PGC1α upregulation, increased mitochondrial biogenesis, and accelerated growth. Taken together, NETs can directly alter the metabolic programming of cancer cells to increase tumor growth. NETs represent a promising therapeutic target to halt cancer progression. SIGNIFICANCE: Neutrophils through the release of NETs facilitate the growth of stressed cancer cells by altering their bioenergetics, the inhibition of which induces cell death.


Assuntos
Proliferação de Células/fisiologia , Armadilhas Extracelulares/fisiologia , Homeostase/fisiologia , Mitocôndrias/fisiologia , Neutrófilos/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Mitocondrial/metabolismo , Armadilhas Extracelulares/metabolismo , Células HCT116 , Humanos , Elastase de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo
20.
J Clin Invest ; 129(9): 3657-3669, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31380807

RESUMO

Fibroblastic reticular cells (FRCs), a subpopulation of stromal cells in lymphoid organs and fat-associated lymphoid clusters (FALCs) in adipose tissue, play immune-regulatory roles in the host response to infection and may be useful as a form of cell therapy in sepsis. Here, we found an unexpected major role of TLR9 in controlling peritoneal immune cell recruitment and FALC formation at baseline and after sepsis induced by cecal ligation and puncture (CLP). TLR9 regulated peritoneal immunity via suppression of chemokine production by FRCs. Adoptive transfer of TLR9-deficient FRCs more effectively decreased mortality, bacterial load, and systemic inflammation after CLP than WT FRCs. Importantly, we found that activation of TLR9 signaling suppressed chemokine production by human adipose tissue-derived FRCs. Together, our results indicate that TLR9 plays critical roles in regulating peritoneal immunity via suppression of chemokine production by FRCs. These data form a knowledge basis upon which to design new therapeutic strategies to improve the therapeutic efficacy of FRC-based treatments for sepsis and immune dysregulation diseases.


Assuntos
Tecido Adiposo/metabolismo , Fibroblastos/metabolismo , Peritônio/imunologia , Reticulina/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Linfócitos B/metabolismo , Quimiocina CXCL13/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritônio/patologia , Sepse/metabolismo , Transdução de Sinais
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