RESUMEN
The expression of some proteins in the autophagy pathway declines with age, which may impact neurodegeneration in diseases, including Alzheimer's Disease. We have identified a novel non-canonical function of several autophagy proteins in the conjugation of LC3 to Rab5+, clathrin+ endosomes containing ß-amyloid in a process of LC3-associated endocytosis (LANDO). We found that LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic ß-amyloid. This inflammation and ß-amyloid deposition were associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from ß-amyloid deposition.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Endocitosis , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia/deficiencia , Proteína 5 Relacionada con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/genética , Antígenos CD36/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microglía/citología , Microglía/metabolismo , Células RAW 264.7 , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.
Asunto(s)
Linfocitos T CD8-positivos , Factor 4F Eucariótico de Iniciación , Diferenciación Celular , Activación de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.
Asunto(s)
Caspasa 8/genética , Susceptibilidad a Enfermedades , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Necroptosis/genética , Animales , Apoptosis/genética , Biomarcadores , Caspasa 8/química , Caspasa 8/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Inflamasomas/metabolismo , Inflamación/mortalidad , Inflamación/patología , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Mortalidad , Fenotipo , Multimerización de ProteínaRESUMEN
Fungi represent a significant proportion of the gut microbiota. Aberrant immune responses to fungi are frequently observed in inflammatory bowel diseases (IBD) and colorectal cancer (CRC), and mutations in the fungal-sensing pathways are associated with the pathogenesis of IBD. Fungal recognition receptors trigger downstream signaling via the common adaptor protein CARD9 and the kinase SYK. Here we found that commensal gut fungi promoted inflammasome activation during AOM-DSS-induced colitis. Myeloid cell-specific deletion of Card9 or Syk reduced inflammasome activation and interleukin (IL)-18 maturation and increased susceptibility to colitis and CRC. IL-18 promoted epithelial barrier restitution and interferon-γ production by intestinal CD8+ T cells. Supplementation of IL-18 or transfer of wild-type myeloid cells reduced tumor burden in AOM-DSS-treated Card9-/- and Sykfl/flLysMCre/+ mice, whereas treatment with anti-fungal agents exacerbated colitis and CRC. CARD9 deletion changes the gut microbial landscape, suggesting that SYK-CARD9 signaling maintains a microbial ecology that promotes inflammasome activation and thereby restrains colitis and colon tumorigenesis.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Colitis/inmunología , Neoplasias del Colon/inmunología , Hongos/inmunología , Microbioma Gastrointestinal/inmunología , Inflamasomas/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/fisiología , Células Mieloides/fisiología , Quinasa Syk/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Células Cultivadas , Colitis/inducido químicamente , Modelos Animales de Enfermedad , Humanos , Interleucina-18/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Dodecil Sulfato de Sodio , Quinasa Syk/genéticaRESUMEN
T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.
Asunto(s)
Inmunidad Humoral , Fosfatidiletanolaminas/metabolismo , Receptores CXCR5/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/inmunología , Sistemas CRISPR-Cas , Diferenciación Celular , Citidina Difosfato , Femenino , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfotransferasas (Aceptor de Grupo Alcohol) , ARN Nucleotidiltransferasas , Transducción de SeñalRESUMEN
Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.
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Proteína ADAMTS4/metabolismo , Fibroblastos/enzimología , Fibroblastos/patología , Virus de la Influenza A/patogenicidad , Pulmón/patología , Pulmón/fisiopatología , Proteína ADAMTS4/antagonistas & inhibidores , Animales , Aves/virología , Matriz Extracelular/enzimología , Perfilación de la Expresión Génica , Humanos , Gripe Aviar/virología , Gripe Humana/patología , Gripe Humana/terapia , Gripe Humana/virología , Interferones/inmunología , Interferones/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Pulmón/enzimología , Pulmón/virología , Ratones , Síndrome de Dificultad Respiratoria/enzimología , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria/terapia , Síndrome de Dificultad Respiratoria/virología , Estaciones del Año , Análisis de la Célula Individual , Células del Estroma/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Histotripsy is a relatively new therapeutic ultrasound technology to mechanically liquefy tissue into subcellular debris using high-amplitude focused ultrasound pulses. In contrast to conventional high-intensity focused ultrasound thermal therapy, histotripsy has specific clinical advantages: the capacity for real-time monitoring using ultrasound imaging, diminished heat sink effects resulting in lesions with sharp margins, effective removal of the treated tissue, a tissue-selective feature to preserve crucial structures, and immunostimulation. The technology is being evaluated in small and large animal models for treating cancer, thrombosis, hematomas, abscesses, and biofilms; enhancing tumor-specific immune response; and neurological applications. Histotripsy has been recently approved by the US Food and Drug Administration to treat liver tumors, with clinical trials undertaken for benign prostatic hyperplasia and renal tumors. This review outlines the physical principles of various types of histotripsy; presents major parameters of the technology and corresponding hardware and software, imaging methods, and bioeffects; and discusses the most promising preclinical and clinical applications.
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Ultrasonido Enfocado de Alta Intensidad de Ablación , Humanos , Animales , Ultrasonido Enfocado de Alta Intensidad de Ablación/métodos , Masculino , Neoplasias/terapia , Neoplasias/diagnóstico por imagen , Diseño de Equipo , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/diagnóstico por imagenRESUMEN
The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.
Asunto(s)
Complejo CD3/inmunología , Citocinas/biosíntesis , Motivo de Activación del Inmunorreceptor Basado en Tirosina/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Complejo CD3/metabolismo , Línea Celular , Proliferación Celular , Células HEK293 , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.
Asunto(s)
Muerte Celular/genética , ARN Helicasas DEAD-box/metabolismo , Inflamasomas/genética , Macrófagos/citología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Fisiológico/genética , Animales , Línea Celular , Supervivencia Celular/genética , ARN Helicasas DEAD-box/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Humanos , Inflamasomas/inmunología , Macrófagos/inmunología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genéticaRESUMEN
Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.
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Angiotensina II , Bacteriemia/inmunología , Células Mieloides/metabolismo , Sepsis/inmunología , Angiotensina II/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Norepinefrina/metabolismo , Receptor de Angiotensina Tipo 1 , Sepsis/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. METHODS: In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. RESULTS: At 48 h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1ß, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILß+ neutrophils and ILß+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1ß, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1ß+ neutrophils, IL-1ß+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. CONCLUSION: Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.
Asunto(s)
Citocinas , Piridinas , Sepsis , Tiadiazoles , Masculino , Ratones , Animales , Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6 , Linfocitos T CD8-positivos/metabolismo , Quimiocina CCL3 , Quimiocinas , Punciones , Endotoxinas , Encéfalo/metabolismo , Ligadura , Colinérgicos , Factor Estimulante de Colonias de Granulocitos , Ratones Endogámicos C57BL , Ciego/metabolismo , Modelos Animales de EnfermedadRESUMEN
OBJECTIVES: To identify research priorities in the management, epidemiology, outcome, and pathophysiology of sepsis and septic shock. DESIGN: Shortly after publication of the most recent Surviving Sepsis Campaign Guidelines, the Surviving Sepsis Research Committee, a multiprofessional group of 16 international experts representing the European Society of Intensive Care Medicine and the Society of Critical Care Medicine, convened virtually and iteratively developed the article and recommendations, which represents an update from the 2018 Surviving Sepsis Campaign Research Priorities. METHODS: Each task force member submitted five research questions on any sepsis-related subject. Committee members then independently ranked their top three priorities from the list generated. The highest rated clinical and basic science questions were developed into the current article. RESULTS: A total of 81 questions were submitted. After merging similar questions, there were 34 clinical and ten basic science research questions submitted for voting. The five top clinical priorities were as follows: 1) what is the best strategy for screening and identification of patients with sepsis, and can predictive modeling assist in real-time recognition of sepsis? 2) what causes organ injury and dysfunction in sepsis, how should it be defined, and how can it be detected? 3) how should fluid resuscitation be individualized initially and beyond? 4) what is the best vasopressor approach for treating the different phases of septic shock? and 5) can a personalized/precision medicine approach identify optimal therapies to improve patient outcomes? The five top basic science priorities were as follows: 1) How can we improve animal models so that they more closely resemble sepsis in humans? 2) What outcome variables maximize correlations between human sepsis and animal models and are therefore most appropriate to use in both? 3) How does sepsis affect the brain, and how do sepsis-induced brain alterations contribute to organ dysfunction? How does sepsis affect interactions between neural, endocrine, and immune systems? 4) How does the microbiome affect sepsis pathobiology? 5) How do genetics and epigenetics influence the development of sepsis, the course of sepsis and the response to treatments for sepsis? CONCLUSIONS: Knowledge advances in multiple clinical domains have been incorporated in progressive iterations of the Surviving Sepsis Campaign guidelines, allowing for evidence-based recommendations for short- and long-term management of sepsis. However, the strength of existing evidence is modest with significant knowledge gaps and mortality from sepsis remains high. The priorities identified represent a roadmap for research in sepsis and septic shock.
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Sepsis , Choque Séptico , Humanos , Choque Séptico/terapia , Choque Séptico/diagnóstico , Sepsis/diagnóstico , Resucitación , Respiración Artificial , Cuidados CríticosRESUMEN
Interleukin 35 (IL-35) belongs to the IL-12 family of heterodimeric cytokines but has a distinct functional profile. IL-35 suppresses T cell proliferation and converts naive T cells into IL-35-producing induced regulatory T cells (iTr35 cells). Here we found that IL-35 signaled through a unique heterodimer of receptor chains IL-12Rß2 and gp130 or homodimers of each chain. Conventional T cells were sensitive to IL-35-mediated suppression in the absence of one receptor chain but not both receptor chains, whereas signaling through both chains was required for IL-35 expression and conversion into iTr35 cells. Signaling through the IL-35 receptor required the transcription factors STAT1 and STAT4, which formed a unique heterodimer that bound to distinct sites in the promoters of the genes encoding the IL-12 subunits p35 and Ebi3. This unconventional mode of signaling, distinct from that of other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.
Asunto(s)
Receptores de Interleucina-1/inmunología , Receptores de Interleucina/inmunología , Transducción de Señal , Animales , Receptor gp130 de Citocinas/inmunología , Interleucinas/inmunología , Ratones , Ratones Noqueados , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/deficiencia , Receptores de Interleucina/metabolismo , Receptores de Interleucina-1/química , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-12/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT4/inmunologíaRESUMEN
Histotripsy is the first noninvasive, non-ionizing, and non-thermal ablation technique that mechanically fractionates target tissue into acellular homogenate via controlled acoustic cavitation. Histotripsy has been evaluated for various preclinical applications requiring noninvasive tissue removal including cancer, brain surgery, blood clot and hematoma liquefaction, and correction of neonatal congenital heart defects. Promising preclinical results including local tumor suppression, improved survival outcomes, local and systemic anti-tumor immune responses, and histotripsy-induced abscopal effects have been reported in various animal tumor models. Histotripsy is also being investigated in veterinary patients with spontaneously arising tumors. Research is underway to combine histotripsy with immunotherapy and chemotherapy to improve therapeutic outcomes. In addition to preclinical cancer research, human clinical trials are ongoing for the treatment of liver tumors and renal tumors. Histotripsy has been recently approved by the FDA for noninvasive treatment of liver tumors. This review highlights key learnings from in vivo shock-scattering histotripsy, intrinsic threshold histotripsy, and boiling histotripsy cancer studies treating cancers of different anatomic locations and discusses the major considerations in planning in vivo histotripsy studies regarding instrumentation, tumor model, study design, treatment dose, and post-treatment tumor monitoring.
Asunto(s)
Ultrasonido Enfocado de Alta Intensidad de Ablación , Neoplasias Renales , Neoplasias Hepáticas , Animales , Recién Nacido , Humanos , Ultrasonido Enfocado de Alta Intensidad de Ablación/métodos , Modelos Animales , Proyectos de InvestigaciónRESUMEN
BACKGROUND: Sepsis is characterized as an insulin resistant state. However, the effects of sepsis on insulin's signal transduction pathway are unknown. The molecular activity driving insulin signaling is controlled by tyrosine phosphorylation of the insulin receptor ß-subunit (IRß) and of insulin receptor substrate molecules (IRS) -1 and IRS-2. HYPOTHESIS: Cecal ligation and puncture (CLP) attenuates IRß, IRS-1 and IRS-2 phosphorylation. METHODS: IACUC-approved studies conformed to ARRIVE guidelines. CLP was performed on C57BL/6 mice; separate cohorts received intraperitoneal insulin at baseline (T0) or at 23 or 47 h. post-CLP, 1 h before mice were euthanized. We measured levels of (1) glucose and insulin in serum, (2) IRß, IRS-1 and IRS-2 in skeletal muscle and liver homogenate and (3) phospho-Irß (pIRß) in liver and skeletal muscle, phospho-IRS-1 (pIRS-1) in skeletal muscle and pIRS-2 in liver. Statistical significance was determined using ANOVA with Sidak's post-hoc correction. RESULTS: CLP did not affect the concentrations of IRß, IRS-1or IRS-2 in muscle or liver homogenate or of IRS-1 in liver. Muscle IRS-1 concentration at 48 h. post-CLP was higher than at T0. Post-CLP pIRS-1 levels in muscle and pIRß and pIRS-2 levels in liver were indistinguishable from T0 levels. At 48 h. post-CLP pIRß levels in muscle were higher than at T0. Following insulin administration, the relative abundance of pIRß in muscle and liver at T0 and at both post-CLP time points was significantly higher than abundance in untreated controls. In T0 controls, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was higher than in untreated mice. However, at both post-CLP time points, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was not distinguishable from the abundance in untreated mice at the same time point. Serum glucose concentration was significantly lower than T0 at 24 h., but not 48 h., post-CLP. Glucose concentration was lower following insulin administration to T0 mice but not in post-CLP animals. Serum insulin levels were significantly higher than baseline at both post-CLP time points. CONCLUSIONS: CLP impaired insulin-induced tyrosine phosphorylation of both IRS-1 in muscle and IRS-2 in liver. These findings suggest that the molecular mechanism underlying CLP-induced insulin resistance involves impaired IRS-1/IRS-2 phosphorylation.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina , Receptor de Insulina , Sepsis , Animales , Ratones , Glucosa/metabolismo , Insulina/metabolismo , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Fosforilación , Punciones , Receptor de Insulina/metabolismo , Sepsis/metabolismo , Tirosina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismoRESUMEN
Sepsis, a clinical syndrome occurring in patients following infection or injury, is a leading cause of morbidity and mortality worldwide. Current immunological mechanisms do not explain the basis of cellular dysfunction and organ failure, the ultimate cause of death. Here we review current dogma and argue that it is time to delineate novel immunometabolic and neurophysiological mechanisms underlying the altered cellular bioenergetics and failure of epithelial and endothelial barriers that produce organ dysfunction and death. These mechanisms might hold the key to future therapeutic strategies.
Asunto(s)
Endotelio/inmunología , Epitelio/inmunología , Sepsis/inmunología , Animales , Endotelio/microbiología , Metabolismo Energético , Epitelio/microbiología , Humanos , Mediadores de Inflamación/metabolismo , Insuficiencia Multiorgánica , Neuroinmunomodulación , Transmisión Sináptica/inmunologíaRESUMEN
Focused Ultrasound (FUS) is emerging as a promising primary and adjunct therapy for the treatment of cancer. This includes histotripsy, which is a noninvasive, non-ionizing, non-thermal ultrasound guided ablation modality. As histotripsy has progressed from bench-to-bedside, it has become evident that this therapy has benefits beyond local tumor ablation. Specifically, histotripsy has the potential to shift the local tumor microenvironment from immunologically 'cold' to 'hot'. This is associated with the production of damage associated molecular patterns, the release of a selection of proinflammatory mediators, and the induction of inflammatory forms of cell death in cells just outside of the treatment zone. In addition to the induction of this innate immune response, histotripsy can also improve engagement of the adaptive immune system and promote systemic anti-tumor immunity targeting distal tumors and metastatic lesions. These tantalizing observations suggest that, in settings of widely metastatic disease burden, selective histotripsy of a limited number of accessible tumors could be a means of maximizing responsiveness to systemic immunotherapy. More work is certainly needed to optimize treatment strategies that best synergize histotripsy parameters with innate and adaptive immune responses. Likewise, rigorous clinical studies are still necessary to verify the presence and repeatability of these phenomena in human patients. As this technology nears regulatory approval for clinical use, it is our expectation that the insights and immunomodulatory mechanisms summarized in this review will serve as directional guides for rational clinical studies to validate and optimize the potential immunotherapeutic role of histotripsy tumor ablation.
Asunto(s)
Ultrasonido Enfocado de Alta Intensidad de Ablación , Neoplasias , Humanos , Microambiente Tumoral , Neoplasias/patología , Ultrasonografía , InmunidadRESUMEN
In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.