RESUMEN
The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here, we show that, during necroptosis, MLKL-dependent calcium (Ca2+) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM "bubbles" with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules and promote antigenic cross-priming of CD8+ T cells.
Asunto(s)
Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Necrosis/metabolismo , Animales , Calcio/metabolismo , Supervivencia Celular , Células HT29 , Humanos , Células Jurkat , Ratones , Células 3T3 NIH , Fosfatidilserinas , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.
Asunto(s)
Caspasa 8/metabolismo , Genes Letales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Caspasa 8/genética , Muerte Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Fibroblastos/metabolismo , Inflamación/metabolismo , Interferones/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
The plasma membrane is crucial to the survival of animal cells, and damage to it can be lethal, often resulting in necrosis. However, cells possess multiple mechanisms for repairing the membrane, which allows them to maintain their integrity to some extent, and sometimes even survive. Interestingly, cells that survive a near-necrosis experience can recognize sub-lethal membrane damage and use it as a signal to secrete chemokines and cytokines, which activate the immune response. This review will present evidence of necrotic cell survival in both in vitro and in vivo systems, including in C. elegans, mouse models, and humans. We will also summarize the various membrane repair mechanisms cells use to maintain membrane integrity. Finally, we will propose a mathematical model to illustrate how near-death experiences can transform dying cells into innate immune modulators for their microenvironment. By utilizing their membrane repair activity, the biological effects of cell death can extend beyond the mere elimination of the cells.
Asunto(s)
Caenorhabditis elegans , Inmunidad Innata , Humanos , Animales , Ratones , Necrosis/metabolismo , Muerte Celular , Membrana Celular/metabolismoRESUMEN
Identifying and quantifying cell death is the basis for all cell death research. Current methods for obtaining these quantitative measurements rely on established biomarkers, yet the marker-based approach suffers from limited marker specificity, high cost of reagents, lengthy sample preparation, and fluorescence imaging. Based on the morphological difference, we developed a Live, Apoptotic, and Necrotic Cell Explorer (LANCE) to categorize cell death status in a label-free manner, by incorporating machine learning and image processing. The LANCE workflow includes cropping individual cells from microscopic images having hundreds of cells, formation of an image database of around 5000 events, training and validation of the convolutional neural network models using multiple cell lines, and treatment conditions. With LANCE, we precisely categorized live, apoptotic, and necrotic cells with a high accuracy of 96.3 ± 0.5%. More importantly, the nondestructive label-free LANCE method allows for tracking time dynamics of the cell death process, which enhances the understanding of subtle cell death regulation at the molecular level. Hence, LANCE is a fast, low-cost, and nondestructive label-free method to distinguish cell status, which can be applied to cell death studies as well as many other biomedical applications.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Redes Neurales de la Computación , Bases de Datos Factuales , Procesamiento de Imagen Asistido por Computador/métodos , Aprendizaje Automático , Imagen Óptica , ApoptosisRESUMEN
Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Inmunidad Innata/inmunología , Inflamasomas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Burkholderia cenocepacia/metabolismo , Caspasa 1/metabolismo , Línea Celular , Clostridioides difficile/metabolismo , Proteínas del Citoesqueleto/genética , Humanos , Inmunidad Innata/genética , Ratones , Ratones Endogámicos , Mutación , Unión Proteica , Pirina , Receptores de Reconocimiento de Patrones/metabolismo , Células U937RESUMEN
Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Sistemas de Secreción Bacterianos/inmunología , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Flagelina/inmunología , Inflamasomas/inmunología , Animales , Caspasa 1/metabolismo , Línea Celular , Chromobacterium/genética , Chromobacterium/inmunología , Chromobacterium/fisiología , Humanos , Inmunidad Innata/inmunología , Inflamasomas/metabolismo , Legionella pneumophila/inmunología , Legionella pneumophila/fisiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Proteína Inhibidora de la Apoptosis Neuronal/inmunología , Proteína Inhibidora de la Apoptosis Neuronal/metabolismoRESUMEN
NF-κB is crucial for innate immune defence against microbial infection. Inhibition of NF-κB signalling has been observed with various bacterial infections. The NF-κB pathway critically requires multiple ubiquitin-chain signals of different natures. The question of whether ubiquitin-chain signalling and its specificity in NF-κB activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-κB signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-κB signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-κB activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cisteína/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Factores de Virulencia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Sistemas de Secreción Bacterianos , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Quinasas Quinasa Quinasa PAM/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Metilación , Metiltransferasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Especificidad por Sustrato , Factor 6 Asociado a Receptor de TNF , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Dedos de ZincRESUMEN
Legionella pneumophila, the causative agent of Legionnaires' pneumonia, resides in a distinct vacuole structure called Legionella-containing vacuole (LCV). The LCV resists fusion with the lysosome and permits efficient bacterial replication in host macrophages, which requires a Dot/Icm type IVB secretion system. Dot/Icm-translocated effector SdhA is critical for L. pneumophila intracellular growth and functions to prevent host cell death. Here, we show that the absence of SdhA resulted in elevated caspase-1 activation and IL-1ß secretion as well as macrophage pyroptosis during Legionella infection. These inflammasome activation phenotypes were independent of the established flagellin-NAIP5-NLRC4 axis, but relied on the DNA-sensing AIM2 inflammasome. We further demonstrate that Legionella DNA was released into macrophage cytosol, and this effect was significantly exaggerated by the absence of SdhA. SdhA bears a functional Golgi-targeting GRIP domain that is required for preventing AIM2 inflammasome activation. Ectopically expressed SdhA formed a unique ring-shape membrane structure, further indicating a role in membrane trafficking and maintaining LCV membrane integrity. Our data together suggest a possible link, mediated by the function of SdhA, between LCV trafficking/maturation and suppression of host innate immune detection.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Flavoproteínas/metabolismo , Inflamasomas/metabolismo , Legionella pneumophila/metabolismo , Proteínas Nucleares/metabolismo , Animales , Apoptosis , Proteínas Bacterianas/genética , Transporte Biológico , Caspasa 1/metabolismo , Línea Celular , ADN Bacteriano/genética , Proteínas de Unión al ADN , Flavoproteínas/genética , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Membranas Intracelulares/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/fisiología , Lisosomas/metabolismo , Lisosomas/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Proteínas Nucleares/genética , Interferencia de ARN , Células U937 , Vacuolas/metabolismo , Vacuolas/microbiologíaRESUMEN
Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP-Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.
Asunto(s)
Apoptosis/fisiología , Macrófagos/citología , Macrófagos/fisiología , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Línea Celular , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Proteína NEDD8 , Homología de Secuencia de Aminoácido , Electricidad Estática , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
BACKGROUND: Identifying cells engaged in fundamental cellular processes, such as proliferation or living/death statuses, is pivotal across numerous research fields. However, prevailing methods relying on molecular biomarkers are constrained by high costs, limited specificity, protracted sample preparation, and reliance on fluorescence imaging. METHODS: Based on cellular morphology in phase contrast images, we developed a deep-learning model named Detector of Mitosis, Apoptosis, Interphase, Necrosis, and Senescence (D-MAINS). RESULTS: D-MAINS utilizes machine learning and image processing techniques, enabling swift and label-free categorization of cell death, division, and senescence at a single-cell resolution. Impressively, D-MAINS achieved an accuracy of 96.4 ± 0.5% and was validated with established molecular biomarkers. D-MAINS underwent rigorous testing under varied conditions not initially present in the training dataset. It demonstrated proficiency across diverse scenarios, encompassing additional cell lines, drug treatments, and distinct microscopes with different objective lenses and magnifications, affirming the robustness and adaptability of D-MAINS across multiple experimental setups. CONCLUSIONS: D-MAINS is an example showcasing the feasibility of a low-cost, rapid, and label-free methodology for distinguishing various cellular states. Its versatility makes it a promising tool applicable across a broad spectrum of biomedical research contexts, particularly in cell death and oncology studies.
Asunto(s)
Apoptosis , Senescencia Celular , Aprendizaje Profundo , Interfase , Mitosis , Necrosis , Humanos , Línea Celular Tumoral , Neoplasias/patología , Neoplasias/metabolismo , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Despite recent advances in cancer treatment, refining therapeutic agents remains a critical task for oncologists. Precise evaluation of drug effectiveness necessitates the use of 3D cell culture instead of traditional 2D monolayers. Microfluidic platforms have enabled high-throughput drug screening with 3D models, but current viability assays for 3D cancer spheroids have limitations in reliability and cytotoxicity. This study introduces a deep learning model for non-destructive, label-free viability estimation based on phase-contrast images, providing a cost-effective, high-throughput solution for continuous spheroid monitoring in microfluidics. Microfluidic technology facilitated the creation of a high-throughput cancer spheroid platform with approximately 12 000 spheroids per chip for drug screening. Validation involved tests with eight conventional chemotherapeutic drugs, revealing a strong correlation between viability assessed via LIVE/DEAD staining and phase-contrast morphology. Extending the model's application to novel compounds and cell lines not in the training dataset yielded promising results, implying the potential for a universal viability estimation model. Experiments with an alternative microscopy setup supported the model's transferability across different laboratories. Using this method, we also tracked the dynamic changes in spheroid viability during the course of drug administration. In summary, this research integrates a robust platform with high-throughput microfluidic cancer spheroid assays and deep learning-based viability estimation, with broad applicability to various cell lines, compounds, and research settings.
Asunto(s)
Supervivencia Celular , Aprendizaje Profundo , Esferoides Celulares , Humanos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas Analíticas Microfluídicas/instrumentación , Dispositivos Laboratorio en un ChipRESUMEN
The inflammatory response plays an important role in the onset, development and prognosis of inflammatory diseases and a variety of chronic diseases. Long-term uncontrolled inflammatory response may lead to dysfunction or loss of organ tissue function. Clinical practice and evidence-based medicine suggest that acupuncture can effectively alleviate the inflammatory status of various inflammatory diseases and chronic diseases. Stimulation of acupoints related to internal organs and target organs can initiate neuromodulation by modulating the microenvironment of acupoints. After integration of acupuncture stimulus information in the central nervous system, neurotransmitters, hormones, etc. are released and ultimately act on immune cells through neuro-endocrine-immune pathways, such as the vagus-mediated cholinergic anti-inflammatory pathway, vagus nerve-adrenal medullary-dopamine pathway, somatic sympathetic nerve pathway, and hypothalamic-pituitary-adrenal axis, etc. Ultimately, the intracellular signaling pathways and polarization balance of monocytes/macrophages and T cell subsets are regulated and the immune homeo-stasis of target organs of the body realizes. Therefore, we proposed that the anti-inflammatory action of acupuncture may be one of the universal therapeutic strategies for multiple diseases, and a powerful interpretation of acupuncture in regulating the balance of yin and yang. The elucidation of its anti-inflammatory action rules and mechanism may better realize the clinical transformation of acupuncture and moxibustion precision treatment.
Asunto(s)
Terapia por Acupuntura , Moxibustión , Humanos , Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , InflamaciónRESUMEN
Rheumatoid arthritis (RA) is an autoimmune disease that generally affects the joints. In the face of inflammation-induced cartilage and bone damage, RA treatment remains insufficient. While research evidence indicates that acupuncture can exert anti-inflammatory and analgesic effects, improve the joint function of RA patients, and delay the disease, data on whether it can promote RA repair are lacking. Findings from the present work demonstrated that both the antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) models can simulate joint swelling of RA. The AIA model was more stable than the CIA model, with a higher incidence of successful arthritis modeling. Moreover, the AIA mice model could simulate the signal molecules and related pathological processes of the autoimmune response in RA, as well as major pathways related to RA and antigen immune response mechanisms. Manual acupuncture (MA) at Zusanli (ST36) significantly improved paw redness and swelling, pain, and inflammatory cell infiltration in the joints in AIA mice. The therapeutic effect of MA on AIA is achieved primarily through the regulation of steroid hormone biosynthesis, cell metabolism, and tissue repair processes. MA at ST36 can increase the gene contents of tissue repair growth factors, including PEG3, GADD45A, GDF5, FGF5, SOX2, and ATP6V1C2 in the inflammatory side joints of AIA mice, as well as the gene expression of the anti-inflammatory cytokine IL-10. In conclusion, acupuncture may alleviate RA in the joints via modulating the tissue healing process.
Asunto(s)
Terapia por Acupuntura , Artritis Experimental , Artritis Reumatoide , Ratones , Animales , Inflamación/patología , Citocinas/uso terapéutico , Antiinflamatorios/farmacología , Antígenos/efectos adversos , Edema/tratamiento farmacológicoRESUMEN
Although overwhelming plasma membrane integrity loss leads to cell lysis and necrosis, cells can tolerate a limited level of plasma membrane damage, undergo ESCRT-III-mediated repair, and survive. Here, we find that cells which undergo limited plasma membrane damage from the pore-forming actions of MLKL, GSDMD, perforin, or detergents experience local activation of PKCs through Ca2+ influx at the damage sites. S660-phosphorylated PKCs subsequently activate the TAK1/IKKs axis and RelA/Cux1 complex to trigger chemokine expressions. We observe that in late-stage cancers, cells with active MLKL show expression of CXCL8. Similar expression induction is also found in ischemia-injured kidneys. Chemokines generated in this manner are also indispensable for recruiting immune cells to the dead and dying cells. This plasma membrane integrity-sensing pathway is similar to the well-established yeast cell wall integrity signaling pathway at molecular level, and this suggests an evolutionary conserved mechanism to respond to the cellular barrier damage.
Asunto(s)
Membrana Celular/metabolismo , Quimiocinas/fisiología , Proteína Quinasa C/fisiología , Animales , Apoptosis/fisiología , Membrana Celular/fisiología , Quimiocinas/genética , Quimiocinas/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Necrosis/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Transducción de SeñalRESUMEN
The autonomic nervous system (ANS) is a diffuse network that regulates physiological systems to maintain body homeostasis by integrating inputs from the internal and external environment, including the sympathetic, parasympathetic, and enteric nervous systems (ENS). Recent evidence suggests that ANS is one of the key neural pathways for acupuncture signal transduction, which has attracted worldwide attention in the acupuncture field. Here, we reviewed the basic and clinical research published in PubMed over the past 20 years on the effects of acupuncture on ANS regulation and homeostasis maintenance. It was found that acupuncture effectively alleviates ANS dysfunction-associated symptoms in its indications, such as migraine, depression, insomnia, functional dyspepsia, functional constipation. Acupuncture stimulation on some specific acupoints activates sensory nerve fibers, the spinal cord, and the brain. Using information integration and efferents from a complex network of autonomic nuclei of the brain, such as the insular cortex (IC), prefrontal cortex, anterior cingulate cortex (ACC), amygdala (AMG), hypothalamus, periaqueductal gray (PAG), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM), nucleus ambiguus (AMB), acupuncture alleviates visceral dysfunction, inflammation via efferent autonomic nerves, and relieves pain and pain affect. The modulating pattern of sympathetic and parasympathetic nerves is associated with acupuncture stimulation on specific acupoints, intervention parameters, and disease models, and the relationships among them require further exploration. In conclusion, ANS is one of the therapeutic targets for acupuncture and mediates acupuncture's actions, which restores homeostasis. A systemic study is needed to determine the rules and mechanisms underlying the effects of acupoint stimulation on corresponding organs mediated by specific central nervous networks and the efferent ANS.
RESUMEN
In "healthy" tumor cells, phosphatidylserine (PS) is predominately localized in the inner plasma membrane leaflet. During apoptosis, PS relocates to the outer leaflet. Herein, we established PSout tumor models with tumor cells lacking PS flippase component CDC50A, constantly exposing PS but alive. PSout tumors developed bigger than wild-type (WT) tumors, featuring M2 polarized tumor-associated macrophages (TAMs) and fewer tumor-antigen-specific T cells. The PS receptor TIM-3 is responsible for PS recognition. Employing an opposite tumor model, PSin, with tumor cells lacking the PS scramblase Xkr8 and unable to expose PS during otherwise normal apoptosis, we find that the accumulated apoptotic tumor cells produce and release cyclic GAMP (cGAMP) to immune cells to activate the STING pathway, leading to TAM M1 polarization, suppressed interleukin (IL)-10 secretion, and natural killer (NK) cell cytotoxicity. Silencing Xkr8 in vivo by either short hairpin RNA (shRNA) or small interfering RNA (siRNA) to achieve a PS externalization blockade provides robust therapeutic anti-tumor efficiency.
Asunto(s)
Neoplasias , Fosfatidilserinas , Humanos , Fosfatidilserinas/metabolismo , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Apoptosis/fisiología , Neoplasias/terapia , Neoplasias/metabolismo , InmunoterapiaRESUMEN
ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. ATR kinase inhibitors (ATRi's) combine with radiation to generate CD8+ T cell-dependent responses in mouse models of cancer. We show that ATRi's induce cyclin-dependent kinase 1 (CDK1)-dependent origin firing across active replicons in CD8+ T cells activated ex vivo while simultaneously decreasing the activity of rate-limiting enzymes for nucleotide biosynthesis. These pleiotropic effects of ATRi induce deoxyuridine (dU) contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and interferon-α/ß (IFN-α/ß). Remarkably, thymidine rescues ATRi-induced dU contamination and partially rescues death and IFN-α/ß expression in proliferating CD8+ T cells. Thymidine also partially rescues ATRi-induced cancer cell death. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell-dependent anti-tumor responses in mouse models. We conclude that ATR is essential to limit dU contamination in genomic DNA and IFN-α/ß expression.
Asunto(s)
Linfocitos T CD8-positivos , Proteína Quinasa CDC2 , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proteína Quinasa CDC2/metabolismo , Muerte Celular , Línea Celular Tumoral , ADN , Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiuridina , Genómica , Interferón-alfa/metabolismo , Interferón beta , Ratones , Nucleótidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN , Timidina/farmacologíaRESUMEN
Tumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis. Neuropilin-2 (NRP2), consisting of the NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality, with NRP2b promoting increased Akt signaling via receptor tyrosine kinases including VEGFRs, MET, and PDGFR. Although NRP2 has been shown to regulate macrophage/TAM biology, the role of the individual NRP2a/NRP2b isoforms in TAMs has yet to be evaluated. Using transcriptional profiling and spectral flow cytometry, we show that NRP2 isoform expression was significantly higher in TAMs from murine mammary tumors. NRP2a/NRP2b levels in human breast cancer metastasis were dependent upon the anatomic location of the tumor and significantly correlated with TAM infiltration in both primary and metastatic breast cancers. We define distinct phenotypes of NRP2 isoform-expressing TAMs in mouse models of breast cancer and within malignant pleural effusions from breast cancer patients which were exclusive of neuropilin-1 expression. Genetic depletion of either NRP2 isoform in macrophages resulted in a dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, depletion of NRP2b, but not NRP2a, inhibited production of IL-6. These results suggest that NRP2 isoforms regulate both shared and unique functionality in macrophages and are associated with distinct TAM subsets in breast cancer.
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Neoplasias de la Mama , Neuropilina-2 , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Ratones , Neuropilina-1/genética , Neuropilina-2/genética , Neuropilina-2/metabolismo , Isoformas de Proteínas , Macrófagos Asociados a TumoresRESUMEN
Due to its own internal laws of development, Chinese medicine (CM) seems more inclined to empirical medicine in a relatively long historical period. It is considered to be lacking objective and unified clinical practice guidelines (CPGs), and the difficulties in diagnosis and therapeutic effect evaluation comes with it, have restricted its further inheritance, development and international communication. Over the years, our research group has been committed to improving the standardization theory and methodology of CM, also perfecting relative techniques for further application, which are all based on the stratified evidence scoring method. We have already applied this method to 45 issued guidelines, including 5 national guidelines, 3 industrial guidelines, and 37 formulation/revision social organization guidelines. The stratified evidence scoring method has been recognized and used widely. It helps scholars and applicators to study, formulate, publish and popularize the acupuncture therapy clinical practice guidelines better, thus further promotes the development of acupuncture therapy.
Asunto(s)
Terapia por Acupuntura , Acupuntura , Medicina Tradicional de Asia Oriental , Guías de Práctica Clínica como Asunto , Proyectos de InvestigaciónRESUMEN
Although the basic mechanism of acupuncture-moxibustion has been revealed from many aspects, there are still many shackles in the transformation of the related research achievements. The transformation of academic achievements of experimental acupunctology is an urgent issue to be solved at present. Network regulation is the basic action mode of acupuncture therapy. In the present paper, we proposed that the "acupuncture network drug" could carry a variety of effective active ingredients which may be the core component of network regulation of acupuncture therapy. The "exosomes", polyvesicle derived from the intracellular lysosomal microparticles invagination, contain complex RNAs and proteins and exist in the body fluids and function in secreting abundant activate substances to participate in intercellular communication, which is the research hotspot in the field of frontier life science in the world. They play an important role in the diagnosis and treatment of diseases, and drug development, etc.. Our studies using rats with adjuvant arthritis and mice with sepsis displayed that after intraperitoneal administration of serum exosomes derived from normal animals receiving acupuncture intervention, an acupuncture-like analgesic effect and an anti-inflammatory effect were achieved, respectively. It is thus possible that acupuncture network drugs could be developed from serum exosomes secreted by exosome autogenous living cells after acupuncture intervention by virtue of the characteristics of low immunogenicity and may have great advantages in drug development and modification. It is also expected to provide new ideas for the transformation of experimental research results and to in depth give explanations about the underlying mechanisms of acupuncture therapy.