RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Shuangdan Jiedu Decoction (SJD) is a formula composed of six Chinese herbs with heat-removing and detoxifying, antibacterial, and anti-inflammatory effects, which is clinically used in the therapy of various inflammatory diseases of the lungs including COVID-19, but the therapeutic material basis of its action as well as its molecular mechanism are still unclear. AIM OF THE STUDY: The study attempted to determine the therapeutic effect of SJD on LPS-induced acute lung injury (ALI), as well as to investigate its mechanism of action and assess its therapeutic potential for the cure of inflammation-related diseases in the clinical setting. MATERIALS AND METHODS: We established an ALI model by tracheal drip LPS, and after the administration of SJD, we collected the bronchoalveolar lavage fluid (BALF) and lung tissues of mice and examined the expression of inflammatory factors in them. In addition, we evaluated the effects of SJD on the cyclic guanosine monophosphate-adenosine monophosphate synthase -stimulator of interferon genes (cGAS-STING) and inflammasome by immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: We demonstrated that SJD was effective in alleviating LPS-induced ALI by suppressing the levels of pro-inflammatory cytokines in the BALF, improving the level of lung histopathology and the number of neutrophils, as well as decreasing the inflammatory factor-associated gene expression. Importantly, we found that SJD could inhibit multiple stimulus-driven activation of cGAS-STING and inflammasome. Further studies showed that the Chinese herbal medicines in SJD had no influence on the cGAS-STING pathway and inflammasome alone at the formulated dose. By increasing the concentration of these herbs, we observed inhibitory effects on the cGAS-STING pathway and inflammasome, and the effect exerted was maximal when the six herbs were combined, indicating that the synergistic effects among these herbs plays a crucial role in the anti-inflammatory effects of SJD. CONCLUSIONS: Our research demonstrated that SJD has a favorable protective effect against ALI, and its mechanism of effect may be associated with the synergistic effect exerted between six Chinese medicines to inhibit the cGAS-STING and inflammasome abnormal activation. These results are favorable for the wide application of SJD in the clinic as well as for the development of drugs for ALI from herbal formulas.
Assuntos
Lesão Pulmonar Aguda , Medicamentos de Ervas Chinesas , Inflamassomos , Lipopolissacarídeos , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Animais , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lipopolissacarídeos/toxicidade , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Nucleotidiltransferases/metabolismo , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Masculino , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismo , Líquido da Lavagem Broncoalveolar/citologiaRESUMO
The immune resistance of tumor microenvironment (TME) causes immune checkpoint blockade therapy inefficient to hepatocellular carcinoma (HCC). Emerging strategies of using chemotherapy regimens to reverse the immune resistance provide the promise for promoting the efficiency of immune checkpoint inhibitors. The induction of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) in tumor cells evokes the adaptive immunity and remodels the immunosuppressive TME. In this study, we report that mitoxantrone (MIT, a chemotherapeutic drug) activates the cGAS-STING signaling pathway of HCC cells. We provide an approach to augment the efficacy of MIT using a signal transducer and activator of transcription 3 (STAT3) inhibitor called napabucasin (NAP). We prepare an aminoethyl anisamide (AEAA)-targeted polyethylene glycol (PEG)-modified poly (lactic-co-glycolic acid) (PLGA)-based nanocarrier for co-delivery of MIT and NAP. The resultant co-nanoformulation can elicit the cGAS-STING-based immune responses to reshape the immunoresistant TME in the mice orthotopically grafted with HCC. Consequently, the resultant co-nanoformulation can promote anti-PD-1 antibody for suppressing HCC development, generating long-term survival, and inhibiting tumor recurrence. This study reveals the potential of MIT to activate the cGAS-STING signaling pathway, and confirms the feasibility of nano co-delivery for MIT and NAP on achieving HCC chemo-immunotherapy.
Assuntos
Carcinoma Hepatocelular , Imunoterapia , Neoplasias Hepáticas , Proteínas de Membrana , Mitoxantrona , Nucleotidiltransferases , Fator de Transcrição STAT3 , Mitoxantrona/farmacologia , Mitoxantrona/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Animais , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Humanos , Nucleotidiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Fator de Transcrição STAT3/metabolismo , Camundongos , Imunoterapia/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Benzofuranos , NaftoquinonasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fetal neuromuscular disorder characterized by the gradual deterioration of motor neurons. Semen Strychni pulveratum (SSP), a processed version of Semen Strychni (SS) powder, is widely used to treat ALS in China. Vomicine is one of the most primary components of SS. However, their pharmacological effects and mechanisms for ALS remain elusive. AIM OF THE STUDY: This study aimed to evaluate the neuroprotective and anti-neuroinflammatory effects of SSP and vomicine, as well as to explore their protective roles in ALS and the underlying mechanisms. MATERIALS AND METHODS: In vivo, 8-week-old hSOD1-WT mice and hSOD1-G93A mice were orally administered different concentrations of SSP (SSP-L = 5.46 mg/ml, SSP-M = 10.92 mg/ml or SSP-H = 16.38 mg/ml) once every other day for 8 weeks. A series of experiments, including body weight measurement, footprint tests, Hematoxylin & Eosin staining, and Nissl staining, were performed to evaluate the preventive effect of SSP. Immunofluorescence staining, western blotting, and RT-qPCR were subsequently performed to evaluate activation of the cGAS-STING-TBK1 pathway in the spinal cord. In vitro, hSOD1G93A NSC-34 cells were treated with vomicine to further explore the pharmacological mechanism of vomicine in the treatment of ALS via the cGAS-STING-TBK1 pathway. RESULTS: SSP improved motor function, body weight loss, gastrocnemius muscle atrophy, and motor neuron loss in the spine and cortex of hSOD1-G93A mice. Furthermore, the cGAS-STING-TBK1 pathway was activated in the spinal cord of hSOD1-G93A mice, with activation predominantly observed in neurons and microglia. However, the levels of cGAS, STING, and pTBK1 proteins and cGAS, IRF3, IL-6, and IL-1ß mRNA were reversed following intervention with SSP. Vomicine not only downregulated the levels of cGAS, TBK1, IL-6 and IFN-ß mRNA, but also the levels of cGAS and STING protein in hSOD1G93A NSC-34 cells. CONCLUSION: This study demonstrated that SSP and vomicine exert neuroprotective and anti-neuroinflammatory effects in the treatment of ALS. SSP and vomicine may reduce neuroinflammation by regulating the cGAS-STING-TBK1 pathway, and could thereby play a role in ALS treatment.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Membrana , Fármacos Neuroprotetores , Nucleotidiltransferases , Proteínas Serina-Treonina Quinases , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Camundongos , Proteínas de Membrana/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nucleotidiltransferases/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Camundongos Transgênicos , Doenças Neuroinflamatórias/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Modelos Animais de DoençasRESUMO
Beyond its role as the bearer of genetic material, DNA also plays a crucial role in the activation phase of innate immunity. Pathogen recognition receptors (PRRs) and their homologs, pathogen-associated molecular patterns (PAMPs), form the foundation for driving innate immune activation and the induction of immune responses during infection. In the context of DNA viruses or bacterial infections, specific DNA sequences are recognized and bound by DNA sensors, marking the DNA as a PAMP for host recognition and subsequent activation of innate immunity. The primary DNA sensor pathway known to date is cGAS-STING, which can induce Type I interferons (IFN) and innate immune responses against viruses and bacteria. Additionally, the cGAS-STING pathway has been identified to mediate functions in autophagy and senescence. Herein, we introduce methods for using DNA PAMPs as molecular tools to study the role of cGAS-STING and its signaling pathway in regulating innate immunity, both in vitro and in vivo.
Assuntos
DNA , Imunidade Inata , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , DNA/metabolismo , DNA/genética , Animais , Moléculas com Motivos Associados a Patógenos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , CamundongosRESUMO
With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Imunidade Inata , Camundongos Knockout , RNA Guia de Sistemas CRISPR-Cas , Animais , Imunidade Inata/genética , Camundongos , RNA Guia de Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Viroses/imunologia , Viroses/genéticaRESUMO
cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.
Assuntos
Vírus da Dengue , Nucleotidiltransferases , Proteínas não Estruturais Virais , Humanos , Proteínas não Estruturais Virais/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Proteólise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Nucleotídeos Cíclicos/metabolismo , Dengue/virologia , Dengue/metabolismoRESUMO
Background: Chemical modifications on RNA profoundly affect RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods: Liquid chromatographytandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m6A in tubular cells and explore the biological functions of m6A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m6A modification in an animal kidney disease model. Results: By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphateAMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions: Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.
Assuntos
Adenosina , Fibrose , Proteínas de Membrana , Metiltransferases , Nucleotidiltransferases , RNA Mensageiro , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , RNA Mensageiro/metabolismo , Animais , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Humanos , Transdução de Sinais , Camundongos , Rim/patologia , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
Maintaining a well-functioning mitochondrial network through the mitochondria quality control (MQC) mechanisms, including biogenesis, dynamics and mitophagy, is crucial for overall health. Mitochondrial dysfunction caused by oxidative stress and further exacerbated by impaired quality control can trigger inflammation through the release of the damage-associated molecular patterns (mtDAMPs). mtDAMPs act by stimulating the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway. Recently, aberrant signalling of the cGAS-STING axis has been recognised to be closely associated with several sterile inflammatory diseases (e.g. non-alcoholic fatty liver disease, obesity). This may fit the pathophysiology of hypothyroidism, an endocrine disorder characterised by the reduction of thyroid hormone production associated with impaired metabolic fluxes, oxidative balance and inflammatory status. Both 3,5,3'-triiodo-L-tyronine (T3) and its derivative 3,5-diiodo-L-thyronine (3,5-T2), are known to mitigate processes targeting mitochondria, albeit the underlying mechanisms are not yet fully understood. Therefore, we used a chemically induced hypothyroidism rat model to investigate the effect of 3,5-T2 or T3 administration on inflammation-related factors (inflammatory cytokines, hepatic cGAS-STING pathway), oxidative stress, antioxidant defence enzymes, mitochondrial DNA (mtDNA) damage, release and repair, and the MQC system in the liver. Hypothyroid rats showed: i) increased oxidative stress, ii) accumulation of mtDNA damage, iii) high levels of circulating cytokines, iv) hepatic activation of cGAS-STING pathways and v) impairment of MQC mechanisms and autophagy. Both iodothyronines restored oxidative balance by enhancing antioxidant defence, preventing mtDNA damage through the activation of mtDNA repair mechanisms (OGG1, APE1, and POLγ) and promoting autophagy progression. Concerning MQC, both iodothyronines stimulated mitophagy and dynamics, with 3,5-T2 activating fusion and T3 modulating both fusion and fission processes. Moreover, only T3 enhanced mitochondrial biogenesis. Notably, 3,5-T2, but not T3, reversed the hypothyroidism-induced activation of the cGAS-STING inflammatory cascade. In addition, it is noteworthy that 3,5-T2 seems more effective than T3 in reducing circulating pro-inflammatory cytokines IL-6 and IL-1B and in stimulating the release of IL-10, a known anti-inflammatory cytokine. These findings reveal novel molecular mechanisms of hepatic signalling pathways involved in hypothyroidism, which could be targeted by natural iodothyronines, particularly 3,5-T2, paving the way for the development of new treatment strategies for inflammatory diseases.
Assuntos
Di-Iodotironinas , Hipotireoidismo , Inflamação , Fígado , Proteínas de Membrana , Nucleotidiltransferases , Estresse Oxidativo , Animais , Ratos , Hipotireoidismo/metabolismo , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/patologia , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Nucleotidiltransferases/metabolismo , Masculino , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Di-Iodotironinas/farmacologia , Proteínas de Membrana/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tri-Iodotironina , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Ratos Wistar , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated diseases. The pathophysiology of COVID-19 uses the following three mechanisms: (1) inflammasome activation mechanism; (2) cGAS-STING signaling mechanism; and (3) SAMHD1 tetramerization mechanism, which leads to IFN-I production. Interactions between the host and virus govern induction, resulting in multiorgan impacts. The NLRP3 with cGAS-STING constitutes the primary immune response. The expression of SARS-CoV-2 ORF3a, NSP6, NSP7, and NSP8 blocks innate immune activation and facilitates virus replication by targeting the RIG-I/MDA5, TRIF, and cGAS-STING signaling. SAMHD1 has a target motif for CDK1 to protect virion assembly, threonine 592 to modulate a catalytically active tetramer, and antiviral IFN responses to block retroviral infection. Plastic and allosteric nucleic acid binding of SAMHD1 modulates the antiretroviral activity of SAMHD1. Therefore, inflammasome activation, cGAS-STING signaling, and SAMHD1 tetramerization explain acute kidney injury, hepatic, cardiac, neurological, and gastrointestinal injury of COVID-19. It might be necessary to effectively block the pathological courses of diverse diseases.
Assuntos
COVID-19 , Imunidade Inata , Inflamassomos , SARS-CoV-2 , Transdução de Sinais , Humanos , COVID-19/virologia , COVID-19/imunologia , COVID-19/metabolismo , Inflamassomos/metabolismo , Inflamassomos/imunologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Nucleotidiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Replicação ViralRESUMO
Immunotherapy has shown marked progress in promoting systemic anti-colorectal cancer (CRC) clinical effects. For further effectively sensitizing CRC to immunotherapy, we have engineered a pH-sensitive zeolitic imidazolate framework-8 (CS/NPs), capable of efficient cGAS-STING pathway activation and immune checkpoint blockade, by encapsulating the chemotherapeutic mitoxantrone (MTX) and immunomodulator thymus pentapeptide (TP5) and tailoring with tumor-targeting chondroitin sulfate (CS). In this nanoframework, CS endows CS/NPs with specific tumor-targeting activity and reduced systemic toxicity. Of note, the coordinated Zn2+ disrupts glycolytic processes and downregulates the expression of glucose transporter type 1 (GLUT1), thus depriving the cancer cells of their energy. Zn2+ further initiates the adenosine 5'-monophosphate activated protein kinase (AMPK) pathway, which leads to PD-L1 protein degradation and sensitizes CRC cells to immunotherapy. Moreover, the damaged double-stranded DNA during MTX treatment activates the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which works together with TP5 induced the proliferation and differentiation of T lymphocytes and dendritic cells to further enhance the anti-CRC immune response. Therefore, CS/NPs efficiently sensitize cells to chemotherapy and stimulate systemic antitumor immune responses both in vitro and in vivo, representing a promising strategy to increase the feasibility of CRC immunotherapy.
Assuntos
Neoplasias Colorretais , Inibidores de Checkpoint Imunológico , Imunoterapia , Proteínas de Membrana , Estruturas Metalorgânicas , Mitoxantrona , Nucleotidiltransferases , Neoplasias Colorretais/tratamento farmacológico , Imunoterapia/métodos , Animais , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Humanos , Camundongos , Nucleotidiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Mitoxantrona/farmacologia , Mitoxantrona/química , Inibidores de Checkpoint Imunológico/farmacologia , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Antígeno B7-H1/metabolismo , Feminino , ImidazóisRESUMO
African swine fever (ASF) is a highly contagious and lethal disease of swine caused by African swine fever virus (ASFV), and the mortality rate caused by virulent stains can approach 100%. Many ASFV viral proteins suppress the interferon production to evade the host's innate immune responses. However, whether ASFV MGF360-4L could inhibit type I interferon (IFN-I) signaling pathway and the underlying molecular mechanisms remain unknown. Our study, indicated that ASFV MGF360-4L could negatively regulates the cGAS-STING mediated IFN-I signaling pathway. Overexpressing ASFV MGF360-4L could inhibit the cGAS/STING signaling pathway by inhibiting the interferon-ß promoter activity, which was induced by cGAS/STING, TBK1, and IRF3-5D, and further reduced the transcriptional levels of ISG15, ISG54, ISG56, STAT1, STAT2, and TYK2. Confocal microscopy and immunoprecipitation revealed that MGF360-4L co-localized and interacted with IRF3, and WB revealed that ASFV MGF360-4L suppressed the phosphorylation of IRF3. 4L-F2 (75-162 aa) and 4L-F3 (146-387 aa) were the crucial immunosuppressive domains and sites. Altogether, our study reveals ASFV MGF360-4L inhibited cGAS-STING mediated IFN-I signaling pathways, which provides insights into an evasion strategy of ASFV involving in host's innate immune responses.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Fator Regulador 3 de Interferon , Interferon Tipo I , Transdução de Sinais , Proteínas Virais , Fator Regulador 3 de Interferon/metabolismo , Vírus da Febre Suína Africana/imunologia , Fosforilação , Animais , Suínos , Interferon Tipo I/metabolismo , Humanos , Proteínas Virais/metabolismo , Febre Suína Africana/virologia , Febre Suína Africana/imunologia , Imunidade Inata , Células HEK293 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune , Nucleotidiltransferases/metabolismoRESUMO
Abnormal glial activation promotes neurodegeneration in Alzheimer's disease (AD), the most common cause of dementia. Stimulation of the cGAS-STING pathway induces microglial dysfunction and sterile inflammation, which exacerbates AD. We showed that inhibiting STING activation can control microglia and ameliorate a wide spectrum of AD symptoms. The cGAS-STING pathway is required for the detection of ectopic DNA and the subsequent immune response. Amyloid-ß (Aß) and tau induce mitochondrial stress, which causes DNA to be released into the cytoplasm of microglia. cGAS and STING are highly expressed in Aß plaque-associated microglia, and neuronal STING is upregulated in the brains of AD model animals. The presence of the APOE ε4 allele, an AD risk factor, also upregulated both proteins. STING activation was necessary for microglial NLRP3 activation, proinflammatory responses, and type-I-interferon responses. Pharmacological STING inhibition reduced a wide range of AD pathogenic features in AppNL-G-F/hTau double-knock-in mice. An unanticipated transcriptome shift in microglia reduced gliosis and cerebral inflammation. Significant reductions in the Aß load, tau phosphorylation, and microglial synapse engulfment prevented memory loss. To summarize, our study describes the pathogenic mechanism of STING activation as well as its potential as a therapeutic target in AD.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Proteínas de Membrana , Microglia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/etiologia , Animais , Microglia/metabolismo , Microglia/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Humanos , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Transdução de SinaisRESUMO
The 2024 Albert Lasker Basic Medical Research Award was attributed to Zhijian (James) Chen for "the discovery of the cGAS enzyme that senses foreign and self DNA, solving the mystery of how DNA stimulates immune and inflammatory responses." Bringing to bear an ingenious in vitro complementation system, an astute insight, and superlative biochemistry, Chen and colleagues identified cGAS (cGAMP synthase) as both the molecule that perceives cytosolic DNA in infected, stressed, or dying cells and the enzyme that catalyzes the synthesis of cGAMP (cyclic GMP-AMP), a critical second messenger along the route to inflammatory cytokine production. These findings cleared up the reigning confusion surrounding a major mechanism of inciting the innate immune system, with therapeutic implications for fighting infections, containing tumors, and extinguishing autoimmune and inflammatory diseases.
Assuntos
Citosol , DNA , Imunidade Inata , Nucleotidiltransferases , Humanos , DNA/metabolismo , Citosol/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Animais , Nucleotídeos Cíclicos/metabolismoRESUMO
Patients with small-cell lung cancer (SCLC) have poor prognosis and typically experience only transient benefits from combined immune checkpoint blockade (ICB) and chemotherapy. Here, we show that inhibition of ataxia telangiectasia and rad3 related (ATR), the primary replication stress response activator, induces DNA damage-mediated micronuclei formation in SCLC models. ATR inhibition in SCLC activates the stimulator of interferon genes (STING)-mediated interferon signaling, recruits T cells, and augments the antitumor immune response of programmed death-ligand 1 (PD-L1) blockade in mouse models. We demonstrate that combined ATR and PD-L1 inhibition causes improved antitumor response than PD-L1 alone as the second-line treatment in SCLC. This study shows that targeting ATR up-regulates major histocompatibility class I expression in preclinical models and SCLC clinical samples collected from a first-in-class clinical trial of ATR inhibitor, berzosertib, with topotecan in patients with relapsed SCLC. Targeting ATR represents a transformative vulnerability of SCLC and is a complementary strategy to induce STING-interferon signaling-mediated immunogenicity in SCLC.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Neoplasias Pulmonares , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Animais , Humanos , Nucleotidiltransferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Camundongos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Interferons/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Topotecan/farmacologia , Pirazinas/farmacologia , Pirazinas/uso terapêutico , IsoxazóisRESUMO
Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Proteínas de Membrana , Nucleotidiltransferases , SARS-CoV-2 , Transdução de Sinais , Replicação Viral , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Humanos , SARS-CoV-2/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , COVID-19/imunologia , COVID-19/virologia , COVID-19/metabolismo , Linhagem Celular , Interferons/metabolismo , Imunidade Inata , Animais , Interferon Tipo I/metabolismoRESUMO
Cytoplasmic mRNA decay is effected by exonucleolytic degradation in either the 5' to 3' or 3' to 5' direction. Pervasive terminal uridylation is implicated in mRNA degradation, however, its functional relevance for bulk mRNA turnover remains poorly understood. In this study, we employ genome-wide 3'-RACE (gw3'-RACE) in the model system fission yeast to elucidate the role of uridylation in mRNA turnover. We observe widespread uridylation of shortened poly(A) tails, promoting efficient 5' to 3' mRNA decay and ensuring timely and controlled mRNA degradation. Inhibition of this uridylation process leads to excessive deadenylation and enhanced 3' to 5' mRNA decay accompanied by oligouridylation. Strikingly we found that uridylation of poly(A) tails and oligouridylation of non-polyadenylated substrates are catalysed by different terminal uridyltransferases Cid1 and Cid16 respectively. Our study sheds new light on the intricate regulatory mechanisms underlying bulk mRNA turnover, demonstrating the role of uridylation in modulating mRNA decay pathways.
Assuntos
Estabilidade de RNA , RNA Mensageiro , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Poli A/metabolismo , Uridina/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Fúngico/metabolismo , RNA Fúngico/genética , NucleotidiltransferasesRESUMO
BACKGROUND: The cGAS-STING pathway is an important component of the innate immune system and plays significant role in acetaminophen-induced liver injury (AILI). Pentagalloylglucose (PGG) is a natural polyphenolic compound with various beneficial effects, including anti-cancer, antioxidant, anti-inflammatory, and liver-protective properties; however, whether it can be used for the treatment of AILI and the specific mechanism remain unclear. MATERIALS AND METHODS: A cell culture model was created to study the effect of PGG on cGAS-STING pathway activation using various techniques including western blotting (WB), real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence (IF), and immunoprecipitation (IP). The effect of PGG was investigated in vivo by establishing a dimethylxanthenone acetic acid (DMXAA)-mediated activation model. An AILI model was used to evaluate the hepatoprotective and therapeutic effects of PGG by detecting liver function indicators, liver histopathology, and cGAS-STING pathway-related indicators in mice with AILI. RESULTS: PGG blocked cGAS-STING pathway activation in bone marrow-derived macrophages (BMDMs), THP-1 cells, and peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, PGG inhibited the generation of type I interferons (IFN-I) and the secretion of inflammatory factors in DMXAA-induced in vivo experiments. In addition, PGG also reduced serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), improved liver tissue damage and apoptosis, and inhibited the cGAS-STING pathway activation caused by acetaminophen. In terms of the mechanism, PGG disrupted the connection between STING and TBK1. CONCLUSIONS: PGG exerts a protective effect against AILI by blocking the cGAS-STING pathway, offering a promising treatment strategy.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Taninos Hidrolisáveis , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Nucleotidiltransferases/metabolismo , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Acetaminofen/efeitos adversos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Humanos , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/uso terapêutico , Masculino , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologiaRESUMO
Protein-protein interactions (PPIs) play pivotal roles in biological processes and are closely linked with human diseases. Research on small molecule inhibitors targeting PPIs provides valuable insights and guidance for novel drug development. The cGAS-STING pathway plays a crucial role in regulating human innate immunity and is implicated in various pathological conditions. Therefore, modulators of the cGAS-STING pathway have garnered extensive attention. Given that this pathway involves multiple PPIs, modulating PPIs associated with the cGAS-STING pathway has emerged as a promising strategy for modulating this pathway. In this review, we summarize an overview of recent advancements in medicinal chemistry insights into cGAS-STING PPI-based modulators and propose alternative strategies for further drug discovery based on the cGAS-STING pathway.
[Box: see text].
Assuntos
Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Humanos , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Química Farmacêutica , Ligação Proteica , Descoberta de Drogas , Imunidade Inata/efeitos dos fármacosRESUMO
Mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) gastric cancer (GC) exhibits an immune-active tumor microenvironment (TME) compared to MMR proficient (pMMR)/microsatellite stable/Epstein-Barr virus-negative [EBV (-)] GC. The tumor cell-intrinsic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been considered a key regulator of immune cell activation in the TME. However, its significance in regulating the immune-active TME in dMMR/MSI-H GC remains unclear. Here, we demonstrated that tumor cell-intrinsic cGAS-STING was highly expressed in dMMR GC compared to pMMR/EBV (-) GC. The expression of tumor cell-intrinsic STING was significantly and positively associated with the number of CD8+ tumor-infiltrating lymphocytes in GC. Analysis of TCGA datasets revealed that the expression of interferon-stimulated genes and STING downstream T-cell attracting chemokines was significantly higher in MSI-H GC compared to other subtypes of GC with EBV (-). These results suggest that tumor cell-intrinsic STING signaling plays a key role in activating immune cells in the dMMR/MSI-H GC TME and might serve as a novel biomarker predicting the efficacy of immunotherapy for GC treatment.
Assuntos
Linfócitos T CD8-Positivos , Linfócitos do Interstício Tumoral , Proteínas de Membrana , Instabilidade de Microssatélites , Transdução de Sinais , Neoplasias Gástricas , Microambiente Tumoral , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Microambiente Tumoral/imunologia , Masculino , Feminino , Reparo de Erro de Pareamento de DNA/genética , Pessoa de Meia-Idade , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Regulação Neoplásica da Expressão Gênica , IdosoRESUMO
Triggering ferroptosis represents a promising anticancer therapeutic strategy, but the development of a selective ferroptosis inducer for cancer-specific therapy remains a great challenge. Herein, a H2S-responsive iridium(III) complex NA-Ir has been well-designed as a ferroptosis inducer. NA-Ir could selectively light up H2S-rich cancer cells, primarily localize in mitochondria, intercalate into mitochondrial DNA (mtDNA), and induce mtDNA damage, exhibiting higher anticancer activity under light irradiation. Mechanistic studies showed that NA-Ir-mediated PDT triggered lipid peroxidation and glutathione peroxidase 4 downregulation through ROS production and GSH depletion, resulting in ferroptosis through multiple pathways. Moreover, the intense mtDNA damage can activate the cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) pathway, leading to ferritinophagy and further ferroptosis. RNA-sequencing analysis showed that NA-Ir-mediated PDT mainly affects the expression of genes related to ferroptosis, autophagy, and cancer immunity. This study demonstrates the first cancer-specific example with ferroptosis and cGAS-STING activation, which provides a new strategy for multimodal synergistic therapy.