RESUMEN

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.

Asunto(s)

ADN , Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , ADN/metabolismo , ADN/genética , Animales , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Ratones

RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Inmunidad Innata , Ratones Noqueados , ARN Guía de Sistemas CRISPR-Cas , Animales , Inmunidad Innata/genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Virosis/inmunología , Virosis/genética

RESUMEN

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.

Asunto(s)

Virus del Dengue , Nucleotidiltransferasas , Proteínas no Estructurales Virales , Humanos , Proteínas no Estructurales Virales/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Proteolisis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Nucleótidos Cíclicos/metabolismo , Dengue/virología , Dengue/metabolismo

RESUMEN

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.

Asunto(s)

Carcinoma Hepatocelular , Inmunoterapia , Neoplasias Hepáticas , Proteínas de la Membrana , Mitoxantrona , Nucleotidiltransferasas , Factor de Transcripción STAT3 , Mitoxantrona/farmacología , Mitoxantrona/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Animales , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Humanos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Factor de Transcripción STAT3/metabolismo , Ratones , Inmunoterapia/métodos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Benzofuranos , Naftoquinonas

RESUMEN

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.

Asunto(s)

Esclerosis Amiotrófica Lateral , Proteínas de la Membrana , Fármacos Neuroprotectores , Nucleotidiltransferasas , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Ratones , Proteínas de la Membrana/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nucleotidiltransferasas/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Transgénicos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Modelos Animales de Enfermedad

RESUMEN

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.

Asunto(s)

Lesión Pulmonar Aguda , Medicamentos Herbarios Chinos , Inflamasomas , Lipopolisacáridos , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Animales , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lipopolisacáridos/toxicidad , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Nucleotidiltransferasas/metabolismo , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Endogámicos C57BL , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Líquido del Lavado Bronquioalveolar/citología

RESUMEN

Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1.

Asunto(s)

Autoinmunidad , Células Dendríticas , Exodesoxirribonucleasas , Ratones Noqueados , Nucleotidiltransferasas , Fosfoproteínas , Animales , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/deficiencia , Exodesoxirribonucleasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/deficiencia , Células Dendríticas/inmunología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/inmunología , Ratones , Autoinmunidad/inmunología , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/patología , Inflamación/inmunología , Inflamación/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/genética

RESUMEN

Arsenic is a toxic metal-like element widely used in the pesticide, preservative and semiconductor industries. However, accumulation of arsenic through the food chain can cause serious damage to animal and human health. However, the toxic mechanism of arsenic-induced hepatotoxicity in chickens is not clear, and the present study aimed to investigate the potential role of cGAS-STING and NF-κB pathways on inflammatory injury in chicken liver. In this study, 75 white-feathered broilers were divided into a control group, a low-dose arsenic group (4 mg/kg) and a high-dose arsenic group (8 mg/kg) to investigate the toxic effects of arsenic on chicken liver. In this study, we found that pathological changes such as inflammatory cell infiltration and vesicular degeneration occurred in the liver when exposed to ATO. Crucially, exposure to ATO triggered the cGAS-STING pathway and markedly raised the levels of mRNA and protein expression of cGAS, STING, TBK1, and IRF7. The type I interferon response was also triggered. Simultaneously, STING induced the activation of the conventional NF-κB signaling pathway and stimulated the expression of genes associated with inflammation, such as IL-6, TNF-α and IL-1ß. In summary, the induction of inflammatory responses via cGAS-STING and NF-κB signaling pathways under high ATO exposure provides new ideas for further studies on the toxicological mechanisms of arsenic.

Asunto(s)

Trióxido de Arsénico , Pollos , Inmunidad Innata , Hígado , FN-kappa B , Nucleotidiltransferasas , Transducción de Señal , Animales , Trióxido de Arsénico/toxicidad , FN-kappa B/metabolismo , Inmunidad Innata/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/inmunología , Transducción de Señal/efectos de los fármacos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Inflamación/inducido químicamente , Enfermedad Hepática Inducida por Sustancias y Drogas , Proteínas Aviares/metabolismo , Proteínas Aviares/genética

RESUMEN

BACKGROUND: Manganese ions (Mn2+) combined with adjuvants capable of damaging and lysing tumor cells form an antitumor nano-modulator that enhances the immune efficacy of cancer therapy through the cascade activation of the cyclic GMP-AMP interferon gene synthase-stimulator (cGAS-STING) pathway, which underscores the importance of developing antitumor nano-modulators, which induce DNA damage and augment cGAS-STING activity, as a critical future research direction. METHODS AND RESULTS: We have successfully synthesized an antitumor nano-modulator, which exhibits good dispersibility and biosafety. This nano-modulator is engineered by loading manganese dioxide nanosheets (M-NS) with zebularine (Zeb), known for its immunogenicity-enhancing effects, and conducting targeted surface modification using hyaluronic acid (HA). After systemic circulation to the tumor site, Mn2+, Zeb, and reactive oxygen species (ROS) are catalytically released in the tumor microenvironment by H+ and H2O2. These components can directly or indirectly damage the DNA or mitochondria of tumor cells, thereby inducing programmed cell death. Furthermore, they promote the accumulation of double-stranded DNA (dsDNA) in the cytoplasm, enhancing the activation of the cGAS-STING signalling pathway and boosting the production of type I interferon and the secretion of pro-inflammatory cytokines. Additionally, Zeb@MH-NS enhances the maturation of dendritic cells, the infiltration of cytotoxic T lymphocytes, and the recruitment of natural killer cells at the tumor site. CONCLUSIONS: This HA-modified manganese-based hybrid nano-regulator can enhance antitumor therapy by boosting innate immune activity and may provide new directions for immunotherapy and clinical translation in cancer.

Asunto(s)

Inmunidad Innata , Compuestos de Manganeso , Proteínas de la Membrana , Nucleotidiltransferasas , Óxidos , Transducción de Señal , Microambiente Tumoral , Nucleotidiltransferasas/metabolismo , Microambiente Tumoral/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Animales , Compuestos de Manganeso/química , Compuestos de Manganeso/farmacología , Proteínas de la Membrana/metabolismo , Transducción de Señal/efectos de los fármacos , Ratones , Óxidos/química , Óxidos/farmacología , Manganeso/química , Manganeso/farmacología , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Femenino , Ratones Endogámicos C57BL

RESUMEN

Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.

Asunto(s)

Dominio Catalítico , Mycobacterium tuberculosis , Nucleotidiltransferasas , Fosforilación , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Modelos Moleculares , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Cristalografía por Rayos X

RESUMEN

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.

Asunto(s)

Linfocitos T CD8-positivos , Linfocitos Infiltrantes de Tumor , Proteínas de la Membrana , Inestabilidad de Microsatélites , Transducción de Señal , Neoplasias Gástricas , Microambiente Tumoral , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Microambiente Tumoral/inmunología , Masculino , Femenino , Reparación de la Incompatibilidad de ADN/genética , Persona de Mediana Edad , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Anciano

RESUMEN

Immune checkpoint blockade (ICB) has emerged as a promising therapeutic option for hepatocellular carcinoma (HCC), but resistance to ICB occurs and patient responses vary. Here, we uncover protein arginine methyltransferase 3 (PRMT3) as a driver for immunotherapy resistance in HCC. We show that PRMT3 expression is induced by ICB-activated T cells via an interferon-gamma (IFNγ)-STAT1 signaling pathway, and higher PRMT3 expression levels correlate with reduced numbers of tumor-infiltrating CD8+ T cells and poorer response to ICB. Genetic depletion or pharmacological inhibition of PRMT3 elicits an influx of T cells into tumors and reduces tumor size in HCC mouse models. Mechanistically, PRMT3 methylates HSP60 at R446 to induce HSP60 oligomerization and maintain mitochondrial homeostasis. Targeting PRMT3-dependent HSP60 methylation disrupts mitochondrial integrity and increases mitochondrial DNA (mtDNA) leakage, which results in cGAS/STING-mediated anti-tumor immunity. Lastly, blocking PRMT3 functions synergize with PD-1 blockade in HCC mouse models. Our study thus identifies PRMT3 as a potential biomarker and therapeutic target to overcome immunotherapy resistance in HCC.

Asunto(s)

Carcinoma Hepatocelular , Chaperonina 60 , Neoplasias Hepáticas , Proteínas de la Membrana , Nucleotidiltransferasas , Proteína-Arginina N-Metiltransferasas , Transducción de Señal , Animales , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Humanos , Ratones , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Chaperonina 60/metabolismo , Chaperonina 60/genética , Línea Celular Tumoral , Metilación , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Mitocondrias/metabolismo , Ratones Endogámicos C57BL , ADN Mitocondrial/genética , ADN Mitocondrial/inmunología , ADN Mitocondrial/metabolismo , Interferón gamma/metabolismo , Interferón gamma/inmunología , Masculino

RESUMEN

OBJECTIVE: Pancreatic cancer is an incurable malignant disease with extremely poor prognosis and a complex tumor microenvironment. We sought to characterize the role of Annexin A1 (ANXA1) in pancreatic cancer, including its ability to promote efferocytosis and antitumor immune responses. METHODS: The tumor expression of ANXA1 and cleaved Caspase-3 (c-Casp3) and numbers of tumor-infiltrating CD68+ macrophages in 151 cases of pancreatic cancer were examined by immunohistochemistry and immunofluorescence. The role of ANXA1 in pancreatic cancer was investigated using myeloid-specific ANXA1-knockout mice. The changes in tumor-infiltrating immune cell populations induced by ANXA1 deficiency in macrophages were assessed by single-cell RNA sequencing and flow cytometry. RESULTS: ANXA1 expression in pancreatic cancer patient samples correlated with the number of CD68+ macrophages. The percentage of ANXA1+ tumor-infiltrating macrophages negatively correlated with c-Casp3 expression and was significantly associated with worse survival. In mice, myeloid-specific ANXA1 deficiency inhibited tumor growth and was accompanied by the accumulation of apoptotic cells in pancreatic tumor tissue caused by inhibition of macrophage efferocytosis, which was dependent on cGAS-STING pathway-induced type I interferon signaling. ANXA1 deficiency significantly remodeled the intratumoral lymphocyte and macrophage compartments in tumor-bearing mice by increasing the number of effector T cells and pro-inflammatory macrophages. Furthermore, combination therapy of ANXA1 knockdown with gemcitabine and anti-programmed cell death protein-1 antibody resulted in synergistic inhibition of pancreatic tumor growth. CONCLUSION: This research uncovers a novel role of macrophage ANXA1 in pancreatic cancer. ANXA1-mediated regulation of efferocytosis by tumor-associated macrophages promotes antitumor immune response via STING signaling, suggesting potential treatment strategies for pancreatic cancer.

Asunto(s)

Anexina A1 , Macrófagos , Proteínas de la Membrana , Nucleotidiltransferasas , Neoplasias Pancreáticas , Microambiente Tumoral , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Animales , Humanos , Ratones , Microambiente Tumoral/inmunología , Anexina A1/metabolismo , Anexina A1/genética , Macrófagos/metabolismo , Macrófagos/inmunología , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Transducción de Señal , Femenino , Masculino , Ratones Noqueados , Eferocitosis

RESUMEN

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.

Asunto(s)

Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Fibroblastos , Glucosa , Respuesta de Proteína Desplegada , Animales , Ratones , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Glucosa/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Transducción de Señal

RESUMEN

Activation of the cGAS-STING pathway plays a key role in the innate immune response to cancer through Type-1 Interferon (IFN) production and T cell priming. Accumulation of cytosolic double-stranded DNA (dsDNA) within tumor cells and dying cells is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) to create the secondary messenger cGAMP, which in turn activates STING (STimulator of INterferon Genes), resulting in the subsequent expression of IFN-related genes. This process is regulated by Three-prime Repair EXonuclease 1 (TREX1), a 3' → 5' exonuclease that degrades cytosolic dsDNA, thereby dampening activation of the cGAS-STING pathway, which in turn diminishes immunostimulatory IFN secretion. Here, we characterize the activity of VB-85680, a potent small-molecule inhibitor of TREX1. We first demonstrate that VB-85680 inhibits TREX1 exonuclease activity in vitro in lysates from both human and mouse cell lines. We then show that treatment of intact cells with VB-85680 results in activation of downstream STING signaling, and activation of IFN-stimulated genes (ISGs). THP1-Dual™ cells cultured under low-serum conditions exhibited an enhanced ISG response when treated with VB-85680 in combination with exogenous DNA. Collectively, these findings suggest the potential of a TREX1 exonuclease inhibitor to work in combination with agents that generate cytosolic DNA to enhance the acquisition of the anti-tumor immunity widely associated with STING pathway activation.

Asunto(s)

Exodesoxirribonucleasas , Fosfoproteínas , Exodesoxirribonucleasas/metabolismo , Humanos , Fosfoproteínas/metabolismo , Ratones , Animales , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Interferones/metabolismo , Inmunidad Innata/efectos de los fármacos

RESUMEN

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.

Asunto(s)

Citosol , Estrés del Retículo Endoplásmico , Interferón beta , Proteínas de la Membrana , Nucleotidiltransferasas , Respuesta de Proteína Desplegada , Humanos , Animales , Ratones , Nucleotidiltransferasas/metabolismo , Citosol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interferón beta/metabolismo , ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo , Endorribonucleasas/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Tapsigargina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional , ADN Mitocondrial/metabolismo

RESUMEN

Inflammation responses have identified as a key mediator of in various liver diseases with high morbidity and mortality. cGAS-STING signalling is essential in innate immunity since it triggers release of type I interferons and various of proinflammatory cytokines. The potential connection between cGAS-STING pathway and liver inflammatory diseases has recently been reported widely. In our review, the impact of cGAS-STING on liver inflammation and regulatory mechanism are summarized. Furthermore, many inhibitors of cGAS-STING signalling as promising agents to cure liver inflammation are also explored in detail. A comprehensive knowledge of molecular mechanisms of cGAS-STING signalling in liver inflammation is vital for exploring novel treatments and providing recommendations and perspectives for future utilization.

Asunto(s)

Inmunidad Innata , Hepatopatías , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Humanos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Transducción de Señal/inmunología , Hepatopatías/metabolismo , Hepatopatías/inmunología , Animales , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología

RESUMEN

DNA damage is typically caused during cell growth by DNA replication stress or exposure to endogenous or external toxins. The accumulation of damaged DNA causes genomic instability, which is the root cause of many serious disorders. Multiple cellular organisms utilize sophisticated signaling pathways against DNA damage, collectively known as DNA damage response (DDR) networks. Innate immune responses are activated following cellular abnormalities, including DNA damage. Interestingly, recent studies have indicated that there is an intimate relationship between the DDR network and innate immune responses. Diverse kinds of cytosolic DNA sensors, such as cGAS and STING, recognize damaged DNA and induce signals related to innate immune responses, which link defective DDR to innate immunity. Moreover, DDR components operate in immune signaling pathways to induce IFNs and/or a cascade of inflammatory cytokines via direct interactions with innate immune modulators. Consistently, defective DDR factors exacerbate the innate immune imbalance, resulting in severe diseases, including autoimmune disorders and tumorigenesis. Here, the latest progress in understanding crosstalk between the DDR network and innate immune responses is reviewed. Notably, the dual function of innate immune modulators in the DDR network may provide novel insights into understanding and developing targeted immunotherapies for DNA damage-related diseases, even carcinomas.

Asunto(s)

Daño del ADN , Inmunidad Innata , Humanos , Animales , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Neoplasias/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas de la Membrana

RESUMEN

cGAS/STING pathway, which is highly related to tumor hypoxia, is considered as a potential target for remodeling the immunosuppressive microenvironment of solid tumors. Metal ions, such as Mn2+, activate the cGAS/STING pathway, but their efficacy in cancer therapy is limited by insufficient effect on immunogenic tumor cell death of a single ion. Here, we evaluate the association between tumor hypoxia and cGAS/STING inhibition and report a polymetallic-immunotherapy strategy based on large mesoporous trimetal-based nanozyme (AuPdRh) coordinated with Mn2+ (Mn2+@AuPdRh) to activate cGAS/STING signaling for robust adaptive antitumor immunity. Specifically, the inherent CAT-like activity of this polymetallic Mn2+@AuPdRh nanozyme decomposes the endogenous H2O2 into O2 to relieve tumor hypoxia induced suppression of cGAS/STING signaling. Moreover, the Mn2+@AuPdRh nanozyme displays a potent near-infrared-II photothermal effect and strong POD-mimic activity; and the generated hyperthermia and •OH radicals synergistically trigger immunogenic cell death in tumors, releasing abundant dsDNA, while the delivered Mn2+ augments the sensitivity of cGAS to dsDNA and activates the cGAS-STING pathway, thereby triggering downstream immunostimulatory signals to kill primary and distant metastatic tumors. Our study demonstrates the potential of metal-based nanozyme for STING-mediated tumor polymetallic-immunotherapy and may inspire the development of more effective strategies for cancer immunotherapy.

Asunto(s)

Inmunoterapia , Rayos Infrarrojos , Proteínas de la Membrana , Animales , Ratones , Proteínas de la Membrana/metabolismo , Manganeso/química , Manganeso/farmacología , Nucleotidiltransferasas/metabolismo , Porosidad , Transducción de Señal/efectos de los fármacos , Humanos , Hipoxia Tumoral/efectos de los fármacos , Oro/química , Oro/farmacología , Línea Celular Tumoral , Paladio/química , Paladio/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Femenino

RESUMEN

Prostate cancer presents as a challenging disease, as it is often characterized as an immunologically "cold" tumor, leading to suboptimal outcomes with current immunotherapeutic approaches in clinical settings. Photodynamic therapy (PDT) harnesses reactive oxygen species generated by photosensitizers (PSs) to disrupt the intracellular redox equilibrium. This process induces DNA damage in both the mitochondria and nucleus, activating the process of immunogenic cell death (ICD) and the cGAS-STING pathway. Ultimately, this cascade of events leads to the initiation of antitumor immune responses. Nevertheless, existing PSs face challenges, including suboptimal tumor targeting, aggregation-induced quenching, and insufficient oxygen levels in the tumor regions. To this end, a versatile bionic nanoplatform has been designed for the simultaneous delivery of the aggregation-induced emission PS TPAQ-Py-PF6 and paclitaxel (PTX). The cell membrane camouflage of the nanoplatform leads to its remarkable abilities in tumor targeting and cellular internalization. Upon laser irradiation, the utilization of TPAQ-Py-PF6 in conjunction with PTX showcases a notable and enhanced synergistic antitumor impact. Additionally, the nanoplatform has the capability of initiating the cGAS-STING pathway, leading to the generation of cytokines. The presence of damage-associated molecular patterns induced by ICD collaborates with these aforementioned cytokines lead to the recruitment and facilitation of dendritic cell maturation. Consequently, this elicits a systemic immune response against tumors. In summary, this promising strategy highlights the use of a multifunctional biomimetic nanoplatform, combining chemotherapy, PDT, and immunotherapy to enhance the effectiveness of antitumor treatment.

Asunto(s)

Muerte Celular Inmunogénica , Inmunoterapia , Proteínas de la Membrana , Nucleotidiltransferasas , Fotoquimioterapia , Fármacos Fotosensibilizantes , Neoplasias de la Próstata , Humanos , Nucleotidiltransferasas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata/patología , Muerte Celular Inmunogénica/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de la radiación , Proteínas de la Membrana/metabolismo , Masculino , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Ratones , Paclitaxel/química , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Porfirinas/química , Porfirinas/farmacología