RESUMEN
As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.
Asunto(s)
Proteína Adaptadora de Señalización NOD1 , Salmonella typhimurium , Transducción de Señal , Ubiquitinación , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Humanos , Salmonella typhimurium/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Células HEK293 , Inmunidad Innata , Inflamación/metabolismo , Inflamación/microbiología , FN-kappa B/metabolismo , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/inmunologíaRESUMEN
BACKGROUND: Nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 are involved in carcinogenic processes by recognizing bacterial cell wall components and triggering inflammation. This study explored the association between genetic variations in NOD1 and NOD2 and susceptibility to hepatocellular carcinoma (HCC) and its progression in a Moroccan population. METHODS: Genotyping of NOD1 rs2075820 (C>T) and NOD2 rs718226 (A>G) was performed using the TaqMan allelic discrimination assay in 467 Moroccan individuals. The cohort included 156 patients with hepatocellular carcinoma (HCC), 155 patients with liver cirrhosis (LC) diagnosed with HBV, HCV, or MASLD, and 156 controls. RESULTS: The NOD1 rs2075820 variant showed no association with HCC susceptibility or progression, which is consistent with in silico predictions. However, the NOD2 rs718226 G allele and GG genotype were more common in the HCC group compared to the cirrhosis and control groups. Individuals with the homozygous G variant had a 2-fold higher risk for HCC (ORad = 2.12; CI=1.01-4.44; Pad = 0.04). Those with the GG genotype also had an increased risk of HCC (GG vs. AG+AA ORad = 2.28; CI=1.15-4.54; Pad = 0.016). Furthermore, GG genotype carriers had a significantly higher risk of HCC progression (ORad = 2.58; CI=1.26-5.31; Padâ = 0.031). Individuals with the rs718226 minor allele had a significantly elevated risk of progressing from LC to HCC (ORad = 1.50; CI=1.07-2.09; Pad = 0.016). Stratification analysis indicated that men had a higher risk of HCC progression compared to women (ORad = 4.63; CI=1.53-14.00 vs. ORad = 2.73; CI=1.05-7.09). CONCLUSION: The NOD1 rs2075820 polymorphism does not appear to be a genetic risk factor for susceptibility to HCC. In contrast, the non-coding NOD2 rs718226 variant significantly increases HCC susceptibility and promotes liver cancer progression in the Moroccan population. Further studies involving larger cohorts are warranted to definitively confirm or refute the effects of NOD1 and NOD2 genetic variants on liver cancer susceptibility and progression.
Asunto(s)
Carcinoma Hepatocelular , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD2 , Polimorfismo de Nucleótido Simple , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína Adaptadora de Señalización NOD2/genética , Masculino , Femenino , Proteína Adaptadora de Señalización NOD1/genética , Persona de Mediana Edad , Marruecos , Progresión de la Enfermedad , Estudios de Casos y Controles , Adulto , Anciano , Genotipo , Cirrosis Hepática/genética , AlelosAsunto(s)
Proteína Adaptadora de Señalización NOD1 , Nodo Sinoatrial , Animales , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiopatología , Ratones , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Potenciales de Acción , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Ratones Endogámicos C57BLRESUMEN
Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.
Asunto(s)
Escherichia coli , Vesículas Extracelulares , Inflamación , Interleucina-1beta , Mucosa Intestinal , MicroARNs , Probióticos , Serotonina , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Vesículas Extracelulares/metabolismo , Probióticos/farmacología , Serotonina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células CACO-2 , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Inflamación/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/terapia , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Células Epiteliales/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Estrés Oxidativo , Regulación de la Expresión GénicaRESUMEN
NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.
Asunto(s)
Proteínas de Peces , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD2 , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Transducción de Señal , Animales , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteínas de Peces/inmunología , Percas/genética , Percas/inmunología , Percas/metabolismo , Interferones/metabolismo , Interferones/genética , Regiones Promotoras Genéticas , Transcripción Genética , Inmunidad Innata/genética , Unión ProteicaRESUMEN
OBJECTIVE: Poorly controlled diabetes frequently exacerbates lung infection, thereby complicating treatment strategies. Recent studies have shown that exendin-4 exhibits not only hypoglycemic but also anti-inflammatory properties. This study aimed to explore the role of exendin-4 in lung infection with diabetes, as well as its association with NOD1/NF-κB and the T1R2/T1R3 sweet taste receptor. METHODS: 16HBE human bronchial epithelial cells cultured with 20 mM glucose were stimulated with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA). Furthermore, SpragueâDawley rats were fed a high-fat diet, followed by intraperitoneal injection of streptozotocin and intratracheal instillation of PA. The levels of TNF-α, IL-1ß and IL-6 were evaluated using ELISAs and RTâqPCR. The expression of T1R2, T1R3, NOD1 and NF-κB p65 was assayed using western blotting and immunofluorescence staining. Pathological changes in the lungs of the rats were observed using hematoxylin and eosin (H&E) staining. RESULTS: At the same dose of LPS, the 20 mM glucose group produced more proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and had higher levels of T1R2, T1R3, NOD1 and NF-κB p65 than the normal control group (with 5.6 mM glucose). However, preintervention with exendin-4 significantly reduced the levels of the aforementioned proinflammatory cytokines and signaling molecules. Similarly, diabetic rats infected with PA exhibited increased levels of proinflammatory cytokines in their lungs and increased expression of T1R2, T1R3, NOD1 and NF-κB p65, and these effects were reversed by exendin-4. CONCLUSIONS: Diabetic hyperglycemia can exacerbate inflammation during lung infection, promote the increase in NOD1/NF-κB, and promote T1R2/T1R3. Exendin-4 can ameliorate PA-related pneumonia with diabetes and overexpression of NOD1/NF-κB. Additionally, exendin-4 suppresses T1R2/T1R3, potentially through its hypoglycemic effect or through a direct mechanism. The correlation between heightened expression of T1R2/T1R3 and an intensified inflammatory response in lung infection with diabetes requires further investigation.
Asunto(s)
Diabetes Mellitus Experimental , Exenatida , Proteína Adaptadora de Señalización NOD1 , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Ratas Sprague-Dawley , Animales , Exenatida/farmacología , Exenatida/uso terapéutico , Humanos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Masculino , Infecciones por Pseudomonas/tratamiento farmacológico , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Citocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , FN-kappa B/metabolismo , Pulmón/patología , Pulmón/efectos de los fármacos , Pulmón/microbiología , Línea Celular , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Ratas , Lipopolisacáridos , Péptidos/farmacología , Péptidos/uso terapéuticoRESUMEN
The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-κB and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells.
Asunto(s)
COVID-19 , Células Epiteliales , Pulmón , Proteína Adaptadora de Señalización NOD1 , SARS-CoV-2 , Humanos , Células A549 , Antivirales/farmacología , COVID-19/inmunología , COVID-19/virología , Tratamiento Farmacológico de COVID-19 , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/farmacología , Células Epiteliales/virología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Inmunidad Innata/efectos de los fármacos , Interleucina-8/metabolismo , Pulmón/inmunología , Pulmón/virología , Pulmón/metabolismo , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/metabolismo , SARS-CoV-2/fisiología , SARS-CoV-2/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo. METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR. RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05). CONCLUSION: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.
Asunto(s)
Glucosa , Legionella pneumophila , Macrófagos , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Animales , Humanos , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Legionella pneumophila/inmunología , Glucosa/metabolismo , Cobayas , Masculino , Interleucina-6/metabolismo , Enfermedad de los Legionarios/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células U937 , Factor de Necrosis Tumoral alfa/metabolismo , RatonesRESUMEN
Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.
Asunto(s)
Neisseria , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD2 , Peptidoglicano , Animales , Humanos , Ratones , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Transporte de Membrana , Neisseria/genética , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Peptidoglicano/metabolismoRESUMEN
Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.
Tuberculosis is the leading cause of death from an infectious disease worldwide, partially due to a lack of access to drug treatments in certain countries where the disease is common. The only available tuberculosis vaccine known as the BCG vaccine is useful for preventing cases in young children, but is ineffective in teenagers and adults. So, there is a need to develop new vaccines that offer better, and longer lasting, durable protection in people of all ages. During an infection, our immune system recognizes markers known as PAMPs on the surface of bacteria, viruses or other disease-causing pathogens. The recognition of PAMPs by the immune system enables the body to distinguish foreign invading organisms from its own cells and tissues, thus triggering a response that fights the infection. If the body encounters the infectious agent again in the future, the immune system is able to quickly recognize and eliminate it before it can cause disease. Vaccines protect us by mimicking the appearance of the pathogen to trigger the first immune response without causing the illness. The BCG vaccine contains live bacteria that are closely related to the bacterium responsible for tuberculosis called Mycobacterium tuberculosis. Both M. tuberculosis and the live bacteria used in the BCG vaccine are able to hide an important PAMP, known as the NOD-1 ligand, from the immune system, making it harder for the body to detect them. The NOD-1 ligand forms part of the bacterial cell wall and modifying the BCG bacterium so it cannot disguise this PAMP may lead to a new, more effective vaccine. To investigate this possibility, Shaku et al. used a gene editing approach to develop a modified version of the BCG bacterium which is unable to hide its NOD-1 ligand when treated with a specific drug. Immune cells trained with the modified BCG vaccine were more effective at controlling the growth of M. tuberculosis than macrophages trained using the original vaccine. Furthermore, mice vaccinated with the modified BCG vaccine were better able to limit M. tuberculosis growth in their lungs than mice that had received the original vaccine. These findings offer a new candidate vaccine in the fight against tuberculosis. Further studies will be needed to modify the vaccine for use in humans. More broadly, this work demonstrates that gene editing can be used to expose a specific PAMP present in a live vaccine. This may help develop more effective vaccines for other diseases in the future.
Asunto(s)
Vacuna BCG , Mycobacterium tuberculosis , Peptidoglicano , Tuberculosis , Animales , Peptidoglicano/metabolismo , Ratones , Vacuna BCG/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Tuberculosis/inmunología , Tuberculosis/microbiología , Humanos , Ratones Endogámicos C57BL , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Femenino , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Modelos Animales de Enfermedad , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ⧠100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.
Asunto(s)
Secuencia de Aminoácidos , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Proteína Adaptadora de Señalización NOD1 , Filogenia , Alineación de Secuencia , Vibrio alginolyticus , Animales , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD1/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Inmunidad Innata/genética , Enfermedades de los Peces/inmunología , Alineación de Secuencia/veterinaria , Vibrio alginolyticus/fisiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae/fisiología , Regulación de la Expresión Génica/inmunología , Perfilación de la Expresión Génica/veterinaria , Vibriosis/inmunología , Vibriosis/veterinaria , Ácido Diaminopimélico/química , Ácido Diaminopimélico/análogos & derivados , Perciformes/inmunología , Perciformes/genética , Peces/inmunología , Peces/genéticaRESUMEN
Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.
Asunto(s)
Toxinas Bacterianas , Neoplasias de la Mama , Resistencia a Antineoplásicos , Células Madre Neoplásicas , Proteína Adaptadora de Señalización NOD1 , Animales , Femenino , Humanos , Ratones , Toxinas Bacterianas/farmacología , Bacteroides fragilis/química , Neoplasias de la Mama/microbiología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Metaloendopeptidasas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Proteína Adaptadora de Señalización NOD1/antagonistas & inhibidores , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismoRESUMEN
The overuse of antibiotics has led to the enhanced resistance of many pathogenic bacteria, posing a threat to human health. Therefore, there is a need to develop green and safe alternatives to antibiotics. Beta-defensins play a crucial role in host defense against pathogens and have multifunctional properties, exerting key roles in innate and adaptive immunity, as well as non-immune processes. In this study, a 210 bp long cDNA sequence of yak DEFB114 gene was amplified and successfully expressed in a prokaryotic system. The DEFB114 protein exhibited significant inhibitory effects on the growth of Aspergillus fumigatus in vitro. When co-cultured with yak macrophages, DEFB114 protein enhanced macrophage phagocytic activity and increased nucleic acid fluorescence intensity (P < 0.05). DEFB114 protein also enhanced the activity of yak macrophages stimulated by inactivated Aspergillus fumigatus spores, increased the release of nitric oxide (NO), and promoted the expression of genes such as γ-actin, Lgals, Man2b, and Capg (P < 0.05). In mice experiments, DEFB114 protein promoted resistance against Aspergillus fumigatus infection, by regulating the NOD1/2-ATG16L1-NF-κB pathway to modulate the host immune response and exert its anti-infective effects. In summary, the yak DEFB114 protein could inhibit the growth of Aspergillus fumigatus and enhance the animal's resistance to pathogenic microorganisms, thereby having significant implications in the treatment and prevention of fungal infections.
Asunto(s)
Aspergilosis , FN-kappa B , Animales , Ratones , Antibacterianos , Aspergilosis/tratamiento farmacológico , Aspergillus fumigatus , Proteínas Relacionadas con la Autofagia/metabolismo , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Transducción de SeñalRESUMEN
NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.
Asunto(s)
Inmunidad Innata , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD2/metabolismo , Indoles/química , Indoles/metabolismoRESUMEN
BACKGROUND: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally, the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells, a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction. METHODS: We generated NOD1-/--Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow-retina axis. RESULTS: Elevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1-/--Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration, as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations, including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation, and the results further indicated that NOD1 had a protective effect against DR. Mechanistically, loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina, subsequently leading to diminished neutrophil chemoattraction and NETosis. CONCLUSIONS: The results of our study unveil, for the first time, the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation, culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR.
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Diabetes Mellitus , Retinopatía Diabética , Proteína Adaptadora de Señalización NOD1 , Degeneración Retiniana , Animales , Ratones , Médula Ósea/metabolismo , Diabetes Mellitus/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/terapia , Células Madre Hematopoyéticas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Ratones Endogámicos C57BL , Retina/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismoRESUMEN
As an ancient species with both conservation and commercial value, Sturgeon's inflammatory regulation mechanism is a research point. Nucleotide-binding and oligomerization domain-containing proteins 1 and 2 (NOD1/2) are classical intracellular pattern recognition receptors (PRRs) in immunity of anti-bacterial infection. However, the characterization and function of NOD1/2 in Sturgeon are still unclear. In this study, we analyzed the synteny relationship of NOD1/2 genes between Acipenser ruthenus and representative fishes at the genome-level. Results showed that the ArNOD2 collinear genes pair was present in all representative fishes. The duplicated ArNOD1/2 genes were under purifying selection during evolution as indicated by their Ka/Ks values. To explore the function of NOD1/2, we further investigated their expression patterns and the effects of pathogenic infection, PAMPs treatment, and siRNA interference in Acipenser baerii, the sibling species of A. ruthenus. Results showed that both AbNOD1/2 were expressed at early developmental stages and in different tissues. Pathogenic infection in vivo and PAMPs treatment in vitro demonstrated that AbNOD1/2 could respond to pathogen stimulation. siRNA interference with AbNOD1/2 inhibited expression levels of RIPK2 and inflammatory cytokines compared to the control group after iE-DAP or MDP treatment. This study hinted that the AbNOD1/2 could stimulate the inflammatory cytokines response during evolutionary processes.
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Infecciones Bacterianas , Moléculas de Patrón Molecular Asociado a Patógenos , Animales , Peces/genética , Citocinas , ARN Interferente Pequeño , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/genéticaRESUMEN
Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.
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Proteínas Adaptadoras de Señalización NOD , Proteína Adaptadora de Señalización NOD1 , Humanos , Proteínas Adaptadoras de Señalización NOD/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Inflamación , Nucleótidos/metabolismoRESUMEN
NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.
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Proteína Adaptadora de Señalización NOD1 , Proteínas Tirosina Quinasas Receptoras , Ácido Diaminopimélico/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismoRESUMEN
Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a downstream signaling molecule essential for the activation of several innate immune receptors, including the NOD-like receptors (NOD1 and NOD2). Recognition of pathogen-associated molecular pattern proteins by NOD1/2 leads to their interaction with RIPK2, which induces release of pro-inflammatory cytokines through the activation of NF-κB and MAPK pathways, among others. Thus, RIPK2 has emerged as a key mediator of intracellular signal transduction and represents a new potential therapeutic target for the treatment of various conditions, including inflammatory diseases and cancer. In this Perspective, first, an overview of the mechanisms that underlie RIPK2 function will be presented along with its role in several diseases. Then, the existing inhibitors that target RIPK2 and different therapeutic strategies will be reviewed, followed by a discussion on current challenges and outlook.
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Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Transducción de Señal , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , FN-kappa B/metabolismo , Citocinas/metabolismo , Proteína Adaptadora de Señalización NOD1 , Proteína Adaptadora de Señalización NOD2/metabolismoRESUMEN
BACKGROUND: Fusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance. METHODS: Fn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments. RESULTS: Tissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively. CONCLUSIONS: Our findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.