RESUMEN
Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) has been a potential target in the management of pathophysiology and clinical sequelae of atherosclerosis. LR12 peptide effectively blocks ligand-TREM-1 interaction; however, the short half-life of LR12 is a major hurdle in its translational application in atherosclerosis management warranting new methods for sustained bioavailability in clinical applications. The present study reports a novel method of packing the coding sequence of LR12 in a lentiviral system to ensure a sustained expression and bioavailability for effective TREM-1 inhibition. Lentivirus vector systems (LV-LR12 and LV-SP) for the expression of LR12 peptide and SP (scrambled peptide) were successfully designed, constructed, and tested in vitro in smooth muscle cells (SMCs). Viral amounts obtained were 703.6 ± 145.12 and 609.3 ± 145.93 ng/ml p24 for LV-LR12 and LV-SP, respectively which correspond to ~ 107 IFU/ml for both vectors. Dot blot assay revealed significantly increased expression of LR12-FLAG and SP-FLAG in 125 µg total protein which was doubled in 250 µg protein with respect to un-transduced SMCs suggesting the sustained release of LR12/SP as confirmed by ELISA. Cellular expression of LR12-FLAG and SP-FLAG displayed 8.44-fold and 7.55-fold increase, respectively compared to the control SMCs. The findings demonstrated a promising strategy for packing the LR12 coding sequence in lentiviral vector for TREM-1 inhibition for the management of atherosclerosis and other inflammatory diseases.
Asunto(s)
Aterosclerosis , Terapia Genética , Lentivirus/genética , Transducción Genética , Receptor Activador Expresado en Células Mieloides 1 , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/terapia , Porcinos , Porcinos Enanos , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Receptor Activador Expresado en Células Mieloides 1/genéticaRESUMEN
Triggering receptor expressed on myeloid cells 1 (TREM1) participates in the development of endometritis. This study aims at identifying the effects and interaction of TREM1 and upstream stimulatory factor 2 (USF2) in endometritis by using a model of lipopolysaccharide (LPS)-induced human endometrial epithelial cells (HEnEpCs). ELISA was performed to determine the levels of interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF-α) after LPS stimulation. TREM1 and USF2 expression was examined with RT-qPCR and Western blot. The JASPAR database was employed to predict the binding site between USF2 and TREM1, which was confirmed by luciferase reporter and chromatin immunoprecipitation assays. After TREM1 overexpression, IL-6, IL-1ß, and TNF-α expression was detected by ELISA. Next, the binding of TREM1 to toll-like receptor (TLR) 2/4 was examined with co-immunoprecipitation. Then, proteins in TLR2/4-nuclear factor-kappaB (NF-κB) signaling in HEnEpCs under LPS condition were assessed by Western blot or immunofluorescence before and after TREM1 knockdown. Finally, TLR2 or TLR4 was silenced to explore whether intervene TLR2/4-NF-κB signaling pathway could rescue TREM1-overexpression-induced inflammation in LPS-induced HEnEpCs. Results revealed that upregulated TREM1 was observed in LPS-challenged HEnEpCs. Next, USF2 was found to have transcriptionally active TREM1 expression. Additionally, USF2 knockdown decreased the levels of IL-6, IL-1ß, and TNF-α, whereas this effect was rescued after TREM1 overexpression. Besides, TREM1 could bind to TLR2/4 to regulate NF-κB signaling. Moreover, the intervention of TLR2/4-NF-κB signaling pathway rescued TREM1-overexpression-induced inflammation in LPS-stimulated HEnEpCs. Collectively, USF2 promotes endometritis by upregulating TREM1, thereby activating TLR2/4-NF-κB pathway.
Asunto(s)
Endometritis/metabolismo , Endometrio/metabolismo , Células Epiteliales/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Regulación hacia Arriba , Factores Estimuladores hacia 5'/metabolismo , Femenino , HumanosRESUMEN
Hepatopathy is frequently observed in patients with severe malaria but its pathogenesis remains unclear. Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses, and exhibit pivotal roles during Plasmodium spp. infection. Here, we analyzed the impact of blockage of galectin-receptor interactions by treatment with alpha (α)-lactose on liver immunopathology during the erythrocytic stage of malaria in mice infected with Plasmodium berghei ANKA (PbANKA). Our results found that compared with PbANKA-infected mice (malarial mice), blockage of galectin-receptor interactions led to decreased host survival rate and increased peripheral blood parasitemia; exacerbated liver pathology, increased numbers of CD68+ macrophages and apoptotic cells, and increased parasite burden in the livers on days 5 and 7 post infection (p.i.) as well as increased mRNA expression levels of galectin-9 (Gal-9) and its receptor, the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), interferon (IFN)α, IFNγ, and the triggering receptor expressed on myeloid cells (TREM)-1 in the livers or spleens of PbANKA-infected mice on day 7 p.i. Observed by transmission electron microscopy, the peritoneal macrophages isolated from malarial mice with α-lactose treatment had more pseudopodia than those from malarial mice. Measured by using quantitative real-time reverse transcription-polymerase chain reaction assay, the mRNA expression levels of Gal-9, IFNα, IFNß, IFNγ, and TREM-1 were increased in the peritoneal macrophages isolated from malarial mice with α-lactose treatment in comparison of those from malarial mice. Furthermore, significant positive correlations existed between the mRNA levels of Gal-9 and Tim-3/IFNγ/TREM-1 in both the livers and the peritoneal macrophages, and between Gal-9 and Tim-3/TREM-1 in the spleens of malarial mice; significant positive correlations existed between the mRNA levels of Gal-9 and IFNγ in the livers and between Gal-9 and IFNα in the peritoneal macrophages from malarial mice treated with α-lactose. Our data suggest a potential role of galectin-receptor interactions in limiting liver inflammatory response and parasite proliferation by down-regulating the expressions of IFNα, IFNγ, and TREM-1 during PbANKA infection.
Asunto(s)
Eritrocitos/parasitología , Galectinas/fisiología , Hígado/patología , Malaria/patología , Parasitemia/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Femenino , Galectinas/antagonistas & inhibidores , Receptor 2 Celular del Virus de la Hepatitis A/antagonistas & inhibidores , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Lactosa/farmacología , Lactosa/toxicidad , Hígado/parasitología , Pulmón/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Malaria/sangre , Ratones , Plasmodium berghei/crecimiento & desarrollo , Seudópodos/ultraestructura , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Receptor Activador Expresado en Células Mieloides 1/genéticaRESUMEN
Background: Triggering receptor expressed on myeloid cells-1 (TREM-1) has been reported as a biomarker in many cancers. However, the biological function of TREM-1 in papillary thyroid carcinoma (PTC) remains unknown. Methods: We obtained TREM-1 expression data from The Cancer Genome Atlas (TCGA) database. Enrichment analysis of coexpressed genes and TREM-1 methylation analysis were performed via LinkedOmics. The correlations between TREM-1 and immune infiltrates were investigated via ESTIMATE, TIMER and TISIDB. We analyzed the association of TREM-1 expression with pan-cancer overall survival via Gene Expression Profiling Interactive Analysis (GEPIA). Results: TREM-1 has lower methylation levels and higher expression levels in PTC tissues compared to normal tissues. TREM-1 expression is significantly associated with poor prognosis, advanced T classification, advanced N classification, and an increased incidence of BRCA2 and BRAF mutations. Genes coexpressed with TREM-1 primarily participate in immune-related pathways. TREM-1 expression is positively correlated with immune infiltration, tumor progression and poor overall survival across cancers. Conclusions: TREM-1 is a good prognostic and diagnostic biomarker in PTC. TREM-1 may promote thyroid cancer progression through immune-related pathways. Methylation may act as an upstream regulator of TREM-1 expression and biological function. Additionally, TREM-1 has broad prognostic value in a pan-cancer cohort.
Asunto(s)
Biología Computacional/métodos , Cáncer Papilar Tiroideo/metabolismo , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Anciano , Biomarcadores de Tumor/metabolismo , Metilación de ADN , Bases de Datos Factuales , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Mapeo de Interacción de Proteínas , Proteómica , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
Gefitinib (Iressa), is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), used in the targeted treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC). Skin toxicity is the major adverse effect observed in patients treated with EGFR-targeted TKIs such as gefitinib and erlotinib. To date, a corresponding skin animal model has not been established to address the mechanisms of these effects. Therefore, we analyzed the skin rash phenotype and its pathological features in Brown Norway (BN) rats treated with gefitinib 2.5 mg, 5.0 mg, or 10 mg/100 g/day for 4 weeks. We found that treatment with gefitinib led to weight loss, rash, itching, and hair loss in a dose-dependent manner. We also investigated the skin pathology and found that the animal model showed thickening of the epidermis, loss of moisture, and apoptosis of keratinocytes. Immunohistochemistry, flow cytometry, and analysis of monocytes and leukocytes in the blood revealed increased macrophage infiltration was associated with the cutaneous toxicities induced by gefitinib in the BN rats. Finally, we found that gefitinib-induced cutaneous toxicity is significantly associated with three inflammatory cytokines known to be secreted by activated macrophages, TREM-1, CINC-2, and CINC-3.
Asunto(s)
Gefitinib/toxicidad , Macrófagos/efectos de los fármacos , Piel/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Quimiocina CXCL2/biosíntesis , Quimiocinas CXC/biosíntesis , Modelos Animales de Enfermedad , Femenino , Cabello/efectos de los fármacos , Inmunohistoquímica , Inflamación , Leucocitos , Macrófagos/metabolismo , Fenotipo , Ratas , Ratas Endogámicas BN , Receptor Activador Expresado en Células Mieloides 1/biosíntesisRESUMEN
Tryptophanyl-tRNA synthetase 1 (WARS1) is an endogenous ligand of mammalian Toll-like receptors (TLR) 2 and TLR4. Microarray data, using mRNA from WARS1-treated human peripheral blood mononuclear cells (PBMCs), had indicated WARS1 to mainly activate innate inflammatory responses. However, exact molecular mechanism remains to be understood. The triggering receptor expressed on myeloid cells (TREM)-1 is an amplifier of pro-inflammatory processes. We found WARS1 to significantly activate TREM-1 at both mRNA and protein levels, along with its cell surface expression and secretion in macrophages. WARS1 stimulated TREM-1 production via TLR2 and TLR4, mediated by both MyD88 and TRIF, since targeted deletion of TLR4, TLR2, MyD88, and TRIF mostly abrogated TREM-1 activation. Furthermore, WARS1 promoted TREM-1 downstream phosphorylation of DAP12, Syk, and AKT. Knockdown of TREM-1 and inhibition of Syk kinase significantly suppressed the activation of inflammatory signaling loop from MyD88 and TRIF, leading to p38 MAPK, ERK, and NF-κB inactivation. Finally, MyD88, TRIF, and TREM-1 signaling pathways were shown to be cooperatively involved in WARS1-triggered massive production of IL-6, TNF-α, IFN-ß, MIP-1α, MCP-1, and CXCL2, where activation of Syk kinase was crucial. Taken together, our data provided a new insight into WARS1's strategy to amplify innate inflammatory responses via TREM-1.
Asunto(s)
Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Triptófano-ARNt Ligasa/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/fisiología , Quinasa Syk/metabolismo , Receptor Activador Expresado en Células Mieloides 1/biosíntesisRESUMEN
Around a 20-30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders (n = 27) and non-responders to anti-TNF therapy (n = 6). Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2-∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders (p value < 0.05). Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.
Asunto(s)
Adalimumab/farmacología , Antiinflamatorios/farmacología , Antirreumáticos/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Infliximab/farmacología , Transcriptoma/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adalimumab/uso terapéutico , Adolescente , Antiinflamatorios/uso terapéutico , Antirreumáticos/uso terapéutico , Niño , Preescolar , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/genética , Infliximab/uso terapéutico , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/sangre , ARN Mensajero/genética , Receptores Tipo II del Factor de Necrosis Tumoral/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Proteína smad7/biosíntesis , Proteína smad7/genética , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/genética , Resultado del Tratamiento , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Receptor Activador Expresado en Células Mieloides 1/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , alfa-Defensinas/biosíntesis , alfa-Defensinas/genéticaRESUMEN
BACKGROUND: There is a need for rapid and accurate diagnostic biomarker for diagnosis of Salmonella fever. AIMS: The aim of the present study was to assess the importance of procalcitonin (PCT), Soluble Triggering Receptors expressed on Myeloid Cells 1 (sTREM1) and C- reactive protein (CRP) in the diagnosis of enteric fever with positive blood culture for S.typhi. METHODS: Blood samples were withdrawn from 200 patients with suspected enteric fever and subjected for the determination of CRP, PCT and sTREM-1. RESULTS: The sensitivity and specificity for PCT cut off were 97.7% & 82.5%, for CRP the sensitivity and specificity were 95.3% and 77% and for s-TREM-1 the sensitivity and specificity were 95.3% & 77%. CONCLUSION: S-TREM-1 may be considered as a novel biomarker for the diagnosis of enteric fever with good sensitivity and specificity.
Asunto(s)
Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Receptor Activador Expresado en Células Mieloides 1/sangre , Fiebre Tifoidea/sangre , Fiebre Tifoidea/diagnóstico , Adulto , Biomarcadores/sangre , Estudios Transversales , Femenino , Expresión Génica , Humanos , Masculino , Polipéptido alfa Relacionado con Calcitonina/biosíntesis , Polipéptido alfa Relacionado con Calcitonina/sangre , Polipéptido alfa Relacionado con Calcitonina/genética , Salmonella typhi/genética , Salmonella typhi/aislamiento & purificación , Receptor Activador Expresado en Células Mieloides 1/genética , Fiebre Tifoidea/genéticaRESUMEN
We aimed to investigate the impact of the single nucleotide polymorphisms of rs34436714 of the NOD-like receptor protein 12 gene on the production of tumor necrosis factor-alpha (TNFα) in patients with inflammatory bowel disease (IBD)In a matched case-control study 90 patients with IBD, 56 with Crohn disease (CD) and 34 with ulcerative colitis, were genotyped and compared to 98 healthy comparators matched for age and gender. Expression level of TNFα, interleukin (IL)-6, IL-12, and soluble triggering receptor expressed on myeloid cells were measured in patients' sera. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated for TNFα production.Serum TNFα was greater among carriers of GT/TT genotypes than GG genotypes of rs34436714. Stimulated TNFα production was also higher in carriers of GT/TT genotypes. The frequency of CD with fistulizing behavior and with CD involving the small intestine was greater among carriers of GT/TT genotypes than of the GG genotype. Distribution of the GG, GT, and TT genotypes of rs34436714 were in Hardy-Weinberg equilibrium in both groups. The genotype distribution was the same in both groups.Carriage of minor frequency alleles of rs34436714 was accompanied by greater circulating levels of TNFα and by greater capacity for stimulated TNFα production by PBMCs. These alleles had an impact on the phenotype of patients with CD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto , Anciano , Estudios de Casos y Controles , Colitis Ulcerosa/genética , Colitis Ulcerosa/fisiopatología , Enfermedad de Crohn/genética , Enfermedad de Crohn/fisiopatología , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Enfermedades Inflamatorias del Intestino/fisiopatología , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Receptor Activador Expresado en Células Mieloides 1/biosíntesisRESUMEN
Spinal cord injury (SCI) is a devastating event, leading to the progression of chronic neuropathic pain syndrome. Triggering receptor expressed on myeloid cells 1 (TREM1) is an innate immune receptor expressed on neutrophils and monocytes/macrophages. TREM1 enhances inflammatory response in various models of diseases, but its significance in SCI remains unclear. In the present study, we attempted to explore the effects of TREM1 on the regulation of SCI. Spinal cord contusion injury was performed in wild type (WT) and TREM1-knockout (TREM1KO) mice, and real time-quantitative PCR (RT-qPCR), western blot, and immunofluorescent (IF) staining were used to calculate TREM1, inflammation and oxidative stress in spinal cord tissues 42 days after SPII. In vitro, astrocytes (AST) and BV2 cells were transfected TREM siRNA or the negative control (NC) siRNA to knockdown (KD) TREM1 expressions, followed by lipopolysaccharide (LPS) stimulation to verify the role od TREM1 in modulating SPI. The results suggested that TREM1 was highly expressed in the spinal cord tissues of WT mice after SCI. TREMKO mice exhibited improved locomotor function, mechanical and thermal hypersensitivity in the hindpaws after SCI. In addition, peripheral nerve injury-related biomarkers were down-regulated by TREM1KO in SCI mice. TREM1KO increased NeuN-stained cells, and decreased GFAP and Iba-1 expressions in spinal cord tissues of mice after SCI. TREM1KO mice showed reduced expressions of inflammation-related regulators in the injured spinal cord. Further, toll like receptors (TLR-2, -3, -4, and -9), p-IκBα and p-nuclear factor-kappa B (NF-κB) protein expression levels were markedly decreased by TREM1KO in mice after SCI injury. Moreover, TREM-deficiency suppressed oxidative stress markers, while enhanced anti-oxidants, such as superoxide dismutase-1 (SOD1), NAD(P)H:quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf2) in the injured spinal cord. The in vivo results mentioned above were verified in LPS-stimulated AST and BV2 cells in vitro. Of note, the in vitro results also demonstrated that suppressing HO-1 expressions using Zn-protoporphyrin (ZnPP) abrogated TREM1KD-reduced inflammation, oxidative stress and glial cells activation. The results above demonstrated that suppressing TREM1 expressions markedly improved the outcome of SCI, most likely through reducing inflammation and oxidative stress at least partly regulated by HO-1 expressions. TREM1 inhibition might be therefore has potential as a therapeutic target after SCI.