RESUMEN
The recruitment of eosinophils into Leishmania lesions is frequently associated with a favorable evolution. A feasible effector for this process is eosinophil cationic protein (ECP, RNase 3), one of the main human eosinophil granule proteins, endowed with a broad spectrum of antimicrobial activity, including parasites. ECP was active on Leishmania promastigotes and axenic amastigotes (LC50's = 3 and 16 µM, respectively) but, in contrast to the irreversible membrane damage caused on bacteria and reproduced by its N-terminal peptides, it only induced a mild and transient plasma membrane destabilization on Leishmania donovani promastigotes. To assess the contribution of RNase activity to the overall leishmanicidal activity of ECP, parasites were challenged in parallel with a single-mutant version, ECP-H15A, devoid of RNase activity, that fully preserves the conformation and liposome permeabilization ability. ECP-H15A showed a similar uptake to ECP on promastigotes, but with higher LC50's (>25 µM) for both parasite stages. ECP-treated promastigotes showed a degraded RNA pattern, absent in ECP-H15A-treated samples. Moreover ECP, but not ECP-H15A, reduced more than 2-fold the parasite burden of infected macrophages. Altogether, our results suggest that ECP enters the Leishmania cytoplasm by an endocytic pathway, ultimately leading to RNA degradation as a key contribution to the leishmanicidal mechanism. Thus, ECP combines both membrane destabilization and enzymatic activities to effect parasite killing. Taken together, our data highlight the microbicidal versatility of ECP as an innate immunity component and support the development of cell-penetrating RNases as putative leishmanicidal agents.
Asunto(s)
Antiinfecciosos , Leishmania donovani , Antiinfecciosos/farmacología , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/metabolismo , Proteínas en los Gránulos del Eosinófilo/farmacología , Humanos , Ribonucleasas/metabolismo , Ribonucleasas/farmacologíaRESUMEN
BACKGROUND: Eosinophils play a key role in the asthma allergic response by releasing cytotoxic molecules such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) that generate epithelium damages. OBJECTIVE: We sought to identify genetic variants influencing ECP and EDN levels in asthma-ascertained families. METHODS: We performed univariate and bivariate genome-wide association analyses of ECP and EDN levels in 1018 subjects from the EGEA study with follow-up in 153 subjects from the Saguenay-Lac-Saint-Jean study and combined the results of these 2 studies through meta-analysis. We then conducted Bayesian statistical fine mapping together with quantitative trait locus and functional annotation analyses to identify the most likely functional genetic variants and candidate genes. RESULTS: We identified 5 genome-wide significant loci (P < 5 × 10<sup>-8</sup>) including 7 distinct signals associated with ECP and/or EDN levels. The genes targeted by our fine mapping and functional search include RNASE2 and RNASE3 (14q11), which encode EDN and ECP, respectively, and 4 other genes that regulate ECP and EDN levels. These 4 genes were JAK1 (1p31), a transcription factor that plays a key role in the immune response and acts as a potential therapeutic target for eosinophilic asthma; ARHGAP25 (2p13), which is involved in leukocyte recruitment to inflammatory sites; NDUFA4 (7p21), which encodes a component of the mitochondrial respiratory chain and is involved in cellular response to stress; and CTSL (9q22), which is involved in immune response, extracellular remodeling, and allergic inflammation. CONCLUSION: Analysis of specific phenotypes produced by eosinophils allows the identification of genes that play a major role in allergic response and inflammation, and offers potential therapeutic targets for asthma.
Asunto(s)
Asma , Hipersensibilidad , Humanos , Eosinófilos , Estudio de Asociación del Genoma Completo , Teorema de Bayes , Neurotoxina Derivada del Eosinófilo/genética , Neurotoxina Derivada del Eosinófilo/metabolismo , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/metabolismo , Hipersensibilidad/metabolismo , Inflamación/metabolismo , Proteínas en los Gránulos del Eosinófilo/genética , Proteínas en los Gránulos del Eosinófilo/metabolismo , Proteínas Sanguíneas/metabolismoRESUMEN
Background Previous gene expression studies have identified genes IFNγ, TNFα, RNase 3, CXCL9, and CD55 as potential biomarkers for sarcoidosis and/or chronic beryllium disease (CBD). We hypothesized that differential expression of these genes could function as diagnostic biomarkers for sarcoidosis and CBD, and prognostic biomarkers for sarcoidosis. Study Design/Methods We performed RT-qPCR on whole blood samples from CBD (n = 132), beryllium sensitized (BeS) (n = 109), and sarcoidosis (n = 99) cases and non-diseased controls (n = 97) to determine differential expression of target genes. We then performed logistic regression modeling and generated ROC curves to determine which genes could most accurately differentiate: 1) CBD versus sarcoidosis 2) CBD versus BeS 3) sarcoidosis versus controls 4) non-progressive versus progressive sarcoidosis. Results CD55 and TNFα were significantly upregulated, while CXCL9 was significantly downregulated in CBD compared to sarcoidosis (p < 0.05). The ROC curve from the logistic regression model demonstrated high discriminatory ability of the combination of CD55, TNFα, and CXCL9 to distinguish between CBD and sarcoidosis with an AUC of 0.98. CD55 and TNFα were significantly downregulated in sarcoidosis compared to controls (p < 0.05). The ROC curve from the model showed a reasonable discriminatory ability of CD55 and TNFα to distinguish between sarcoidosis and controls with an AUC of 0.86. There was no combination of genes that could accurately differentiate between CBD and BeS or sarcoidosis phenotypes. Interpretation CD55, TNFα and CXCL9 expression levels can accurately differentiate between CBD and sarcoidosis, while CD55 and TNFα expression levels can accurately differentiate sarcoidosis and controls.
Asunto(s)
Beriliosis/diagnóstico , Beriliosis/genética , Regulación de la Expresión Génica/genética , Expresión Génica/genética , Sarcoidosis Pulmonar/diagnóstico , Sarcoidosis Pulmonar/genética , Adulto , Anciano , Biomarcadores/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Enfermedad Crónica , Diagnóstico Diferencial , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/metabolismo , Femenino , Marcadores Genéticos , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory "core-response" independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners.
Asunto(s)
Proteína Catiónica del Eosinófilo/metabolismo , Secuencia de Aminoácidos , Línea Celular , Regulación hacia Abajo , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib/farmacología , Humanos , Inmunidad Innata , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/virología , Mutagénesis Sitio-Dirigida , Mycobacteriaceae/efectos de los fármacos , Mycobacteriaceae/fisiología , Mapas de Interacción de Proteínas , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Virus Sincitial Respiratorio Humano/fisiología , Alineación de Secuencia , Transducción de Señal , Transcriptoma , Regulación hacia ArribaRESUMEN
Severe malaria (SM) caused by Plasmodium falciparum (Pf) infection has been associated with life-threatening anemia, metabolic acidosis, cerebral malaria and multiorgan dysfunction. It may lead to death if not treated promptly. RNASE 3 has been linked to Pf growth inhibition and its polymorphisms found associated with SM and cerebral malaria in African populations. This study aimed to assess the association of RNASE 3 polymorphisms with SM in an Indian population. RNASE 3 gene and flanking regions were amplified followed by direct DNA sequencing in 151 Indian patients who visited Wenlock District Government Hospital, Mangalore, Karnataka, India. Allele, genotype and haplotype frequencies were compared between patients with SM (n = 47) and uncomplicated malaria (UM; n = 104). Homozygous mutant genotype was only found for rs2233860 (+ 499G > C) polymorphism (< 1% frequency). No significant genetic associations were found for RNASE 3 polymorphism genotypes and alleles in Indian SM patients using the Fisher's exact test. C-G-G haplotype of rs2233859 (- 38C > A), rs2073342 (+ 371C > G) and rs2233860 (+ 499G > C) polymorphisms was correlated significantly with SM patients (OR = 3.03; p = 0.008) after Bonferroni correction. A haplotype of RNASE 3 gene was found associated with an increased risk of SM and confirming that RNASE 3 gene plays a role in susceptibility to SM.
Asunto(s)
Proteína Catiónica del Eosinófilo/genética , Predisposición Genética a la Enfermedad/genética , Haplotipos , Malaria Falciparum/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Alelos , Niño , Proteína Catiónica del Eosinófilo/metabolismo , Femenino , Frecuencia de los Genes , Genotipo , Humanos , India , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is associated with childhood asthma. Nevertheless, not all children exposed to RSV develop asthma symptoms, possibly because genes modulate the effects of RSV on asthma exacerbations. OBJECTIVE: The purpose of this study was to identify genes that modulate the effect of RSV latent infection on asthma exacerbations. METHODS: We performed a meta-analysis to investigate differentially expressed genes (DEGs) of RSV infection from Gene Expression Omnibus datasets. Expression quantitative trait loci (eQTL) methods were applied to select single nucleotide polymorphisms (SNPs) that were associated with DEGs. Gene-based analysis was used to identify SNPs that were significantly associated with asthma exacerbations in the Taiwanese Consortium of Childhood Asthma Study (TCCAS), and validation was attempted in an independent cohort, the Childhood Asthma Management Program (CAMP). Gene-RSV interaction analyses were performed to investigate the association between the interaction of SNPs and RSV latent infection on asthma exacerbations. RESULTS: A total of 352 significant DEGs were found by meta-analysis of RSV-related genes. We used 38 123 SNPs related to DEGs to investigate the genetic main effects on asthma exacerbations. We found that eight RSV-related genes (GADD45A, GYPB, MS4A3, NFE2, RNASE3, EPB41L3, CEACAM6 and CEACAM3) were significantly associated with asthma exacerbations in TCCAS and also validated in CAMP. In TCCAS, rs7251960 (CEACAM3) significantly modulated the effect of RSV latent infection on asthma exacerbations (false-discovery rate <0.05). The rs7251960 variant was associated with CEACAM3 mRNA expression in lung tissue (p for trend=1.2×10-7). CEACAM3 mRNA was reduced in nasal mucosa from subjects with asthma exacerbations in two independent datasets. CONCLUSIONS: rs7251960 is an eQTL for CEACAM3, and CEACAM3 mRNA expression is reduced in subjects experiencing asthma exacerbations. CEACAM3 may be a modulator of RSV latent infection on asthma exacerbations.
Asunto(s)
Asma/genética , Asma/virología , Antígeno Carcinoembrionario/genética , ARN Mensajero/metabolismo , Infecciones por Virus Sincitial Respiratorio/complicaciones , Adolescente , Antígenos CD/genética , Asma/fisiopatología , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular/genética , Niño , Progresión de la Enfermedad , Proteína Catiónica del Eosinófilo/genética , Femenino , Proteínas Ligadas a GPI/genética , Perfilación de la Expresión Génica , Genotipo , Glicoforinas/genética , Humanos , Inmunoglobulina M/sangre , Infección Latente/complicaciones , Infección Latente/inmunología , Pulmón/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Subunidad p45 del Factor de Transcripción NF-E2/genética , Polimorfismo de Nucleótido Simple , Mucosa Respiratoria/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Brote de los SíntomasRESUMEN
Positive bronchodilation (BD) tests can be noticed in some stable chronic obstructive pulmonary disease (COPD) patients. The characteristics of airway inflammation in this entity remain unclear. Our study aimed to identify the characteristics of airway inflammation in stable COPD patients with positive BD tests. The airway inflammation was assessed in 88 patients with stable COPD using the examination of induced sputum in the aftermath of lung function and BD tests. Cellular counts and the levels of molecular markers including eosinophil cationic protein (ECP), myeloperoxidase (MPO), interleukin-5 (IL-5), and IL-8 were assayed by Wright's stain, Immuno-CAP system, and ELISA, RT-PCR. Among the 88 patients with stable COPD, 20 (22.7%) showed positive BD tests. The values of eosinophils (4.7%±3.4%) and ECP (90.1±41.6 ng/mL) in induced sputum in stable COPD patients with positive BD tests were markedly elevated as compared with those in stable COPD patients with negative BD tests or in healthy controls (all P>0.05), but significantly lower than those in asthmatic patients (all P<0.01). The IL-5 in sputum supernatant was significantly decreased in stable COPD patients with positive BD tests as compared with the patients with asthma (12.5±7.8 vs. 48.2±26.0 ng/mL;.P<0.01). However, healthy controls exhibited similar concentrations of IL-5 in induced sputum with patients with stable COPD, whether with positive or negative BD tests (all P>0.05). Moreover, the values of neutrophils (61.8%±15.1%), MPO (574.0±111.8 ng/mL), and IL-8 (32.6±13.4 ng/mL) in induced sputum in stable COPD patients with positive BD tests were significantly higher than those in asthmatics or normal controls (all P<0.01). However, the values of the above inflammatory markers in induced sputum were similar among stable COPD patients with positive or negative BD tests (all P>0.05). The stable COPD patients with positive BD tests may present not only eosinophilic airway inflammation but also neutrophilic airway inflammation.
Asunto(s)
Asma/diagnóstico , Biomarcadores , Inflamación/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Adolescente , Adulto , Anciano , Asma/genética , Asma/patología , Broncodilatadores/administración & dosificación , Proteína Catiónica del Eosinófilo/genética , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Humanos , Inflamación/genética , Inflamación/patología , Interleucina-5/genética , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Neutrófilos/patología , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Pruebas de Función Respiratoria , Esputo/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defence and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood. OBJECTIVE: Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion. METHODS: Fresh human and mouse eosinophils were stimulated with CCL11, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors. RESULTS: We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, is required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EAR secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EAR secretion. CONCLUSIONS: Collectively, these data suggest a cytoskeleton-dependent mechanism of EAR secretion from eosinophils, findings that are pertinent to host defence, allergy and other eosinophil-associated diseases.
Asunto(s)
Proteína Catiónica del Eosinófilo/inmunología , Eosinófilos/inmunología , Proteínas de Unión al GTP rac/inmunología , Quinasas Asociadas a rho/inmunología , Animales , Quimiocina CCL11/genética , Quimiocina CCL11/inmunología , Proteína Catiónica del Eosinófilo/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas de Unión al GTP rac/genética , Quinasas Asociadas a rho/genéticaRESUMEN
BACKGROUND: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant and activator that is synthesized not only in inflammatory cells but also in bronchial epithelial cells. The purpose of this study is to clarify whether 5-oxo-ETE can promote the production of eosinophil cation protein (ECP) by eosinophils in nasal polyps (NP) in vitro, and whether normal nasal epithelial cells can produce this lipid mediator in response to oxidative stress. MATERIALS AND METHODS: Nasal biopsy samples were obtained from normal subjects or subjects with chronic rhinosinusitis with NP. The infiltration of eosinophil in NP was detected and cultured. After that, concentrations of ECP in eosinophil and NP cultures were evaluated after the treatment of 5-oxo-ETE or 5-oxo-ETE + its receptor (OXER) antagonist, pertussis toxin (PT). Then we studied the synthesis of 5-oxo-ETE after H2O2 stimulation by normal nasal epithelial cells and by epithelial cells of NP alone in the cultures, and also determined the OXER expression in NP. RESULTS: The number of infiltrative eosinophils in NP was increased. The ECP levels in eosinophil and NP cultures were enhanced after the administration of 5-oxo-ETE, and decreased by the PT treatment. 5-Oxo-ETE was upregulated in the cultures of nasal epithelial cells in the presence of H2O2 and of NP epithelial cells alone. The OXER was expressed in inflammatory cells, and not in epithelial cells. CONCLUSION: 5-Oxo-ETE produced by nasal epithelial cells may play a role in the formation and development of NP.
Asunto(s)
Ácidos Araquidónicos/farmacología , Proteína Catiónica del Eosinófilo/genética , Eosinófilos/efectos de los fármacos , Pólipos Nasales/inmunología , Regulación hacia Arriba , Adulto , Ensayo de Inmunoadsorción Enzimática , Proteína Catiónica del Eosinófilo/metabolismo , Eosinófilos/inmunología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mucosa Nasal/inmunología , Sinusitis/inmunología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: Severe forms of malaria (SM) are an outcome of Plasmodium falciparum infection and can cause death especially in children under 4 years of age. RNASE3 (ECP) has been identified as an inhibitor of Plasmodium parasites growth in vitro, and genetic analysis in hospitalized Ghanaian subjects has revealed the RNASE3 +371G/C (rs2073342) polymorphism as a susceptibility factor for cerebral malaria. The +371 C allele results in an Arg/Thr mutation that abolishes the cytotoxic activity of the ECP protein. The present study aims to investigate RNASE3 gene polymorphisms and their putative link to severe malaria in a malaria cohort from Senegal. METHODS/RESULTS: Patients enrolled from hospitals were classified as having either uncomplicated (UM) or severe malaria (SM). The analysis of the RNASE3 gene polymorphisms was performed in 241 subjects: 178 falciparum infected (96 SM, 82 UM) and 63 non-infected subjects as population control group (CTR). Six frequent SNPs (MAF > 3%) were identified, and one SNP was associated with malaria severity by performing a logistic regression analysis SM vs.UM: RNASE3 +499G/C (rs2233860) under age, sex as covariates and HbS/HbC polymorphisms adjustment (p = 0.003, OR 0.43, CI 95% 0.20-0.92). The polymorphisms: +371G/C (rs2073342), +499G/C (rs2233860) and +577A/T (rs8019343) defined a haplotype risk (G-G-T) for malaria severity (Fisher exact test, p = 0.03) (OR 4.1, IC 95% (1.1-14.9). CONCLUSION: In addition to the previously described association of +371G/C polymorphism in Ghanaians cohort, the RNASE3 +499G/C polymorphism was associated with susceptibility to SM in a Senegalese population. The haplotype +371G/+499G/+577T defined by RNASE3 polymorphisms was associated with severity. The genetic association identified independently in the Senegalese population provide additional evidence of a role of RNASE3 (ECP) in malaria severity.
Asunto(s)
Proteína Catiónica del Eosinófilo/genética , Predisposición Genética a la Enfermedad/genética , Malaria Cerebral , Malaria Falciparum , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Malaria Cerebral/epidemiología , Malaria Cerebral/genética , Malaria Falciparum/epidemiología , Malaria Falciparum/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Senegal/epidemiología , Adulto JovenRESUMEN
Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.
Asunto(s)
Antiinflamatorios/uso terapéutico , Clobetasol/uso terapéutico , Proteína Catiónica del Eosinófilo/genética , Eosinófilos/efectos de los fármacos , Penfigoide Ampolloso/diagnóstico , Penfigoide Ampolloso/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Movimiento Celular/efectos de los fármacos , Proteína Catiónica del Eosinófilo/sangre , Proteína Catiónica del Eosinófilo/inmunología , Proteína Mayor Básica del Eosinófilo/sangre , Proteína Mayor Básica del Eosinófilo/genética , Proteína Mayor Básica del Eosinófilo/inmunología , Neurotoxina Derivada del Eosinófilo/sangre , Neurotoxina Derivada del Eosinófilo/genética , Neurotoxina Derivada del Eosinófilo/inmunología , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Expresión Génica , Humanos , Masculino , Penfigoide Ampolloso/genética , Penfigoide Ampolloso/inmunología , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Recurrencia , Inducción de Remisión , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: Local allergic rhinitis (LAR) is a phenotype of allergic rhinitis characterized by the presence of a localized immune response in the nasal mucosa of patients with negative skin prick test (SPT) results and undetectable serum specific IgE (sIgE). It unknown whether LAR is limited to areas with low or moderate aeroallergen exposure. OBJECTIVE: To explore the presence of LAR and the clinical and immunological characteristics of this entity in geographic areas with high grass pollen loads. METHODS: A cross-sectional observational study was carried out in 2 hospitals in central Spain (Madrid and Ciudad Real). Sixty-one patients with seasonal rhinitis and negative SPT results and undetectable serum sIgE were evaluated using a clinical questionnaire, determination of serum total IgE, and a nasal allergen provocation test (NAPT) with Phleum species. The response to NAPT was monitored using assessment of nasal symptoms, acoustic rhinometry, and determination of sIgE, tryptase, and eosinophil cationic protein in the nasal cavity. RESULTS: Seasonal LAR was detected in 37 patients (61%) using the techniques described above. Eleven percent of patients with LAR were adolescents or children, and 14% reported onset of rhinitis in childhood. Most patients reported persistent-moderate seasonal nasal symptoms, and 41% reported worsening of the disease during the last 2 years. Conjunctivitis was the most common comorbidity, affecting 95% of cases. CONCLUSIONS: LAR to grass pollen is relevant in patients with seasonal symptoms indicative of allergic rhinitis but with a negative skin test result who live in areas with high allergenic pollen loads. This entity should be included the differential diagnosis of rhinitis.
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Alérgenos/inmunología , Conjuntivitis/inmunología , Mucosa Nasal/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Adolescente , Adulto , Anciano , Niño , Conjuntivitis/sangre , Conjuntivitis/complicaciones , Conjuntivitis/patología , Estudios Transversales , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/inmunología , Femenino , Expresión Génica , Humanos , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Mucosa Nasal/patología , Pruebas de Provocación Nasal , Phleum/química , Phleum/inmunología , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/complicaciones , Rinitis Alérgica Estacional/patología , Estaciones del Año , Pruebas Cutáneas , Encuestas y Cuestionarios , Triptasas/genética , Triptasas/inmunologíaRESUMEN
Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.
Asunto(s)
Asma/genética , Proteína Catiónica del Eosinófilo/genética , Eosinófilos/enzimología , ARN Mensajero/biosíntesis , Asma/sangre , Asma/enzimología , Hiperreactividad Bronquial/sangre , Hiperreactividad Bronquial/genética , Niño , Proteína Catiónica del Eosinófilo/biosíntesis , Proteína Catiónica del Eosinófilo/sangre , Femenino , Humanos , Masculino , ARN Mensajero/sangre , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (NPs) in Western populations is associated with TH2 cytokine polarization. IL-25, an IL-17 family cytokine, was recently reported to induce TH2-type immune responses and to contribute to several allergic diseases, such as atopic dermatitis and asthma. However, the role of IL-25 in Asian patients with nasal polyposis remains unclear. OBJECTIVE: We sought to determine the role of IL-25 in Asian patients with nasal polyposis and CRS. METHODS: We investigated IL-25 expression and its cellular origins in NPs of human subjects using immunohistochemistry (IHC), quantitative RT-PCR, and ELISA of NP tissues. Correlations between IL-25 expression and expression of other inflammatory markers in NP tissues were also explored. Anti-IL-25 neutralizing antibody was administered in an ovalbumin- and staphylococcal enterotoxin B-induced murine NP model to confirm the function of IL-25 during nasal polypogenesis. RESULTS: IL-25 expression was upregulated in NP mucosa from patients with CRS with NPs compared with uncinate process tissue from control subjects and those with CRS without NPs. Overexpression of epithelial IL-25 was confirmed by using IHC, and double IHC staining showed that tryptase-positive cells were one of the main sources of IL-25 among immune cells. Furthermore, IL-17 receptor B levels were also increased in immune cells of patients with NPs compared with those in control subjects. In NPs IL-25 mRNA expression positively correlated with the expression of several inflammatory markers, including T-box transcription factor, RAR-related orphan receptor C, GATA3, eosinophil cationic protein, TGF-ß1, and TGF-ß2. IL-25 was more abundant in the murine NP model compared with control mice, and similar correlations between IL-25 and inflammatory markers were observed in murine models. Anti-IL-25 treatment reduced the number of polyps, mucosal edema thickness, collagen deposition, and infiltration of inflammatory cells, such as eosinophils and neutrophils. This treatment also inhibited expression of local inflammatory cytokines, such as IL-4 and IFN-γ. Furthermore, expression of CCL11, CXCL2, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in the nasal mucosa was suppressed in the anti-IL-25-treated group. CONCLUSION: Our results suggest that IL-25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the pathogenesis of CRS with NPs in Asian patients. In addition, our results suggest the novel possibility of treating nasal polyposis with anti-IL-25 therapy.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Expresión Génica/efectos de los fármacos , Interleucina-17/antagonistas & inhibidores , Pólipos Nasales/tratamiento farmacológico , Rinitis/tratamiento farmacológico , Sinusitis/tratamiento farmacológico , Adulto , Animales , Estudios de Casos y Controles , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/inmunología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/inmunología , Expresión Génica/inmunología , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Masculino , Ratones , Persona de Mediana Edad , Pólipos Nasales/complicaciones , Pólipos Nasales/genética , Pólipos Nasales/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/patología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/inmunología , Rinitis/complicaciones , Rinitis/genética , Rinitis/inmunología , Sinusitis/complicaciones , Sinusitis/genética , Sinusitis/inmunología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/inmunologíaRESUMEN
RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil ribonuclease cluster, and the only known RNase A ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ⪠liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished â¼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.
Asunto(s)
Proteína Catiónica del Eosinófilo/metabolismo , Macrófagos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/genética , Inmunidad Innata , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Bazo/citologíaRESUMEN
BACKGROUND: Eosinophil cationic protein (ECP) is one of four basic proteins of the secretory granules of eosinophils. It has a variety of functions associated with inflammatory responses. Little is known about the causes for variation in serum ECP levels. AIM: To identify factors associated with variation in serum ECP and to determine the relative proportion of the variation in ECP due to genetic and non-genetic factors, in an adult twin sample. METHODS: A sample of 575 twins, selected through a proband with self-reported asthma, had serum ECP, lung function, airway responsiveness to methacholine, exhaled nitric oxide, and skin test reactivity, measured. Linear regression analysis and variance component models were used to study factors associated with variation in ECP and the relative genetic influence on ECP levels. RESULTS: Sex (regression coefficient = -0.107, P < 0.001), body mass index (BMI) (0.007, P = 0.028), and airway responsiveness to methacholine (0.074, P = 0.001) were significantly associated with ECP. Adjusted for these factors, ECP correlated 0.53 (P < 0.001) and 0.27 (P = 0.001) in monozygotic and dizygotic twins, respectively (P-value for difference = 0.05). According to the most parsimonious variance component model, genetic factors accounted for 57% (CI: 42-72%, P < 0.001) of the variance in ECP levels, whereas the remainder (43%) was ascribable to non-shared environmental factors. The genetic correlation between ECP and airway responsiveness to methacholine was statistically non-significant (r = -0.11, P = 0.50). CONCLUSION: Around half of all variance in serum ECP is explained by genetic factors. Serum ECP is influenced by sex, BMI, and airway responsiveness. Serum ECP and airway responsiveness seem not to share genetic variance.
Asunto(s)
Proteína Catiónica del Eosinófilo/sangre , Proteína Catiónica del Eosinófilo/genética , Variación Genética , Adulto , Dinamarca , Eosinófilos/metabolismo , Espiración , Femenino , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/diagnóstico , Hipersensibilidad/epidemiología , Hipersensibilidad/genética , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Sistema de Registros , Factores de Riesgo , Pruebas Cutáneas , Encuestas y Cuestionarios , Gemelos , Adulto JovenRESUMEN
Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single-nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene. In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil-derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5'nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non-allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non-allergic and allergic subjects.The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.
Asunto(s)
Proteína Catiónica del Eosinófilo/biosíntesis , Proteína Catiónica del Eosinófilo/genética , Neurotoxina Derivada del Eosinófilo/biosíntesis , Neurotoxina Derivada del Eosinófilo/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Proteína Catiónica del Eosinófilo/inmunología , Neurotoxina Derivada del Eosinófilo/inmunología , Eosinófilos/enzimología , Eosinófilos/inmunología , Femenino , Regulación de la Expresión Génica , Frecuencia de los Genes , Humanos , Hipersensibilidad/enzimología , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
Asunto(s)
Proteína Catiónica del Eosinófilo/metabolismo , Heparina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteína Catiónica del Eosinófilo/química , Proteína Catiónica del Eosinófilo/genética , Eosinófilos/enzimología , Expresión Génica , Heparina/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , TermodinámicaRESUMEN
In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.
Asunto(s)
Biología Computacional , Proteína Catiónica del Eosinófilo/genética , Genoma , MicroARNs/genética , Filogenia , Phytophthora/genética , ARN Interferente Pequeño/genética , Secuencia de Aminoácidos , Elementos Transponibles de ADN , Proteína Catiónica del Eosinófilo/clasificación , Proteína Catiónica del Eosinófilo/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/clasificación , MicroARNs/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Phytophthora/clasificación , Phytophthora/metabolismo , Enfermedades de las Plantas , Interferencia de ARN , ARN Interferente Pequeño/clasificación , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The ribonuclease (RNase) A superfamily lineage includes distant members with antimicrobial properties, suggesting a common ancestral host-defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we applied site-directed mutagenesis on its closest family homolog, the eosinophil-derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as being among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N-terminus involved in the protein antimicrobial action, encompassing residues 8-16 and 34-36. Here, we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double-mutant Q34R/R35W displays enhanced bactericidal activity, particularly towards Gram-negative bacteria, and a significant increase in its affinity towards the bacterial outer membrane lipopolysaccharides. Moreover, we confirmed the direct contribution of residue W35 in lipopolysaccharide binding, membrane interaction and permeabilization processes. Furthermore, additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for protein self-aggregation and triggers bacterial agglutination, thereby increasing the final antimicrobial activity by up to 20-fold. Our results highlight how single selected mutations can reshape the entire protein function. This study provides an example of how structure-guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles.