RESUMEN
Dupilumab is a monoclonal antibody approved for the treatment of atopic dermatitis (AD); however, its effects on molecular, cellular, and immunological levels remain to be elucidated. In this study, blood and dermal interstitial fluid (ISF) from nonlesional (NL) and lesional (L) skin were collected from eight patients with moderate to severe AD, before (visit 2-v2) and at the end of a 16-week treatment with dupilumab (visit 10-v10). Clinical treatment effect was demonstrated by significantly decreased AD severity scores at the end of treatment. At v10 versus v2, the percentages of CD4+ interleukin-producing cells showed a decreasing trend in ISF L and NL, unbound IL-4 levels in plasma were increased, IL-5 levels in ISF L reduced, and levels of factors involved in anti-inflammatory pathways and re-epithelization increased. At v2, ISF L showed that AD lesions might have altered amino acid pathways and lipid signaling compared to ISF NL. At v10, ISF L exhibited raised levels of long- and very-long-chain fatty acids and lipids compared to v2. Furthermore, dupilumab administration caused reduced expression of miR-155-5p and miR-378a-3p in ISF L. In conclusion, results from the present study provided novel knowledge by linking local immune and metabolic alterations to AD pathogenesis and treatment response.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Dermatitis Atópica , Líquido Extracelular , Humanos , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Masculino , Femenino , Adulto , Mediadores de Inflamación/metabolismo , Metaboloma/efectos de los fármacos , Interleucina-4/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Piel/metabolismo , Piel/efectos de los fármacos , Piel/irrigación sanguínea , Persona de Mediana Edad , Interleucina-5RESUMEN
Profiling antibodies to SARS-CoV-2 can help to assess potential immune response after COVID-19 disease. Luciferase IP system (LIPS) assay is a sensitive method for quantitative detection of antibodies to antigens in their native conformation. We here describe LIPS to detect antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in COVID-19 patients. The antibodies targeted both S and N fragments and gave a high assay sensitivity by identifying 26 out of 26 COVID-19 patients with N antigen or with three protein fragments when combined into a single reaction. The assay correlated well with ELISA method and was specific to COVID-19 as we saw no reactivity among uninfected healthy controls. Our results show that LIPS is a rapid and measurable method to screen antibody responses against SARS-CoV-2 antigens.
Asunto(s)
Anticuerpos Antivirales , Betacoronavirus , Infecciones por Coronavirus , Proteínas de la Nucleocápside , Pandemias , Neumonía Viral , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Betacoronavirus/metabolismo , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/metabolismo , Neumonía Viral/sangre , Neumonía Viral/inmunología , SARS-CoV-2RESUMEN
Inappropriate activation or inadequate regulation of CD4+ and CD8+ T cells may contribute to the initiation and progression of multiple autoimmune and inflammatory diseases. Studies on disease-associated genetic polymorphisms have highlighted the importance of biological context for many regulatory variants, which is particularly relevant in understanding the genetic regulation of the immune system and its cellular phenotypes. Here we show cell type-specific regulation of transcript levels of genes associated with several autoimmune diseases in CD4+ and CD8+ T cells including a trans-acting regulatory locus at chr12q13.2 containing the rs1131017 SNP in the RPS26 gene. Most remarkably, we identify a common missense variant in IL27, associated with type 1 diabetes that results in decreased functional activity of the protein and reduced expression levels of downstream IRF1 and STAT1 in CD4+ T cells only. Altogether, our results indicate that eQTL mapping in purified T cells provides novel functional insights into polymorphisms and pathways associated with autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/genética , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Sitios de Carácter Cuantitativo/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Mapeo Cromosómico/métodos , Diabetes Mellitus Tipo 1/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Células HEK293 , Humanos , Factor 1 Regulador del Interferón/genética , Interleucina-27/genética , Mutación , Polimorfismo de Nucleótido Simple , Proteínas Ribosómicas/genética , Factor de Transcripción STAT1/genéticaRESUMEN
Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue-specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF-κB binding sites upstream of the Aire coding region. We show that CNS1-deficient mice lack thymic expression of Aire and share several features of Aire-knockout mice, including downregulation of Aire-dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF-κB activation, and thymic expression of Aire.
Asunto(s)
FN-kappa B/fisiología , Timo/metabolismo , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Células Epiteliales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Activador del Factor Nuclear kappa-B/fisiología , Transducción de Señal , Timo/citología , Factores de Transcripción/genética , Proteína AIRERESUMEN
mRNA-based vaccines and candidate therapeutics have great potential in various medical fields. For the delivery of mRNA into target cells and tissues, lipid formulations are often employed. However, this approach could cause the activation of immune responses, making it unsuitable for the treatment of inflammatory conditions. Therefore, alternative delivery systems are highly demanded. In this study, we evaluated the transport efficiency and characteristics of cell-penetrating peptide PepFect14 (PF14) and mRNA nanoparticles in the presence of different additives. Our results show that all PF14-mRNA formulations entered cultured cells, while calcium chloride enhanced the transport and production of the encoded protein in HeLa and HaCaT cell lines, and polysorbate 80 did so in primary human keratinocytes. All formulations had similar physical properties and did not remarkably affect cell viability. By selectively blocking endocytosis pathways, we show that PF14-mRNA nanoparticles primarily entered HeLa cells via macropinocytosis and HaCaT cells via both macropinocytosis and clathrin-mediated endocytosis, while none of the blockers significantly affected the delivery into primary keratinocytes. Finally, subcutaneous injection of PF14-mRNA nanoparticles before inducing mouse irritant contact dermatitis resulted in the expression of a reporter protein without provoking harmful immune responses in the skin. Together, our findings suggest that PF14-mRNA nanoparticles have the potential for developing mRNA-based therapeutics for treating inflammatory skin conditions.
RESUMEN
The role of NLRP1 inflammasome activation and subsequent production of IL-1 family cytokines in the development of atopic dermatitis (AD) is not clearly understood. Staphylococcus aureus is known to be associated with increased mRNA levels of IL1 family cytokines in the skin and more severe AD. In this study, the altered expression of IL-1 family cytokines and inflammasome-related genes was confirmed, and a positive relationship between mRNA levels of inflammasome sensor NLRP1 and IL1B or IL18 was determined. Enhanced expression of the NLRP1 and PYCARD proteins and increased caspase-1 activity were detected in the skin of patients with AD. The genetic association of IL18R1 and IL18RAP with AD was confirmed, and the involvement of various immune cell types was predicted using published GWAS and expression quantitative trait loci datasets. In keratinocytes, the inoculation with S. aureus led to the increased secretion of IL-1ß and IL-18, whereas small interfering RNA silencing of NLRP1 inhibited the production of these cytokines. Our results suggest that skin colonization with S. aureus may cause the activation of the NLRP1 inflammasome in keratinocytes, which leads to the secretion of IL-1ß and IL-18 and thereby may contribute to the pathogenesis of AD, particularly in the presence of genetic variations in the IL-18 pathway.
Asunto(s)
Dermatitis Atópica , Staphylococcus aureus Resistente a Meticilina , Humanos , Inflamasomas/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Interleucina-18/genética , Staphylococcus aureus/metabolismo , Citocinas/metabolismo , ARN Mensajero , Proteínas NLRRESUMEN
SARS-CoV-2 infection has a risk to develop into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require ICU admission. While essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNγ, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.
Asunto(s)
Apoptosis , Prueba de COVID-19/métodos , COVID-19/sangre , COVID-19/diagnóstico , Caspasa 8/sangre , Quimiocinas/sangre , Proteoma , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , COVID-19/virología , Femenino , Hospitalización , Humanos , Inflamación/sangre , Unidades de Cuidados Intensivos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Proteómica/métodos , Factores de Riesgo , Regulación hacia Arriba , Adulto JovenRESUMEN
High titer autoantibodies produced by B lymphocytes are clinically important features of many common autoimmune diseases. APECED patients with deficient autoimmune regulator (AIRE) gene collectively display a broad repertoire of high titer autoantibodies, including some which are pathognomonic for major autoimmune diseases. AIRE deficiency severely reduces thymic expression of gene-products ordinarily restricted to discrete peripheral tissues, and developing T cells reactive to those gene-products are not inactivated during their development. However, the extent of the autoantibody repertoire in APECED and its relation to thymic expression of self-antigens are unclear. We here undertook a broad protein array approach to assess autoantibody repertoire in APECED patients. Our results show that in addition to shared autoantigen reactivities, APECED patients display high inter-individual variation in their autoantigen profiles, which collectively are enriched in evolutionarily conserved, cytosolic and nuclear phosphoproteins. The APECED autoantigens have two major origins; proteins expressed in thymic medullary epithelial cells and proteins expressed in lymphoid cells. These findings support the hypothesis that specific protein properties strongly contribute to the etiology of B cell autoimmunity.
RESUMEN
The SP100 family members comprise a set of closely related genes on chromosome 2q37.1. The widely expressed SP100 and the leukocyte-specific proteins SP110 and SP140 have been associated with transcriptional regulation and various human diseases. Here, we have characterized the SP100 family member SP140L. The genome sequence analysis showed the formation of SP140L gene through rearrangements of the two neighboring genes, SP100 and SP140, during the evolution of higher primates. The SP140L expression is interferon-inducible with high transcript levels in B cells and other peripheral blood mononuclear cells. Subcellularly, SP140L colocalizes with SP100 and SP140 in nuclear structures that are devoid of SP110, PML, or p300 proteins. Similarly to SP100 and SP140 protein, we detected serum autoantibodies to SP140L in patients with primary biliary cirrhosis using luciferase immunoprecipitation system and immunoblotting assays. In conclusion, our results show that SP140L is phylogenetically recent member of SP100 proteins and acts as an autoantigen in primary biliary cirrhosis patients.