RESUMEN
Fetal extravillous trophoblasts (EVTs) are the most invasive cells of the placenta and play a key role in modulating maternal immune responses. Here, we present a protocol to purify and culture human leukocyte antigen-G (HLA-G)+ EVTs. We describe steps for tissue dissection, tissue digestion, density gradient centrifugation, and cell sorting, and we provide detailed methods to determine EVT function. HLA-G+ EVTs are isolated from two maternal-fetal interfaces: the chorionic membrane and the basalis/villous tissue. This protocol allows in-depth functional investigation of maternal immune interactions with HLA-G+ EVTs. For complete details on the use and execution of this protocol, please refer to Papuchova et al. (2020),1 Salvany-Celades et al. (2019),2 Tilburgs et al. (2015),3 Tilburgs et al. (2015),4 van der Zwan et al. (2018).5.
RESUMEN
PROBLEM: Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses. METHOD OF STUDY: Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (TReg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16- CD56++ and CD16- CD56++ NK transcripts in PBMC and UBMC samples. RESULTS: Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45+ leukocytes. Higher percentages of CD16- CD56++ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45+ CD3- vs. 2.73%, respectively), suggesting that CD16- CD56++ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16+ CD56++ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface. CONCLUSION: Intrauterine leukocytes, especially CD16- CD56++ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16- CD56++ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.
Asunto(s)
Leucocitos Mononucleares , Placenta , Femenino , Humanos , Embarazo , Útero , Células Asesinas Naturales , Inmunofenotipificación , Leucocitos , Antígeno CD56/metabolismo , Receptores de IgG/metabolismoRESUMEN
HLA-C arose during evolution of pregnancy in the great apes 10 to 15 million years ago. It has a dual function on placental extravillous trophoblasts (EVTs) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First-trimester primary EVTs in which HLA-C is highly expressed, as well as JEG3, an EVT model cell line, were employed. Single-cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. Chromatin immunoprecipitation (ChIP)-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced, and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 small interfering RNAs (siRNAs) and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3's coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a superenhancer that spans more than 5 Mb, identified by assay for transposase-accessible chromatin using sequencing (ATAC-seq), as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive-feedback loop that drives ELF3 expression, with down-regulation of HLA-C expression as a consequence.
Asunto(s)
Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Retroalimentación Fisiológica , Antígenos HLA-C/genética , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genética , Trofoblastos/inmunología , Aborto Legal , Adamantano/farmacología , Azepinas/farmacología , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/inmunología , Femenino , Regulación del Desarrollo de la Expresión Génica/inmunología , Antígenos HLA-B/genética , Antígenos HLA-B/inmunología , Antígenos HLA-C/inmunología , Humanos , Inmunidad Materno-Adquirida , Indoles/farmacología , Complejo Mediador/genética , Complejo Mediador/inmunología , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/inmunología , Embarazo , Primer Trimestre del Embarazo , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Factores de Transcripción del Factor Regulador X/genética , Factores de Transcripción del Factor Regulador X/inmunología , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/inmunología , Triazoles/farmacología , Trofoblastos/citología , Trofoblastos/efectos de los fármacosRESUMEN
During pregnancy, invading HLA-G+ extravillous trophoblasts (EVT) play a key role in placental development, uterine spiral artery remodeling, and prevention of detrimental maternal immune responses to placental and fetal antigens. Failures of these processes are suggested to play a role in the development of pregnancy complications, but very little is known about the underlying mechanisms. Here we present validated methods to purify and culture primary HLA-G+ EVT from the placental disk and chorionic membrane from healthy term pregnancy. Characterization of HLA-G+ EVT from term pregnancy compared to first trimester revealed their unique phenotypes, gene expression profiles, and differing capacities to increase regulatory T cells (Treg) during coculture assays, features that cannot be captured by using surrogate cell lines or animal models. Furthermore, clinical variables including gestational age and fetal sex significantly influenced EVT biology and function. These methods and approaches form a solid basis for further investigation of the role of HLA-G+ EVT in the development of detrimental placental inflammatory responses associated with pregnancy complications, including spontaneous preterm delivery and preeclampsia.
Asunto(s)
Antígenos HLA-G/inmunología , Inmunidad Innata/genética , Placentación/inmunología , Preeclampsia/inmunología , Línea Celular , Movimiento Celular/inmunología , Femenino , Regulación del Desarrollo de la Expresión Génica/inmunología , Humanos , Relaciones Materno-Fetales , Placenta/inmunología , Placenta/metabolismo , Preeclampsia/patología , Embarazo , Primer Trimestre del Embarazo , Trofoblastos/inmunologíaRESUMEN
Decidual NK cells (dNK) are the main lymphocyte population in early pregnancy decidual mucosa. Although dNK decrease during pregnancy, they remain present in decidual tissues at term. First trimester dNK facilitate trophoblast invasion, provide protection against infections, and were shown to have many differences in their expression of NKRs, cytokines, and cytolytic capacity compared with peripheral blood NK cells (pNK). However, only limited data are available on the phenotype and function of term pregnancy dNK. In this study, dNK from human term pregnancy decidua basalis and decidua parietalis tissues were compared with pNK and first trimester dNK. Profound differences were found, including: 1) term pregnancy dNK have an increased degranulation response to K562 and PMA/ionomycin but lower capacity to respond to human CMV-infected cells; 2) term pregnancy dNK are not skewed toward recognition of HLA-C, as was previously shown for first trimester dNK; and 3) protein and gene expression profiles identified multiple differences between pNK, first trimester, and term pregnancy dNK, suggesting term pregnancy dNK are a distinct type of NK cells. Understanding the role of dNK throughout pregnancy is of high clinical relevance for studies aiming to prevent placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens.
Asunto(s)
Decidua/inmunología , Células Asesinas Naturales/inmunología , Línea Celular Tumoral , Movimiento Celular/inmunología , Células Cultivadas , Femenino , Expresión Génica/inmunología , Antígenos HLA-C/inmunología , Humanos , Células K562 , Embarazo , Trofoblastos/inmunologíaRESUMEN
Polycystic ovarian syndrome (PCOS) is one of the most common endocrine disorders in women of both developed and developing countries. It is associated with insulin resistance, hyperinsulinemia, hyperandrogenism, oxidative stress and various long-term complications. The present study was undertaken to evaluate the efficacy and safety of the supplementation (Trazer F ForteTM-CORONA Remedies Pvt. Ltd.) providing combination of insulin sensitising agents (myo-inositol, D-chiro-inositol and chromium picolinate), antioxidants (N-acetylcysteine and lycopene) and vitamins (vitamin D, biotin and folic acid) in women with PCOS. After 12 weeks of supplementation, a significant improvement was observed in menstrual cyclicity, acne and hirsutism in both obese and lean PCOS patients. A significant reduction was observed in body weight and BMI of obese subjects. However, both parameters remain unchanged in lean subjects. We suggest that combination therapy of insulin sensitising agents, antioxidants and vitamins may be a fruitful approach for the management of PCOS.Impact statementWhat is already known on this subject? Monotherapy of insulin sensitising agents, antioxidants and vitamins is beneficial in the treatment of PCOS.What do the results of this study add? Combined use of insulin sensitising agents (myo-inositol, D-chiro-inositol and chromium picolinate), antioxidants (N-acetylcysteine and lycopene), and vitamins (vitamin D, biotin and folic acid) is safe and effective in obese and non-obese women with PCOS.What are the implications of these findings for clinical practice and/or further research? Since PCOS is a multifactorial and a complex endocrine disorder, combination therapy can be used for the comprehensive management of PCOS.
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Antioxidantes/administración & dosificación , Suplementos Dietéticos , Inositol/administración & dosificación , Obesidad/terapia , Síndrome del Ovario Poliquístico/terapia , Vitaminas/administración & dosificación , Adulto , Índice de Masa Corporal , Peso Corporal , Terapia Combinada , Femenino , Hirsutismo/etiología , Humanos , Insulina/sangre , Ciclo Menstrual , Obesidad/sangre , Obesidad/complicaciones , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/complicaciones , Complejo Vitamínico B/administración & dosificaciónRESUMEN
Beta 2 microglobulin (beta2m) is an essential subunit of major histocompatibility complex (MHC) type I molecules. In this report, beta2m cDNAs were identified and sequenced from sandbar shark spleen cDNA library. Sandbar shark beta2m gene encodes one amino acid less than most teleost beta2m genes, and 3 amino acids less than mammal beta2m genes. Although sandbar shark beta2m protein contains one beta sheet less than that of human in the predicted protein structure, the overall structure of beta2m proteins is conserved during evolution. Germline gene for the beta2m in sandbar and nurse shark is present as a single locus. It contains three exons and two introns. CpG sites are evenly distributed in the shark beta2m loci. Several DNA repeat elements were also identified in the shark beta2m loci. Sequence analysis suggests that the beta2m locus is not linked to the MHC I loci in the shark genome.
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Regulación de la Expresión Génica , Sitios Genéticos , Tiburones/genética , Microglobulina beta-2/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tiburones/inmunología , Microglobulina beta-2/química , Microglobulina beta-2/inmunologíaRESUMEN
Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.