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IGM+ B LYMPHOCYTES AND IMPACT OF ESTROGENS IN THEIR FUNCTIONS
Autores:
ISABEL CABAS SÁNCHEZ
,
MARÍA DEL CARMEN RODENAS BLEDA
, ALICIA GARCÍA ALCÁRAZ,
JOSE MESEGUER PEÑALVER
,
VICTORIANO MULERO MÉNDEZ
,
ALFONSA GARCÍA AYALA
,
Grupos de investigación:
[GI/IMIB/C060/2011] Inmunidad, inflamación y cáncer
Comunicación:
Antecedentes:
The role of estrogens in immunity is relatively well known. Thus, these sex steroids have been identified as being partially responsible for the sexual dimorphism observed in some chronic inflammatory and autoimmune diseases. Traditionally, estrogens were thought to act via classical nuclear ERs, namely ER-alpha and ER-beta. However, it was observed some rapid or “non-genomic” effects, which are mediated by a membrane anchored receptor called G protein-coupled estrogen receptor (GPER), opening the possibility to explore additional estrogens-mediated effects. Nevertheless, the expression and role of GPER in the immune system has not been exhaustively studied and its relevance in lymphocytes and in their differentiation into plasma cells, a complex and key process in the adaptive immune response, remains unknown. Using G1, a GPER-selective ligand, we recently reported an evolutionarily conserved role of GPER in neutrophils.
Métodos:
The animal model used in this study was the gilthead seabream (Sparus aurata L.). Using magnetic activated cell sorting (MACS), IgM+BLy-enriched cell fractions were obtained from head kidney (the main hematopoyetic organ in teleost) leukocytes. The IgM and GPER expression was determined by immunofluorescence using specific antibodies. IgM+Bly-enriched cell fractions were incubated with genomic DNA from the bacterium Vibrio anguillarum (VaDNA) or the mitogen PWM, or with 0.1, 1, 100 and 100 ?M G1 (GPER agonist) in the presence or absence of VaDNA for 44 h. After the treatments, the cell viability (using propidium iodide) and FSC/SSC parameters were determined by flow cytometry. A cell sorting unit (SONY SH800Z) was used to isolate the populations of interest. IgM+Bly-enriched cell fractions or the sorted populations were processed for transmission electron microscopy (PHILIPS TECNAI 12) and gene expression (RT-qPCR) studies. t-Student or ANOVA + Tukey test were applied to determine statistically significant differences among groups. p <0.05, using the GraphPad Prism software.
Resultados:
In the present study, we show that IgM+-B lymphocytes from the teleost fish gilthead seabream (Sparus aurata L.) consist of two subpopulations with different size, abundance and membrane IgM expression. Moreover, these cells are able to differentiate in vitro in response to bacterial DNA (TLR9 agonist) or the mitogen PWM into a cell type with high granularity (SSC) and size (FSC), as assayed by flow cytometry, which loses the membrane IgM expression. This latter feature allowed us to sort both progenitor and differentiated cell populations and to analyze their gene expression profile by RT-qPCR and ultraestructure by TEM. In addition, we also observed that gilthead seabream IgM+ B cells express a functional GPER, whose expression is modulated by immune stimuli.
Conclusiones:
Collectively, our results provide new insights into the phylogeny of B lymphocytes differentiation and the impact of GPER signaling in this process. Such results may have a potential clinical impact in immune diseases where B lymphocytes and estrogens play an important role
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