Estrogens regulate the innate and the adaptive immune response

  1. Rodenas Bleda, Maria Del Carmen
Supervised by:
  1. Alfonsa García Ayala Director
  2. Victoriano Francisco Mulero Méndez Director
  3. José Meseguer Peñalver Director

Defence university: Universidad de Murcia

Fecha de defensa: 05 May 2017

  1. Antonio Bernabé Salazar Chair
  2. Elena Chaves Pozo Secretary
  3. Francisco José Roca Soler Committee member

Type: Thesis


Endocrine disrupting chemicals (EDCs) are a class of heterogeneous chemicals present in the environment that bind to estrogen receptors (ERs) and interfere with the estrogen-dependent control of body homeostasis. In the present thesis, we present evidence that EDCs modulate the immune system of gilthead seabream (Sparus aurata L.) even when exposure to these chemicals has ended. We evaluate the effect of 17?-ethinylestradiol (EE2), a bio-active estrogen used in oral contraceptive pills, and tamoxifen (Tmx), a drug used worldwide for the treatment of ER-positive breast cancer. Both have become a widespread problem in the environment due to their high resistance to degradation. The gilthead seabream is a marine teleost fish of significant economic value in the Mediterranean area and has been extensively used in research as a protandrous hermaphrodite fish. Furthermore, during the development of the thsesis we identified a membrane-anchored receptor called G protein-coupled estrogen receptor (GPER1) in human neutrophils. The thesis was carried out in four stages. First of all, we confirmed that EE2 and Tmx, incorporated in the diet, promote an estrogenic response in adult fish although the effect of Tmx was seen to be much less pronounced. We also investigated the capacity of both compounds to modulate the immune response induced by an antigenic challenge and its capacity to recover its functionality when the treatments ceased. Both EE2 and Tmx transiently inhibited the induction of interleukin-1? (il-1?) gene expression and transiently increased reactive oxygen species (ROS) in head kidney leukocytes from vaccinated fish. Moreover, EE2 and Tmx increased the antibody titer but only the fish exposed to Tmx showed a higher IgM titer and a higher percentage of IgM+B lymphocytes during the recovery period. After observing that EE2 and Tmx affected the immune response of adult specimens, we attempted to ascertain whether these effects are also produced in juveniles. Moreover, it was seen that treatment with G1, a GPER-selective agonist, also altered the immune response of adult specimens. Only EE2 and Tmx differently increased vtg mARN levels during the treatment and the expression returned to basal levels during recovery. Although none of the three compounds affected ROS production, they all inhibited the induction of il1b gene expression after priming. Significantly, Tmx increased the percentage of IgM+ cells in both head kidney and spleen during the recovery period. More importantly, the antibody response of vaccinated fish was modified by the three compounds but the exact effects depended on the time when the immunization was performed. The previous results led us to investigate the influence of EE2 on the innate immune response, focusing on lymphocytes and in the humoral activity when exposure to this EDC ceased. Significantly, EE2 affects the percentage and proliferation of T and IgM+-B lymphocytes. For the first time it was seen that EE2 significantly induces the production of antibodies, an effect mediated through GPER1 signalling pathway. Finally, we functionally characterized GPER1 in human neutrophils. These cells express a functional GPER1 in the plasma membrane and stimulation of the neutrophils with G1 resulted in a dose-dependent increase in transcript levels of CFOS, a marker of GPER1 activation. Besides, G1 significantly primed the production of O2- and drastically altered their gene expression profile. G1 treatment regulated the activation and life span of human neutrophils through several signaling pathways. To sum up, our results show for the first time that GPER1 activation promotes the polarization of human neutrophils towards a pro-inflammatory phenotype and identify GPER1 as a potential therapeutic target in immune diseases where neutrophils play a key role.