Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Cell volume regulation modulates NLRP3 inflammasome activation
(Elsevier, 2012-09-21) Compan, Vincent; Baroja Mazo, Alberto; López Castejón ∙, Gloria; Gómez, Ana I.; Angosto, Diego; Montero, María T.; Herranz, Antonio S.; Bazán, Eulalia; Reimers, Diana; Martínez Cáceres, Carlos Manuel; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Anatomía y Anatomía Patológica Comparadas
Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here, we report that, from fish to mammals, the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed via the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced a K+-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by activation of the NLRP3 inflammasome and caspase-1, which was controlled by transient receptor potential channels during RVD. Both mechanisms were necessary for interleukin-1β processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data identify cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and reveal a mechanism for NLRP3 inflammasome activation.
Open Access
17α-Ethynylestradiol alters the peritoneal immune response of gilthead seabream
(Elsevier, 2017) Gómez González, Nuria Esther; Cabas, Isabel; Rodenas, María del Carmen; Arizcun, Marta; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Biología Celular e Histología
17α-Ethynylestradiol (EE2), a synthetic estrogen used in most oral contraceptives pills and hormone replacement therapies, is found in many water bodies, where it can modulate the fish immune response. EE2 acts as an endocrine disruptor in gilthead seabream, Sparus aurata L., a marine teleost fish of great economic value in Mediterranean aquaculture, as it induces hepatic vitellogenin gene (vtg) expression. Moreover, EE2 also alters the capacity of gilthead seabream to appropriately respond to infection although it does not behave as an immunosuppressor. Nevertheless, these previous studies have mainly focused on the head kidney leukocytes and no information exists on peritoneal leukocytes, including mast cells. In the present work, juvenile gilthead seabream fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later, and the peritoneal immune response was analyzed. EE2 supplementation induced vtg expression but returned to basal levels by 3 months post-treatment. Interestingly, gilthead seabream peritoneal leukocytes express the genes encoding for the nuclear estrogen receptor α and the G protein-coupled estrogen receptor 1 and the dietary intake of EE2 induced these expression. Moreover, EE2 induced an inflammatory response in the peritoneal cavity in unvaccinated fish, which was largely maintained for several months after the cessation of the treatment. However, the impact of EE2 in vaccinated fish was rather minor and transient. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.
Open Access
Esferoides y esferas líquidas. Cultivos celulares en 3D para mimetizar el ambiente de las células en el organismo
(Universidad de Murcia, 2015) Meseguer, José; Cuesta Peñafiel, Alberto; Esteban Abad, María de los Ángeles; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar
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The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream
(Elsevier, 2005-08-30) Lopez-Castejón, Gloria; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream (sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head-kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.
Open Access
Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins
(Nature Research, 2016-07-01) Martín Sánchez, María Rosario Fátima; Tyrkalska, Sylwia D.; Candel Camacho, Sergio; Angosto, Diego; Gómez Abellán, Victoria; García Moreno, Diana; Zapata Pérez, Rubén; Sánchez Ferrer, Álvaro; Pelegrín Vivancos, Pablo; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Farmacología
Inflammasomes are cytosolic molecular platforms that alert the immune system about thepresence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4),an IFNg-inducible GTPase protein harbouring a C-terminal CARD domain, is required for theinflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo.Despite the presence of the CARD domain, Gbp4 requires the universal inflammasomeadaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophilsare recruited to the infection site through the inflammasome-independent production of thechemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearancethrough the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our resultspoint to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis andbacterial clearance by neutrophils and suggest that transient activation of the inflammasomemay be used to treat bacterial infections.
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Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation
(American Association of Immunologists Oxford University Press, 2009-02-15) Alcaraz-Perez, Francisca; López-Muñoz, Azucena; Meseguer Peñalver, J.; Cayuela Fuentes, Maria Luisa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.
Open Access
Comunicaciones Orales.- Estudio del papel de las ferroxidasas en la virulencia del hongo Mucor circinelloides
(2020-05-06) Navarro Mendoza, María Isabel; López-Muñoz, A.; Hernández-Oñate, M. A.; Herrera-Estrella, A.; Torres-Martínez, S; Ruiz-Vázquez, R. M.; Garre Mula, Victoriano; Nicolás, F. E.; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Genética y Microbiología; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
Open Access
Diseño de un protocolo para el aislamiento de células cebadas del exudado peritoneal de Sparus aurata para la identificación de receptores de estrógenos
(2020-04-13) Gómez-González, Nuria Esther; García-García, Érick; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
RESUMEN. La contaminación de las aguas por disruptores endocrinos, y en concreto de estrógenos sintéticos, crece diariamente pudiendo cambios en el sistema inmunitario en los animales que en ellas habitan al presentar en las células que lo componen receptores de estrógenos. La inflamación es la primera respuesta de los animales frente a una infección o daño en el tejido. La histamina tiene un papel muy importante en la regulación de la inflamación en vertebrados capaces de almacenarla en células especializadas inmunitarias llamadas células cebadas. Sparus aurata presenta histamina en las células cebadas de tejidos mucosos. El exudado peritoneal de S. aurata está enriquecido en células cebadas. Hemos identificado que el exudado peritoneal de dorada está compuesto por linfocitos, granulocitos acidófilos, macrófagos y células cebadas. Hemos diseñado un protocolo de separación de las células cebadas de exudado peritoneal de S. aurata consiguiendo purezas superiores al 95% cuando se mide por citometría. Estos datos fueron confirmados mediante microscopía electrónica. Este protocolo de separación servirá como un paso hacia adelante para realizar estudios in vivo e in vitro orientándonos en la evolución de los mecanismos inflamatorio de vertebrados. También hemos demostrado por citometría de flujo y western-blot utilizando los anticuerpos adecuados que las células cebadas de esta especie presentan el receptor de estrógenos acoplado a la proteína G (GPER). Además, con la idea de poder estudiar en investigaciones futuras el efecto que altas concentraciones de estrógenos causan en el sistema inmune de S. aurata intentamos estudiar la capacidad de liberación de histamina en células cebadas tras su estimulación con diferentes PAMPs (patrones moleculares asociados a patógenos) lipopolisacáridos (LPS), Poly I:C (análogo sintético de la doble cadena de ARN (dsRNA)) y AND genómico de Vibrio angillarum (VaDNA), aunque ninguno era capaz de inducir la liberación de histamina en estas células.
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Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor
(Elsevier, 2008-04-07) Mulero Méndez, Iván; Meseguer Peñalver, J.; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr+) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr+ cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.
Open Access
Efecto de la testosterona en la espermatogénesis de machos de dorada (Sparus aurata L.)
(2020-04-13) Sánchez-Hernández, Miriam; Chaves-Pozo, Elena; Cabas, Isabel; García-Alcázar, Alicia; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología
RESUMEN Los andrógenos pueden inducir la espermatogénesis en peces teleósteos inmaduros. Sin embargo, su función no se conoce con exactitud en los ejemplares adultos. Para conocer el efecto in vivo de la testosterona (T) sobre la espermatogénesis de dorada (Sparus aurata L. 1758), especie hermafrodita protándrica de interés económico alto en la región de Murcia, inyectamos un implante (in situ forming microparticle system) a ejemplares que se encontraban en la etapa de involución testicular del segundo ciclo reproductor. Se trabajó con tres grupos de ejemplares: i) inyectados con implantes con 1 mg T/Kg de peso corporal, ii) inyectados con implantes sin T y iii) no inyectados. Se tomaron muestras de sangre, gónada e hígado a los 7, 14, 21 y 28 días después de haberlos inyectado para realizar un análisis hormonal, morfológico, inmunocitoquímico y de expresión génica. El objetivo de nuestro trabajo era determinar el efecto de la T, y de sus derivados, sobre la fisiología de la gónada analizando la progresión de la espermatogénesis, la tasa de proliferación celular y de apoptosis, la presencia de leucocitos y el perfil de expresión de genes relevantes en la respuesta inmunitaria. Los implantes con T provocaron un aumento en los niveles de T, alcanzando niveles supra-fisiológicos. El perfil de expresión génica, en gónada e hígado de algunas enzimas implicadas en la síntesis y el metabolismo de esteroides, descartó la transformación de T en compuestos estrogénicos. Por otra parte la T parece inhibir la respuesta inmunitaria en la gónada ya que observamos una disminución de los niveles de expresión de los genes que codifican para los TLR, lo que podría provocar una disminución de su capacidad para reconocer y responder a los patógenos. Sin embargo se observó un aumento de granulocitos acidófilos y linfocitos lo que nos sugiere que los andrógenos participan en el flujo migratorio de leucocitos al interior de la gónada.