Pelegrín Vivancos, Pablo

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Pelegrín Vivancos, Pablo

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Pelegrín Vivancos, Pablo

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Universidad de Murcia. Departamento de Bioquímica y Biología Molecular"B" e Inmunología

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Open Access
Pannexin-1 mediates large pore formation and interleukin-1ß release by the ATP-gated P2X7 receptor
(2006) Surprenant, Annmarie; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
P2X(7) receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1beta release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1beta induced by P2X(7) receptor activation.
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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
The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response
(Nature Research, 2014-08) Baroja Mazo, Alberto; Gómez, Ana I.; Amores Iniesta, Joaquín; Compan, Vincent; Barberà Cremades, María; Yagüe, Jordi; Ruiz Ortiz, Estibaliz; Antón, Jordi; Buján, Segundo; Coullin, Isabelle; Brough, David; Arostegui, Juan I.; Martínez Cáceres, Carlos Manuel; Martín Sánchez, María Rosario Fátima; Pelegrín Vivancos, Pablo; Anatomía y Anatomía Patológica Comparadas
Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.
Open Access
Inflammasome-dependent IL-1β release depends upon membrane permeabilisation
(Nature, 2016) Diamon, Catherine; Zeitler, Marcel; Gomez-Sanchez, Ana; Baroja-Mazo, Alberto; Bagnall, James; Spiller, David; White, Michael; Daniels, Michael J. D.; Mortellaro, Alessandra; Peñalver, Marcos; Paszek, Pawel; Steringer, Julia P.; Nickel, Walter; Brough, David; Martín Sánchez, María Rosario Fátima; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
Interleukin (IL)-1β is a critical regulator of the inflammatory response. IL-1β is not secreted through the conventional ER-Golgi route of protein secretion and to-date its mechanism of release has been unknown. Crucially its secretion depends upon the processing of a precursor form following the activation of the multi-molecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1β release process, in combination with biochemical, biophysical and real-time single-cell confocal microscopy with macrophage cells expressing Venus labelled IL-1β, we have discovered that the secretion of IL-1β after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus in macrophages the release of IL-1β in response to inflammasome activation appears to be a secretory process independent of non-specific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1β release is distinct from the unconventional secretory mechanism employed by its structural homologues FGF2 or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into mechanisms of protein release.
Open Access
CD14 release induced by P2X7 receptor restrict inflammation and increases survival during sepsis
(eLife Sciences Publications, 2020-11-26) Alarcón-Vila, Cristina; Baroja-Mazo, Alberto; Torre-Minguela, Carlos de; Martínez-Banaclocha, Helios; Gracia-Palenciano, Carlos; Martínez Cáceres, Carlos Manuel; Pelegrín Vivancos, Pablo; Martínez García, Juan José; Bioquímica y Biología Molecular B e Inmunología
P2X7 receptor activation induces the release of different cellular proteins, such as CD14, a glycosylphosphatidylinositol (GPI)-anchored protein to the plasma membrane important for LPS signaling via TLR4. Circulating CD14 has been found at elevated levels in sepsis, but the exact mechanism of CD14 release in sepsis has not been established. Here we show for first time that P2X7 receptor induces the release of CD14 in extracellular vesicles, resulting in a net reduction in macrophage plasma membrane CD14 that functionally affects LPS, but not monophosphoryl lipid A, pro-inflammatory cytokine production. Also, we found that during a murine model of sepsis, P2X7 receptor activity is important for maintaining elevated levels of CD14 in biological fluids and a decrease in its activity results in higher bacterial load and exacerbated organ damage, ultimately leading to premature deaths. Our data reveal that P2X7 is a key receptor for helping to clear sepsis because it maintains elevated concentrations of circulating CD14 during infection.
Open Access
Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation
(American Association for the Advancement of Science, 2021-09-15) Angosto-Bazarra, Diego; Alarcón-Vila, Cristina; Baños, Maria C; Hafner-Bratkovič, Iva; Oliva, Baldomero; Pelegrín Vivancos, Pablo; Tapia Abellán, Ana; Bioquímica y Biología Molecular B e Inmunología
The NLRP3 inflammasome is activated in response to a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the different NLRP3 activation models. Here we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3 promoting an open conformation as a step preceding activation. This conformational change is facilitated by the presence of the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker is also important to facilitate the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux-specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the N-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.
Open Access
Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process
(Nature Research, 2016-03-03) Carlos de Torre-Minguela; Maria Barberà-Cremades; Ana I. Gómez; Martín Sánchez, María Rosario Fátima; Pelegrín Vivancos, Pablo; Farmacología
The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P2X7R stimulation goes beyond caspase-1 secretome. The characterization of P2X7R-secretome reveals a new function of this receptor through a fine-tuning of protein release. We found that P2X7R stimulation in macrophages is able to release potent anti-inflammatory proteins, such as Annexin A1, independently of their polarization state suggesting for first time a potential role for P2X7R during resolution of the inflammation and not linked to the release of pro-inflammatory cytokines. These results are of prime importance for the development of therapeutics targeting P2X7R.
Open Access
Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production
(Nature Research, 2024-02-06) Molina-López, Cristina; Hurtado-Navarro, Laura; Garcia, Carlos J.; Angosto-Bazarra, Diego; Vallejo, Fernando; Marques-Soares, Joana R.; Vargas, Carmen; Bujan-Rivas, Segundo; Tomás-Barberán, Francisco A.; Arostegui, Juan I.; Pelegrín Vivancos, Pablo; Tapia Abellán, Ana; Bioquímica y Biología Molecular B e Inmunología
Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1b production. Although these are gain-of-function variants characterised by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4A. The constitutively active NLRP3-inflammasome is responsive to the selective NLRP3 inflammasome inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-kB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1b production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host's innate defence against pathogens while also limiting IL-1b–dependent inflammatory episodes through immunometabolism modulation.
Open Access
Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization
(Nature Research, 2019-04-08) Martín Sánchez, María Rosario Fátima; Baroja Mazo, Alberto; Compan, Vince; Tapia Abellán, Ana; Coullin, Isabelle; Pelegrín Vivancos, Pablo; Farmacología
Unconventional protein secretion represents an important process of the inflammatory response.The release of the pro-inflammatory cytokine interleukin (IL)-1β which burst during pyroptosis asa consequence of gasdermin D plasma membrane pore formation, can also occur through otherunconventional secretion pathways dependent on caspase-1 activation. However, how caspase-1mediates cytokine release independently of gasdermin D remains poorly understood. Here weshow that following caspase-1 activation by different inflammasomes, caspase-1 cleaves earlyendosome autoantigen 1 (EEA1) protein at Asp127/132 . Caspase-1 activation also results in the releaseof the endosomal EEA1 protein in a gasdermin D-independent manner. EEA1 knock-down resultsin adecreased release of caspase-1 and IL-1β, but the pyroptotic release of other inflammasomecomponents and lactate dehydrogenase was not affected. This study shows how caspase-1 control therelease of EEA1 and IL-1β in a pyroptotic-independent manner
Open Access
Isolation of functional mature peritoneal macrophages from healthy humans.
(Wiley, 2019-11-06) Martínez Banaclocha, Helios; Marín Sánchez, Pilar; Carmona Martínez, Violeta; Iniesta Albadalejo, Miguel Ángel; Tristán Manzano, María; García Peñarrubia, Pilar; Martínez-Esparza Alvargonzález, María Concepción; Pelegrín Vivancos, Pablo; Ruiz Alcaraz, Antonio José; Tapia Abellán, Ana; Machado Linde, Francisco; Bioquímica y Biología Molecular B e Inmunología
Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.