Functional characterization of purinergic receptors in the differentiation and activation of macrophages = Caracterización funcional de recptores purinérgicos en la diferenciación y activación de macrófagos

Resumen Esta Tesis estudia la caracterizacion de diferentes receptores purinergicos, principalmente P2X7, en celulas presentadoras de antigeno de la estirpe mieloide mononuclear fagocitica (monocitos, macrofagos peritoneales y macrofagos derivados de medula osea). En concreto, se determina como la senalizacion purinergica afecta a la diferenciacion de los macrofagos, la produccion de eicosanoides y de TNF-?, en estados tanto pro-inflamatorias y como de inmunosupresion. Para ello, se emplearon diferentes estimulos celulares; senales de patogenos, principalmente lipopolisacarido (LPS) bacteriano, senal que activa a los macrofagos a traves de los receptores tipo toll 4; y senales de dano celular, principalmente ATP y adenosina. Siendo los objetivos concretos de esta Tesis: 1) Estudiar como afecta la senalizacion purinergica a la diferenciacion de los macrofagos; 2) Analizar la relacion entre la activacion del receptor P2X7 en macrofagos y la produccion de eicosanoides; 3) Determinar como el receptor P2X7 regula la liberacion del factor de necrosis tumoral (TNF)-? en macrofagos extenuados. La metodologia empleo macrofagos peritoneales humanos y monocitos de sangre periferica de donantes sanos y pacientes con un proceso septico severo de origen abdominal; muestras de ratones de la cepa C57BL/6 y deficientes para los receptores a estudio (P2rx7-/- y P2rx4-/-) que se emplearon para estudios tanto in vivo como in vitro. En la mayoria de ensayos se emplearon macrofagos derivados de medula osea de raton. Los diferentes macrofagos fueron sometidos a distintos protocolos de estimulo segun el objetivo abordado: diferentes dosis de LPS para estudiar la respuesta ante situaciones inflamatorias, o polarizacion de macrofagos a M1 con LPS e interferon-??o a M2 con interleuquina (IL)-4, para en todos los casos estimular con un nucleotido. De los estudios in vitro se analizo la cantidad de IL-1?, TNF-?, prostaglandina (PG) E2, tromboxano A2 y leucotrieno B4 liberada al medio mediante la tecnica ELISA, la muerte celular cuantificando la actividad de lactatodehidrogenasa en los sobrenadantes celulares; la viabilidad celular mediante ensayos de reduccion meatabolica del bromuro de 3-(4,5- dimetiltiazol-2-ilo)-2,5-difeniltetrazol; la cuantificacion de la expresion relativa de los genes que codifican para los diferentes receptores y marcadores citoquimicos de interes, empleando la tecnica de PCR-transcriptasa reversa a tiempo real, la cuantificacion de marcadores de membrana especificos de macrofagos mediante citometria de flujo, y finalmente la actividad de la enzima convertidora de TNF-? (TACE) y la actividad catepsina B, mediante tecnicas fluorometricas. Los estudios in vivo realizados en raton analizaron la actividad del receptor P2X7 tras la administracion intraperitoneal de LPS, cuantificando IL-1? y TNF-? en lavados peritoneales empleando la tecnica ELISA, la presencia de ATP extracelular en peritoneo se cuantifico mediante bioluminiscencia, y la medicion de la temperatura rectal en un modelo inducido de sepsis mediante una sonda termica. Resultados y conclusiones: En esta Tesis se demuestra que la senalizacion purinergica es capaz de reducir el numero de macrofagos diferenciados de medula osea, resultando en una regulacion positiva de ciertos marcadores M2, derivando en un fenotipo especifico de macrofago. En los macrofagos maduros, la activacion de P2X7 induce la liberacion del mediador lipidico PGE2, siendo P2X7 importante en la respuesta febril, un signo clasico asociado con el proceso inflamatorio. Ademas, la senalizacion mediante el receptor P2X7 en macrofagos M1 extenuados es capaz de controlar la liberacion clasica de TNF-?. Estos resultados tienen implicaciones clinicas, ya que el receptor P2X7 emerge como una diana terapeutica prometedora para la fiebre y su potenciacion durante inmunosupresion podria aumentar la inmunidad en los procesos septicos. Summary This Thesis studies the characterization of different purinergic receptors, mainly P2X7, analyzing their functions in antigen presenting cells of mononuclear phagocytic myeloid lineage (monocytes, peritoneal macrophages and bone marrow derived macrophages). Precisely, the Thesis determine how purinergic signaling affects macrophage differentiation, eicosanoids and tumor necrosis factor (TNF)-? release, during both proinflammatory and immunosuppressive conditions. Therefore, cells were treated with bacterial products, mainly lipopolysaccharide (LPS), which specifically activates Toll-like receptor 4, and signals of cell damage, mainly ATP and adenosine. Objectives: 1) To study how purinergic signaling affects macrophage differentiation; 2) To analyze the association between the activation of P2X7 in macrophages and the production of eicosanoids; 3) To identify how P2X7 receptor regulates TNF-? release during immune extenuation. Methods: The experiments were performed using peritoneal human macrophages and peripheral blood monocytes of healthy subjects and patients with severe sepsis; mice samples from strain C57BL/6 and the knockout for the genes encoding the receptors to study (P2rx7-/- and P2rx4-/-), these mice were used for both in vivo and in vitro studies. Mice bone marrow derived macrophages were used in almost all the experiments of this thesis. The different macrophages were stimulated with different protocols to address the different objectives: different doses of LPS to mimic inflammatory conditions; LPS and interferon-? to polarize macrophages to M1 and with interleukin (IL)-4 to polarize to M2. The amount of IL-1?, TNF-?, prostaglandin (PG) E2, thromboxane A2 and leukotriene B4 released, were measured by ELISA; cell death quantification was measured by the activity of lactate dehydrogenase in cells supernatants; macrophage viability was measured by the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; relative expression of genes encoding for the desired receptors and markers was quantified by real-time reverse-transcription PCR; the quantification of specific macrophage membrane markers was analyzed by flow cytometry; the converting TNF-? enzyme (TACE) and cathepsin B activity were measured by fluorometric techniques. In vivo mouse studies to study P2X7 receptor activity after intraperitoneal administration of LPS was analyzed by ELISA, measuring IL-1? and TNF-? in peritoneal lavage; detection of extracellular ATP in peritoneum was quantified by bioluminescence; rectal temperature was measured in a sepsis model. Results and Conclusions: this Thesis demonstrates that purinergic signaling is able to reduce the number of differentiated macrophages from bone marrow, which display upregulation of certain M2 markers, resulting in a specific phenotype of macrophage. In mature macrophages, ATP activating P2X7 receptor induces the release of the lipid mediator PGE2, being P2X7 important for the febrile response, a classical signal associated with the inflammatory process. Furthermore, P2X7 receptor signaling in extenuated M1 macrophages is able to control the classical release of TNF-?. These findings have important clinical implications, since P2X7 receptor emerges as a promising target for fever, however its potentiation during immunosuppression could boost immunity in septic processes.