Alteração da homeostase no sistema linfo-hematopoético em animais P2X7-/- e seus efeitos nos processos de proliferação e diferenciação
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Data
2024-03-22
Autores
Souza, Kamylla Fernanda Souza de [UNIFESP]
Orientadores
Paredes-Gamero, Edgar Julian [UNIFESP]
Tipo
Tese de doutorado
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As células-tronco hematopoéticas (CTH) têm o potencial de gerar todas as células do sistema linfo-hematopoético. Compreender seus mecanismos de auto-renovação, expansão e diferenciação, durante a hematopoese, tem grande relevância científica e clínica. Alguns moduladores, como o ATP e os receptores purinérgicos, têm sido estudados para compreender sua atuação nos nichos hematopoéticos, onde podem atuar na regulação e manutenção das CTH e células progenitoras. O receptor purinérgico P2X7 (P2X7R) é um receptor do tipo canal catiônico, que tem o ATP como seu principal agonista fisiológico, cuja atividade tem sido principalmente associada ao sistema imunológico, com poucos estudos mostrando sua participação na hematopoese. Neste estudo foram utilizados camundongos C57BL/6 wild type (WT) e nocaute para o P2X7R (P2X7-/-), para investigar a relevância e a função deste receptor na modulação das CTH e seus efeitos na diferenciação mieloide e linfoide. Ao avaliar a expressão do P2X7R nas células hematopoéticas extraídas da medula óssea, observou-se que o P2X7R é mais expresso nas células primitivas em contexto normal e após sepse. A imunofenotipagem das populações hematopoéticas da medula óssea e do baço mostrou um aumento do número de CTH e de células progenitoras mieloide e linfoide em animais P2X7-/-. A redução de eritrócitos na medula óssea destes animais foi compensada com o aumento desta população no baço e os parâmetros hematológicos não foram alterados. Também não houve alteração na capacidade das células hematopoéticas e progenitoras da medula óssea de formar colônias. Escolheu-se a indução de sepse pelo método de punção e ligadura cecal (CLP) para promover uma resposta inflamatória e modular o sistema linfo-hematopoético. O estímulo inflamatório induziu na medula óssea dos animais WT o aumento de CTH, c-Kit+, progenitores de granulócitos e granulócitos maduros, em contrapartida ao menor número de neutrófilos no sangue periférico. Os animais P2X7-/- apresentaram resposta contrária, com aumento do número de neutrófilos periféricos e redução de CTH e de todos os progenitores na MO. Tanto em animais WT quanto em P2X7-/- a eritropoese foi reduzida e houve menor expressão dos fatores de transcrição NF-E2, Gata-1, na MO. Em animais P2X7-/-, a indução de CLP levou ao aumento da formação de colônias hematopoéticas, especialmente de unidades formadoras de colônias de monócitos, mas no sangue periférico essas células estavam em menor número. Ademais, foram avaliados outros marcadores relacionados à hematopoese e inflamação como espécies reativas de oxigênio (EROs), citocinas pró e anti-inflamatórias, e imunoglobulinas. Animais P2X7-/- sépticos apresentaram altos níveis de EROs na cavidade peritoneal e medula óssea, mas não no plasma, onde houve aumento de interleucina-6 e imunoglobulina M. Em conjunto, nossos resultados destacam uma complexa interação do P2X7R com influência em diferentes aspectos da homeostase hematopoética e na dinâmica de proliferação e diferenciação em modelo inflamatório, abrindo caminho para futuras pesquisas e aplicações clínicas potenciais.
Hematopoietic stem cells (HSCs) possess the potential of generate all cells in lymphohematopoietic system. Understanding the self-renewal, expansion, and differentiation mechanisms in hematopoiesis constitutes a great scientific and clinical relevance. Modulators such as ATP and purinergic receptors have been studied to understand their role in hematopoietic niche maintenance. The P2X7 purinergic receptor (P2X7R) is a cation channel-type receptor that has ATP as its main physiological agonist. Its activity has been primarily associated with the immune system, with few studies showing its influence in hematopoiesis. In this study, C57BL/6 wild-type (WT) and P2X7 knockout (P2X7-/-) mice were employed to investigate the importance and function of this receptor in modulating HSCs and its effects on myeloid and lymphoid differentiation. The evaluation of P2X7R expression in hematopoietic cells from bone marrow shows that P2X7R is more highly expressed in primitive cells. The immunophenotyping of hematopoietic populations from bone marrow and spleen showed an increase in HSC number and myeloid and lymphoid progenitor cells in P2X7-/- animals. The decrease in erythrocytes in bone marrow was compensated by an increase in this population in the spleen, and no changes were found in hematological parameters. There was also no impact on the ability of bone marrow hematopoietic cells and progenitors to form colonies. The sepsis induction using the cecal ligation and puncture (CLP) method was performed to promote an inflammatory response and modulate the lymphohematopoietic system. The inflammatory stimulus induced in WT animals produced an increase in the number of HSCs, as well as an increase in c-Kit+, granulocyte progenitors and mature granulocytes in the bone marrow, contrasting with the small number of neutrophils in peripheral blood. On the other hand, the P2X7-/- mice showed a high number of peripheral neutrophils, a reduction in the number of HSCs, c-Kit+, granulocyte progenitors, and mature granulocytes. In both WT and P2X7-/- mice, erythropoiesis was impaired, and the transcription factors NF-E2, Gata-1 and Gata-2 were downregulated, in bone marrow. In P2X7-/- mice, the CLP induction led to an increased colony formation. Monocyte colony-forming units were specially formed, while these cells were decreased in peripheral blood. Additionally, other markers related to hematopoiesis and inflammation, such as reactive oxygen species (ROS), pro and anti-inflammatory cytokines, and immunoglobulins, were evaluated. Septic P2X7-/- mice exhibited high levels of ROS in the peritoneal cavity and bone marrow, but not in plasma, where increased interleukin-6 and immunoglobulin M were found. Overall, our results highlight a complex crosstalk between P2X7R and a different aspect of hematopoietic homeostasis, the dynamics of proliferation and differentiation in an inflammatory model, providing scope for future research and possible clinical applications.
Hematopoietic stem cells (HSCs) possess the potential of generate all cells in lymphohematopoietic system. Understanding the self-renewal, expansion, and differentiation mechanisms in hematopoiesis constitutes a great scientific and clinical relevance. Modulators such as ATP and purinergic receptors have been studied to understand their role in hematopoietic niche maintenance. The P2X7 purinergic receptor (P2X7R) is a cation channel-type receptor that has ATP as its main physiological agonist. Its activity has been primarily associated with the immune system, with few studies showing its influence in hematopoiesis. In this study, C57BL/6 wild-type (WT) and P2X7 knockout (P2X7-/-) mice were employed to investigate the importance and function of this receptor in modulating HSCs and its effects on myeloid and lymphoid differentiation. The evaluation of P2X7R expression in hematopoietic cells from bone marrow shows that P2X7R is more highly expressed in primitive cells. The immunophenotyping of hematopoietic populations from bone marrow and spleen showed an increase in HSC number and myeloid and lymphoid progenitor cells in P2X7-/- animals. The decrease in erythrocytes in bone marrow was compensated by an increase in this population in the spleen, and no changes were found in hematological parameters. There was also no impact on the ability of bone marrow hematopoietic cells and progenitors to form colonies. The sepsis induction using the cecal ligation and puncture (CLP) method was performed to promote an inflammatory response and modulate the lymphohematopoietic system. The inflammatory stimulus induced in WT animals produced an increase in the number of HSCs, as well as an increase in c-Kit+, granulocyte progenitors and mature granulocytes in the bone marrow, contrasting with the small number of neutrophils in peripheral blood. On the other hand, the P2X7-/- mice showed a high number of peripheral neutrophils, a reduction in the number of HSCs, c-Kit+, granulocyte progenitors, and mature granulocytes. In both WT and P2X7-/- mice, erythropoiesis was impaired, and the transcription factors NF-E2, Gata-1 and Gata-2 were downregulated, in bone marrow. In P2X7-/- mice, the CLP induction led to an increased colony formation. Monocyte colony-forming units were specially formed, while these cells were decreased in peripheral blood. Additionally, other markers related to hematopoiesis and inflammation, such as reactive oxygen species (ROS), pro and anti-inflammatory cytokines, and immunoglobulins, were evaluated. Septic P2X7-/- mice exhibited high levels of ROS in the peritoneal cavity and bone marrow, but not in plasma, where increased interleukin-6 and immunoglobulin M were found. Overall, our results highlight a complex crosstalk between P2X7R and a different aspect of hematopoietic homeostasis, the dynamics of proliferation and differentiation in an inflammatory model, providing scope for future research and possible clinical applications.
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Citação
SOUZA, Kamylla Fernanda Souza de. Alteração da homeostase no sistema linfo-hematopoético em animais P2X7-/- e seus efeitos nos processos de proliferação e diferenciação. 2024. 93 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.