Terapia com ciclofosfamida no Lúpus Eritematoso Sistêmico: heterogeneidade no impacto sobre o sistema imunitário inato
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Data
2024-02-21
Autores
Prado, Mônica Simon [UNIFESP]
Orientadores
Andrade, Luis Eduardo Coelho [UNIFESP]
Tipo
Tese de doutorado
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Introdução: Os pacientes com Lúpus Eritematoso Sistêmico (LES) são frequentemente tratados com medicamentos imunossupressores. Alguns pacientes tornam-se vulneráveis a infecções graves e repetitivas em função da imunodepressão ocasionada pelos medicamentos, enquanto outros pacientes parecem tolerar bem o tratamento. A heterogeneidade na ocorrência de infecções em pacientes lúpicos sob terapia imunossupressora pode estar associada a algum grau de heterogeneidade individual na suscetibilidade dos vários mecanismos imunológicos às drogas imunossupressoras.
Objetivos: Avaliar o efeito da pulsoterapia com ciclofosfamida sobre o sistema imunitário inato de pacientes com LES, analisando o comportamento fenotípico e funcional de monócitos e neutrófilos circulantes.
Materiais e Métodos: Realizamos análise transversal de 60 pacientes com nefrite lúpica em atividade e indicação de pulsoterapia com ciclofosfamida, assim como de 40 controles saudáveis. Também analisamos longitudinalmente 39 pacientes antes e após 4 meses de tratamento com ciclofosfamida. O número de neutrófilos totais, maduros, imaturos e CD62Llow e de monócitos totais, clássicos, intermediários e não clássicos foram avaliados no sangue periférico por citometria de fluxo. A fagocitose de neutrófilos e monócitos foi estudada por citometria de fluxo utilizando zymosan como estímulo. A queima oxidativa de neutrófilos foi analisada por quimiofluorescência utilizando luminol e lucigenina como marcadores, espontânea e com PMA como estímulo. A netose de neutrófilos foi avaliada por imunofluorescência indireta com tripla marcação com e sem estímulo com PMA.
Resultados: Fenotipicamente observamos maior frequência de neutrófilos totais (p<0,001), neutrófilos CD62Llow (p=0,008) e menor de neutrófilos maduros (p=0,021) nos pacientes antes do tratamento em relação aos controles, longitudinalmente tivemos um aumento da frequência de neutrófilos imaturos após o tratamento (p=0,025). Quanto aos monócitos, os pacientes antes do tratamento apresentaram uma menor frequência de monócitos clássicos (p<0,001) e uma maior frequência de monócitos não clássicos (p=0,036) e intermediários (p=0,016) em comparação com os controles e quando avaliados longitudinalmente os pacientes aumentaram a frequência de monócitos totais (p=0,011). Funcionalmente os pacientes com nefrite lúpica antes do tratamento apresentaram uma maior frequência de netose espontânea (p=0,018) quando comparados com os controles, diminuindo significativamente após o tratamento (p<0,001). Identificamos três clusters de pacientes com características semelhantes em relação a elementos e funções da imunidade inata. Os pacientes alocados no cluster 2 apresentaram maior frequência de infecções e de remissão e aumentaram estatisticamente a taxa de fagocitose, tanto para monócitos quanto para neutrófilos no tempo 2. Este cluster também apresentou um aumento significante de neutrófilos imaturos e monócitos intermediários e uma diminuição de monócitos clássicos após o tratamento. Já no cluster 3 tivemos o maior número de pacientes alocados e, neste grupo, a explosão respiratória apresentou valores maiores após o tratamento.
Conclusões: A comparação entre controles hígidos e pacientes lúpicos no tempo basal evidenciou que os pacientes apresentaram maior taxa de netose espontânea, menor frequência de neutrófilos maduros e maior de neutrófilos totais e neutrófilos CD16++CD62low, menor frequência de monócitos totais e clássicos e maior frequência de monócitos intermediários e não clássicos. Após quatro meses de pulsoterapia com ciclofosfamida, o grupo de pacientes lúpicos apresentou queda na taxa de netose espontânea, aumento na frequência de neutrófilos imaturos e de monócitos totais, ficando com valores semelhantes ao grupo controle. Identificamos três clusters de pacientes com características semelhantes em relação a elementos e funções da imunidade inata. Estes clusters foram significantemente diferentes entre si, principalmente em relação às atividades de fagocitose e explosão respiratória, neutrófilos imaturos e monócitos clássicos e intermediários. A acentuada heterogeneidade interindividual no comportamento dos parâmetros de imunidade inata nos pacientes lúpicos, antes e após tratamento com ciclofosfamida, sugere que alguns pacientes podem ser mais suscetíveis ao risco por infecção decorrente do tratamento.
Introduction: Patients with Systemic Lupus Erythematosus (SLE) are often treated with immunosuppressive drugs. Some patients become vulnerable to severe and repetitive infections due to the immunosuppression caused by the drugs, while other patients seem to tolerate the treatment well. The heterogeneity in the occurrence of infections in SLE patients under immunosuppressive therapy may be associated with some degree of individual heterogeneity in the susceptibility of the various immunological mechanisms to immunosuppressive drugs. Objectives: To evaluate the effect of cyclophosphamide pulse therapy on the innate immune system of patients with SLE, analyzing the phenotypic and functional behavior of circulating monocytes and neutrophils. Materials and Methods: We performed a crosssectional analysis of 60 patients with active lupus nephritis and indication for pulse therapy with cyclophosphamide, as well as 40 healthy controls. We also longitudinally analyzed 39 patients before and after 4 months of cyclophosphamide treatment. The number of total, mature, immature and CD62Llow neutrophils and total, classical, intermediate and nonclassical monocytes were evaluated in peripheral blood by flow cytometry. Neutrophil and monocyte phagocytosis was studied by flow cytometry using zymosan as stimulus. Neutrophil oxidative burn was analyzed by chemo fluorescence using luminol and lucigenin as markers and PMA as stimulus. Neutrophil netosis was assessed by indirect immunofluorescence with triple staining, with and without PMA stimulus. Results: Phenotypically, we observed a higher frequency of total neutrophils (p<0.001), CD62Llow neutrophils (p=0.008) and a lower frequency of mature neutrophils (p=0.021) in patients, before treatment, in relation to controls, longitudinally we had an increase in the frequency of immature neutrophils after the treatment (p=0.025). Considering monocytes, patients before treatment had a lower frequency of classic monocytes (p<0.001) and a higher frequency of nonclassical (p=0.036) and intermediate monocytes (p=0.016) compared to controls and when evaluated longitudinally patients had an increased frequency of total monocytes (p=0.011). Functionally, patients with lupus nephritis before treatment showed a higher frequency of spontaneous NETosis (p=0.018) when compared to controls, decreasing significantly after treatment (p<0.001). We identified three clusters of patients with similar characteristics regarding elements and functions of innate immunity. Patients allocated to cluster 2 showed a higher frequency of infections and remission and statistically increased the rate of phagocytosis, both for monocytes and neutrophils at time 2. This cluster also showed a significant increase in immature neutrophils and intermediate monocytes and a decrease in monocytes classics after treatment. In cluster 3 we had the largest number of patients allocated and, in this group, the respiratory burst showed higher values after treatment. Conclusions: The comparison between healthy controls and SLE patients at baseline showed that patients had a higher rate of spontaneous NETosis, a lower frequency of mature neutrophils and a higher frequency of total neutrophils and CD16++CD62low neutrophils, a lower frequency of total and classic monocytes and a higher frequency of intermediate and nonclassical monocytes. After four months of pulse therapy with cyclophosphamide, the group of SLE patients showed a drop in the rate of spontaneous NETosis, an increase in the frequency of immature neutrophils and total monocytes, reaching values similar to the control group. We identified three clusters of patients with similar characteristics regarding elements and functions of innate immunity. These clusters were significantly different from each other, mainly in relation to phagocytosis and respiratory burst activities, immature neutrophils and classic and intermediate monocytes. The marked interindividual heterogeneity in the behavior of innate immunity parameters in SLE patients, before and after treatment with cyclophosphamide, suggests that some patients may be more susceptible to the risk of infection resulting from treatment.
Introduction: Patients with Systemic Lupus Erythematosus (SLE) are often treated with immunosuppressive drugs. Some patients become vulnerable to severe and repetitive infections due to the immunosuppression caused by the drugs, while other patients seem to tolerate the treatment well. The heterogeneity in the occurrence of infections in SLE patients under immunosuppressive therapy may be associated with some degree of individual heterogeneity in the susceptibility of the various immunological mechanisms to immunosuppressive drugs. Objectives: To evaluate the effect of cyclophosphamide pulse therapy on the innate immune system of patients with SLE, analyzing the phenotypic and functional behavior of circulating monocytes and neutrophils. Materials and Methods: We performed a crosssectional analysis of 60 patients with active lupus nephritis and indication for pulse therapy with cyclophosphamide, as well as 40 healthy controls. We also longitudinally analyzed 39 patients before and after 4 months of cyclophosphamide treatment. The number of total, mature, immature and CD62Llow neutrophils and total, classical, intermediate and nonclassical monocytes were evaluated in peripheral blood by flow cytometry. Neutrophil and monocyte phagocytosis was studied by flow cytometry using zymosan as stimulus. Neutrophil oxidative burn was analyzed by chemo fluorescence using luminol and lucigenin as markers and PMA as stimulus. Neutrophil netosis was assessed by indirect immunofluorescence with triple staining, with and without PMA stimulus. Results: Phenotypically, we observed a higher frequency of total neutrophils (p<0.001), CD62Llow neutrophils (p=0.008) and a lower frequency of mature neutrophils (p=0.021) in patients, before treatment, in relation to controls, longitudinally we had an increase in the frequency of immature neutrophils after the treatment (p=0.025). Considering monocytes, patients before treatment had a lower frequency of classic monocytes (p<0.001) and a higher frequency of nonclassical (p=0.036) and intermediate monocytes (p=0.016) compared to controls and when evaluated longitudinally patients had an increased frequency of total monocytes (p=0.011). Functionally, patients with lupus nephritis before treatment showed a higher frequency of spontaneous NETosis (p=0.018) when compared to controls, decreasing significantly after treatment (p<0.001). We identified three clusters of patients with similar characteristics regarding elements and functions of innate immunity. Patients allocated to cluster 2 showed a higher frequency of infections and remission and statistically increased the rate of phagocytosis, both for monocytes and neutrophils at time 2. This cluster also showed a significant increase in immature neutrophils and intermediate monocytes and a decrease in monocytes classics after treatment. In cluster 3 we had the largest number of patients allocated and, in this group, the respiratory burst showed higher values after treatment. Conclusions: The comparison between healthy controls and SLE patients at baseline showed that patients had a higher rate of spontaneous NETosis, a lower frequency of mature neutrophils and a higher frequency of total neutrophils and CD16++CD62low neutrophils, a lower frequency of total and classic monocytes and a higher frequency of intermediate and nonclassical monocytes. After four months of pulse therapy with cyclophosphamide, the group of SLE patients showed a drop in the rate of spontaneous NETosis, an increase in the frequency of immature neutrophils and total monocytes, reaching values similar to the control group. We identified three clusters of patients with similar characteristics regarding elements and functions of innate immunity. These clusters were significantly different from each other, mainly in relation to phagocytosis and respiratory burst activities, immature neutrophils and classic and intermediate monocytes. The marked interindividual heterogeneity in the behavior of innate immunity parameters in SLE patients, before and after treatment with cyclophosphamide, suggests that some patients may be more susceptible to the risk of infection resulting from treatment.
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Citação
PRADO, Mônica Simon, Terapia com ciclofosfamida no Lúpus Eritematoso Sistêmico: heterogeneidade no impacto sobre o sistema imunitário inato. 2024. 124 f. Tese (Doutorado em Reumatologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.