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Immunomodulation in pregnancy and labor
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RESUMO: A gravidez é um desafio para o sistema imunológico, porque tem de tolerar um
feto geneticamente diferente e ao mesmo tempo proteger tanto a mãe como o feto
contra ameaças biológicas. Isto requer uma regulação estreita dos vários tipos de
células do sistema imunitário durante as diferentes fases da gravidez. Estudos
recentes revelaram que determinadas subpopulações de células B e células T têm
funções reguladoras e são essenciais para a manutenção da tolerância materno-fetal
durante as fases iniciais da gravidez. No entanto, não está claro se as células do
sistema imunológico materno, especialmente as recém descritas células reguladoras
B (Breg) e T (Treg), também podem desempenhar um papel relevante no final da
gravidez normal, no parto e no pós-parto.
Com o objetivo de analisar o perfil imunológico desde o final da gravidez até ao
pós-parto, foram efetuados três estudos observacionais que seguiram mulheres
saudáveis, grávidas (n = 43) e não grávidas (como grupo controlo; n = 35), vigiadas
na consulta externa do hospital CUF Descobertas. Em primeiro lugar, as contagens
no sangue periférico das subpopulações de linfócitos B, considerando o perfil
maturativo e incluindo as células Breg, foram analisadas em todas as mulheres
grávidas durante o terceiro trimestre da gravidez, no dia do parto (imediatamente
após o parto), e no pós-parto (pelo menos 6 semanas após o parto), e comparadas
com as do grupo de mulheres não grávidas. Em segundo lugar, a evolução das
subpopulações de células Treg foi avaliada no sangue periférico das mulheres
grávidas nos mesmos pontos temporais do estudo anterior, e comparadas com as do
grupo de controlo. Em terceiro lugar, o efeito do trabalho de parto sobre as
subpopulações circulantes de células T, e sobre as subpopulações de células Treg e
Breg, foi avaliado no dia do parto, comparando o subgrupo de mulheres grávidas que
tiveram o parto por cesariana eletiva (sem trabalho de parto; n = 14), com as que
tiveram um parto espontâneo vaginal (após o trabalho de parto; n = 18).
A quantificação e a caracterização fenotípica de células B e células T foi
efetuada por citometria de fluxo, de acordo com a expressão dos marcadores de
superfície celular presentes nas suas diferentes subpopulações. Especificamente, as subpopulações de células B foram caracterizadas no sangue periférico pela expressão
de CD19 (marcador de células B), e pela expressão de IgD e CD38 para identificar o
perfil maturativo dos linfócitos B de acordo com o sistema de classificação Bm1-5:
células B de transição, células B naïve, células B de memória não switched, células B
de memória switched entretanto divididas em células pós centro germinativo e
células de memória em repouso, e ainda plasmablastos. Os linfócitos Breg foram
caracterizados através da expressão de CD24, CD27, CD38, e a produção de IL-10
foi avaliada após a estimulação com acetato de forbolmiristato (PMA), ionóforo de
cálcio e lipopolissacárido (LPS) bacteriano. Relativamente às subpopulações de
células T de sangue periférico, as células com expressão de CD3 (marcador de
células T) que co-expressam CD4 (células T auxiliadoras) ou CD8 (células T
citotóxicas) foram diferenciadas em naïve, memória central, memória efetoras,
memória efetoras com diferenciação terminal utilizando os marcadores de superfície
de células CD45RA e CD62L. A expressão de marcadores de ativação (HLA-DR e
CD25) foi avaliada na população de linfócitos T totais e nas respetivas
subpopulações de células T CD4 e CD8. Além disso, as células T natural killer
(NKT)-like foram identificados pela co-expressão de CD3, CD16 e CD56.
Finalmente, três estratégias analíticas foram utilizadas para caracterizar as células
Treg (CD4DimCD25Hi, CD4+CD25HiCD127-/dim e CD4+CD25HiFoxp3+). Foi também
efetuada a avaliação da expressão intracelular de Foxp3 na subpopulação de células
Treg CD4DimCD25Hi.
Os resultados mostraram que as contagens absolutas e percentuais dos linfócitos
B foram significativamente mais baixas (p<0,05) nas mulheres grávidas no dia do
parto, em comparação com as mulheres não grávidas. De uma forma geral, as
contagens absolutas e percentagens da maioria das subpopulações de células B foram
significativamente mais baixas (p<0,05) no terceiro trimestre da gravidez e no dia do
parto. Além disso, essas contagens absolutas e percentuais das subpopulações de
células B não tiveram diferenças significativas entre o período pós-parto e as
mulheres não grávidas. No entanto, as exceções mais notáveis foram: as
percentagens de células B naïve, que foram significativamente mais elevadas
(p<0,05) no terceiro trimestre e no dia do parto do que no período pós-parto e o
observado nas mulheres não grávidas; e as percentagens da subpopulação de células
Breg CD24HiCD38Hi, que foram significativamente mais elevadas (p<0,05) no período pós-parto do que no terceiro trimestre, no dia do parto e no grupo das
mulheres não grávidas.
De um modo semelhante às células Breg, as contagens absolutas de todas as
subpopulações de células Treg foram significativamente maiores (p<0,05) no período
pós-parto, em comparação com o terceiro trimestre da gravidez e com o dia do parto.
Além disso, a expressão intracelular do fator de transcrição Foxp3 na subpopulação
de células Treg CD4DimCD25Hi diminuiu significativamente (p<0,001) no terceiro
trimestre da gravidez e no dia do parto em comparação com o observado nas
mulheres não grávidas; e aumentou significativamente (p<0,001) no período pósparto,
comparativamente com o terceiro trimestre de gravidez e com o dia do parto.
Finalmente, verificou-se que as contagens absolutas e percentagens das
subpopulações de células Breg e Treg circulantes não foram significativamente
diferentes entre as mulheres que tiveram cesarianas eletivas (sem trabalho de parto) e
aquelas que tiveram partos vaginais espontâneos (após o trabalho de parto). No
entanto, as contagens absolutas dos linfócitos B e das células NKT-like foram
significativamente menores (p<0,05) no sangue periférico das mulheres que tiveram
parto vaginal.
Concluiu-se que no sangue periférico os compartimentos de células B e T
sofrem alterações quantitativas do final da gravidez normal até ao período pós-parto.
Particularmente, as subpopulações de células Breg e Treg aumentam no período pósparto.
Além disso, o trabalho de parto não parece ter um impacto sobre as
subpopulações de células T e sobre as subpopulações de células Breg e Treg no
sangue periférico materno. Portanto, estes resultados podem permitir uma melhor
compreensão da imunomodulação que ocorre durante a gravidez humana e fornecem
algumas evidências para o desenvolvimento de novas estratégias de diagnóstico e
tratamento de patologias obstétricas. Estes resultados poderão contribuir para o
estudo dos mecanismos de resposta materna à vacinação e à infeção.
ABSTRACT: The immune system is challenged during pregnancy because it must tolerate a genetically foreign fetus and protect both the mother and the fetus against biological threats. This achievement requires a tight regulation of the various immune system cell types during the different stages of pregnancy. Recent studies have reported that subsets of B cells and T cells have regulatory functions that are essential for the maintenance of tolerance during early pregnancy by suppressing the maternal alloreactivity against the fetus. However, it is unclear whether the maternal immune system cells, especially the recently described regulatory B cell (Breg) and T cell (Treg) subsets, can also play a relevant role in normal late pregnancy, labor and in postpartum period. To analyze the immunological profile from late pregnancy to postpartum, three observational studies have been performed encompassing the follow-up of healthy pregnant women (n = 43) and non-pregnant women (as a control group; n = 35) who were attending a hospital-based outpatient clinic (Hospital CUF Descobertas). First, the levels of peripheral blood B cell subsets, considering the different maturational stages and including Breg, were analyzed in all pregnant women during the 3rd trimester of pregnancy, on the day of delivery (immediately after labor), and in the postpartum period (at least 6 weeks after delivery), and compared with those of the non-pregnant women. Second, the variation in Treg subsets was assessed in peripheral blood of pregnant women at the same time points used in the previous study and compared with the control group. Third, the effect of labor on the peripheral blood T cell subsets and on the Treg and Breg subsets was assessed on the day of delivery, comparing the subgroup of pregnant women who gave birth by elective cesarean (no labor; n = 14) with those who had a spontaneous vaginal delivery (after labor; n = 18). Quantification and phenotypic characterization of B cells and T cells according to the expression of specific cell surface markers has been performed by flow cytometry. Specifically, circulating B cell subsets were characterized by the expression of CD19 (B cell marker) and by the expression of IgD and CD38 to identify the different B cell maturational stages according to the Bm1-5 classification system: transitional B cells, naïve B cells, unswitched memory B cells and switched memory B cells, which were subsequently divided into post-germinal memory B cells and resting memory B cells; and plasmablasts. Breg subsets were characterized by the expression of CD24, CD27, CD38, and the production of IL-10 after phorbol 12-myristate 13-acetate (PMA), calcium ionophore and lipopolysaccharide (LPS) stimulation. Regarding the circulating T cell subsets, cells expressing CD3 (T cell marker) and co-expressing either CD4 (helper T cells) or CD8 (cytotoxic T cells) were discriminated into naïve, central memory, effector memory, and terminally differentiated effector memory cells based on the cell surface markers CD45RA and CD62L. The expression of activation markers (HLA-DR and CD25) was assessed in total T lymphocytes and in CD4 and CD8 T cells subsets. In addition, natural killer T (NKT)-like cells were identified by the co-expression of CD3 with CD16 and CD56. Finally, three analytical strategies were used to characterize Treg cells (CD4DimCD25Hi, CD4+CD25HiCD127-/dim and CD4+CD25HiFoxp3+ T cells). Additionally, the expression levels of the transcription factor Foxp3 in the CD4DimCD25Hi Treg cells were analyzed. The results showed that the absolute counts and percentages of B cells were significantly lower (p<0.05) in pregnant women on the day of delivery compared with non-pregnant women. Overall, the absolute counts and percentages of the majority of the B cell subsets were significantly lower (p<0.05) in the 3rd trimester of pregnancy and on the day of delivery. Moreover, these counts and percentages did not differ significantly between the postpartum period and non-pregnant women. However, the most notable exceptions were the percentages of naïve B cells, which were significantly higher (p<0.05) in the 3rd trimester and on the day of delivery compared with the postpartum period and in non-pregnant women, and the percentages of the CD24HiCD38Hi Breg subset, which were significantly higher (p<0.05) in the postpartum period compared with the 3rd trimester, the day of delivery, and in non-pregnant women. Similarly to Breg cells, the absolute counts of all Treg subsets were significantly higher (p<0.05) in the postpartum period compared with the 3rd trimester and the day of delivery. In addition, intracellular expression of Foxp3 in the CD4DimCD25Hi Treg subset significantly decreased (p<0.001) in the 3rd trimester of pregnancy and on the day of delivery compared with non-pregnant women; they also significantly increased (p<0.001) during the postpartum period compared with the 3rd trimester and on the day of delivery. Finally, the absolute counts and percentages of circulating Breg and Treg subsets were not significantly different between women who had elective cesareans (no labor) and those who had spontaneous vaginal deliveries (after labor). Nevertheless, the absolute counts of B cells and of NKT-like cells were significantly lower (p<0.05) in the peripheral blood of women who had vaginal deliveries. These findings indicated that the peripheral blood B cell and T cell compartments undergo quantitative changes from normal late pregnancy to the postpartum period. In particular, the results presented in this thesis suggest that circulating Breg and Treg subsets increase during the postpartum period, despite their well-known involvement in early pregnancy. In addition, labor does not seem to have a major impact on maternal peripheral blood T cell subsets and on Breg and Treg subsets. Therefore, these findings may improve our understanding of immunomodulation during human pregnancy and provide some evidence for the development of new strategies to diagnose and treat pregnancy-associated disturbances. These investigations may also contribute to the study of the mechanisms of maternal responses to vaccination and infection.
ABSTRACT: The immune system is challenged during pregnancy because it must tolerate a genetically foreign fetus and protect both the mother and the fetus against biological threats. This achievement requires a tight regulation of the various immune system cell types during the different stages of pregnancy. Recent studies have reported that subsets of B cells and T cells have regulatory functions that are essential for the maintenance of tolerance during early pregnancy by suppressing the maternal alloreactivity against the fetus. However, it is unclear whether the maternal immune system cells, especially the recently described regulatory B cell (Breg) and T cell (Treg) subsets, can also play a relevant role in normal late pregnancy, labor and in postpartum period. To analyze the immunological profile from late pregnancy to postpartum, three observational studies have been performed encompassing the follow-up of healthy pregnant women (n = 43) and non-pregnant women (as a control group; n = 35) who were attending a hospital-based outpatient clinic (Hospital CUF Descobertas). First, the levels of peripheral blood B cell subsets, considering the different maturational stages and including Breg, were analyzed in all pregnant women during the 3rd trimester of pregnancy, on the day of delivery (immediately after labor), and in the postpartum period (at least 6 weeks after delivery), and compared with those of the non-pregnant women. Second, the variation in Treg subsets was assessed in peripheral blood of pregnant women at the same time points used in the previous study and compared with the control group. Third, the effect of labor on the peripheral blood T cell subsets and on the Treg and Breg subsets was assessed on the day of delivery, comparing the subgroup of pregnant women who gave birth by elective cesarean (no labor; n = 14) with those who had a spontaneous vaginal delivery (after labor; n = 18). Quantification and phenotypic characterization of B cells and T cells according to the expression of specific cell surface markers has been performed by flow cytometry. Specifically, circulating B cell subsets were characterized by the expression of CD19 (B cell marker) and by the expression of IgD and CD38 to identify the different B cell maturational stages according to the Bm1-5 classification system: transitional B cells, naïve B cells, unswitched memory B cells and switched memory B cells, which were subsequently divided into post-germinal memory B cells and resting memory B cells; and plasmablasts. Breg subsets were characterized by the expression of CD24, CD27, CD38, and the production of IL-10 after phorbol 12-myristate 13-acetate (PMA), calcium ionophore and lipopolysaccharide (LPS) stimulation. Regarding the circulating T cell subsets, cells expressing CD3 (T cell marker) and co-expressing either CD4 (helper T cells) or CD8 (cytotoxic T cells) were discriminated into naïve, central memory, effector memory, and terminally differentiated effector memory cells based on the cell surface markers CD45RA and CD62L. The expression of activation markers (HLA-DR and CD25) was assessed in total T lymphocytes and in CD4 and CD8 T cells subsets. In addition, natural killer T (NKT)-like cells were identified by the co-expression of CD3 with CD16 and CD56. Finally, three analytical strategies were used to characterize Treg cells (CD4DimCD25Hi, CD4+CD25HiCD127-/dim and CD4+CD25HiFoxp3+ T cells). Additionally, the expression levels of the transcription factor Foxp3 in the CD4DimCD25Hi Treg cells were analyzed. The results showed that the absolute counts and percentages of B cells were significantly lower (p<0.05) in pregnant women on the day of delivery compared with non-pregnant women. Overall, the absolute counts and percentages of the majority of the B cell subsets were significantly lower (p<0.05) in the 3rd trimester of pregnancy and on the day of delivery. Moreover, these counts and percentages did not differ significantly between the postpartum period and non-pregnant women. However, the most notable exceptions were the percentages of naïve B cells, which were significantly higher (p<0.05) in the 3rd trimester and on the day of delivery compared with the postpartum period and in non-pregnant women, and the percentages of the CD24HiCD38Hi Breg subset, which were significantly higher (p<0.05) in the postpartum period compared with the 3rd trimester, the day of delivery, and in non-pregnant women. Similarly to Breg cells, the absolute counts of all Treg subsets were significantly higher (p<0.05) in the postpartum period compared with the 3rd trimester and the day of delivery. In addition, intracellular expression of Foxp3 in the CD4DimCD25Hi Treg subset significantly decreased (p<0.001) in the 3rd trimester of pregnancy and on the day of delivery compared with non-pregnant women; they also significantly increased (p<0.001) during the postpartum period compared with the 3rd trimester and on the day of delivery. Finally, the absolute counts and percentages of circulating Breg and Treg subsets were not significantly different between women who had elective cesareans (no labor) and those who had spontaneous vaginal deliveries (after labor). Nevertheless, the absolute counts of B cells and of NKT-like cells were significantly lower (p<0.05) in the peripheral blood of women who had vaginal deliveries. These findings indicated that the peripheral blood B cell and T cell compartments undergo quantitative changes from normal late pregnancy to the postpartum period. In particular, the results presented in this thesis suggest that circulating Breg and Treg subsets increase during the postpartum period, despite their well-known involvement in early pregnancy. In addition, labor does not seem to have a major impact on maternal peripheral blood T cell subsets and on Breg and Treg subsets. Therefore, these findings may improve our understanding of immunomodulation during human pregnancy and provide some evidence for the development of new strategies to diagnose and treat pregnancy-associated disturbances. These investigations may also contribute to the study of the mechanisms of maternal responses to vaccination and infection.
Descrição
Palavras-chave
Pregnancy Immune system