RESUMO
During human pregnancy, leukocytes that infiltrate the maternal-fetal interface play a major role in establishing a delicate balance between immune tolerance and functional response and setting the inflammatory process that leads to labor. Here we describe two methods for isolating immune cells from the chorioamniotic membranes (decidua parietalis) and placental blood (decidua basalis) that combine gentle enzymatic digestion, magnetic cell sorting, and density gradient. Isolated leukocytes can be immunophenotypified by flow cytometry, and both isolation methods are compatible with downstream cellular and molecular applications, such as cell culture, transcriptome, and proteome analyses.
Assuntos
Decídua , Placenta , Gravidez , Humanos , Feminino , Imunofenotipagem , Separação Celular/métodos , LeucócitosRESUMO
Leukocyte infiltration into the maternal-fetal interface is a consequence of the robust inflammation in the gestational tissues during term labor and preterm labor with or without infection. During pregnancy, the fetal membranes act as a physical barrier that isolates the fetus into the amniotic cavity, keeping it in an optimal environment for its development. In addition, the fetal membranes possess immunological competencies such as the secretion of cytokines and chemokines in response to different stimuli. Clinical and experimental evidence indicates that these tissues are involved in the extensive chemotaxis of immune cells in normal or pathological conditions.Few studies have evaluated the chemotactic capacities of the fetal membranes considering that this tissue is composed of two adjacent tissues, the amnion and the chorion, which have different characteristics. Although these tissues function as a unit, their response is complex since there is an interaction between them, where each tissue contributes differently. The protocol described here allows us to evaluate the in vitro chemotactic capacities of fetal membranes in response to various applied stimuli, considering the contribution of each of their components (amnion and choriodecidua) using a Boyden chamber assay and phenotyping the chemo-attracted leukocytes by flow cytometry.
Assuntos
Membranas Extraembrionárias , Trabalho de Parto , Gravidez , Recém-Nascido , Feminino , Humanos , Âmnio , Córion , Quimiotaxia de LeucócitoRESUMO
During pregnancy, the fetal membranes composed of the amnion and chorodecidua constitute a selective barrier separating two distinct environments, maternal and fetal. These tissues have the function of delimiting the amniotic cavity. Their histological complexity gives them physical, mechanical, and immunological properties to protect the fetus. Although the study of the amnion, chorion, and decidua separately provides knowledge about the functions of the fetal membranes, the protocol we describe in this chapter has the advantage of maintaining the biological and functional complexity of these tissues. In addition, this experimental model allows the researcher to recreate various pathological scenarios because this model allows for differential stimulation of the amnion or choriodecidua.
Assuntos
Decídua , Membranas Extraembrionárias , Gravidez , Feminino , Humanos , Âmnio , Córion , FetoRESUMO
Prolactin (PRL) is a pleiotropic hormone with a key role in pregnancy. In fetal membranes, PRL can regulate the secretion of pro-inflammatory factors, which induces the activation of matrix metalloproteinases (MMPs). The increase and activation of MMPs deregulate the turnover of the extracellular matrix in the fetal membranes, altering its structure and function, causing premature rupture of the membranes and preterm labor. In this work, we evaluate the effect of PRL upon the secretion of MMP-1, MMP-2, MMP-9, MMP-13, and the tissue inhibitors of metalloproteinases (TIMPs) in human fetal membranes after lipopolysaccharide (LPS) challenge. Nine fetal membranes from healthy non-laboring cesarean deliveries at term were cultured in a 2-independent chamber system and pre-treated with 250, 500, 1000 or 4000 ng/ml of PRL for 24 h, then choriodecidual region was stimulated with 500 ng/ml of LPS plus fresh PRL for 24 h. The MMPs and TIMPs secretion were quantified by ELISA, additionally MMP-2 and MMP-9 gelatinolytic activity was measured by zymography. LPS induced the MMP-9 and MMP-1 secretion, but no MMP-2 or MMP-13 in comparison with basal levels. PRL co-treatment decreased the MMP-2, MMP-9 and MMP-1 secretion induced by LPS. The active forms were present in the tissue extract, showing a response consistent with the secretion profile. TIMP-1 and TIMP-2 secretion was decreased after LPS treatment and the PRL co-treatment reverts this effect. The present results support that PRL may favor the balance between these factors involved in the structural maintenance of fetal membranes in an inflammatory event.
Assuntos
Anti-Inflamatórios , Membranas Extraembrionárias , Inflamação , Metaloproteinase 9 da Matriz , Metaloproteinases da Matriz Secretadas , Prolactina , Anti-Inflamatórios/farmacologia , Regulação para Baixo , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/metabolismo , Feminino , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/terapia , Lipopolissacarídeos/efeitos adversos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Gravidez , Prolactina/farmacologia , Técnicas de Cultura de Tecidos , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
BACKGROUND: Human labor is associated with an inflammatory process that takes place at the maternal-fetal interface, where leukocytes infiltrate and contribute to the local production of effector molecules such as cytokines, chemokines, MMPs, etc. This process may be altered by a low-grade chronic inflammation, characteristic of obesity, resulting in adverse pregnancy outcomes. In this cross-sectional pilot study, we analyzed the relationship between maternal adiposity and inflammation-related gene expression in leukocytes from six healthy women with term pregnancies without labor. METHODS: We estimated maternal adiposity and examined the relative expression of 211 inflammation-related genes in maternal peripheral blood leukocytes (MAT), placental intervillous blood leukocytes (PLA), and choriodecidual leukocytes (CHD) by real-time qPCR. Finally, we analyzed the correlation between maternal adiposity and gene expression. RESULTS: Participants' adiposity ranged from 27.6% to 61.1% (n = 6). The expression of 23 genes significantly differed (p < 0.05) in MAT, PLA, and CHD leukocytes, most of which code for chemokines and proinflammatory cytokines. Importantly, increasing maternal adiposity correlated (r > 0.7) mostly positively with the expression of genes related to activation, migration, infiltration, and proinflammation in MAT (36 genes) and PLA (31 genes). In contrast, in CHD leukocytes maternal adiposity correlated only negatively with seven genes, involved in migration and infiltration. CONCLUSION: Our findings suggest that during term pregnancy, increased maternal adiposity may enhance the priming of peripheral leukocytes, while in choriodecidua it may alter leukocyte recruitment and proinflammatory activity. Maternal adiposity must be considered an important variable in further studies that analyze inflammation-related gene expression in pregnant women.
Assuntos
Adiposidade , Citocinas/genética , Leucócitos/metabolismo , Placenta/metabolismo , Gravidez/metabolismo , Adulto , Citocinas/metabolismo , Feminino , Humanos , Placenta/citologia , Gravidez/genética , TranscriptomaRESUMO
PROBLEM: Human parturition is associated with an intrauterine pro-inflammatory environment in the choriodecidua. Evidence that some mediators of this signaling cascade also elicit responses leading to labor prompted us to characterize the cellular sources of these mediators in the human choriodecidua. METHOD OF STUDY: Leukocyte-enriched preparations from human choriodecidua (ChL) and intervillous placental blood leukocytes (PL) were maintained in culture. Secretions of inflammatory cytokines, chemokines, and MMP-9 were documented. Leukocyte phenotype of ChL and PL was determined by flow cytometry using specific fluorochrome-conjugated antibodies. RESULTS AND CONCLUSIONS: ChL showed a distinct pro-inflammatory secretion pattern of cytokines and chemokines when compared with PL, including higher amounts of TNF-α and IL-6, and decreased secretions of IL-4 and IL-1ra. ChL also secreted more MIP-1α and MCP-1 and MMP-9 than PL. No significant differences were found in leukocytes subsets between compartments. Based on our findings, we propose that ChL isolated from fetal membranes at term are functionally different from PL and may collaborate to modulate the microenvironment linked to induction and progression of human labor.