Characterization of murine amniotic fluid B cells in normal pregnancy and in preterm birth

in Reproduction
Correspondence should be addressed to F Jensen; Email: fjensen@unaj.edu.ar
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The amniotic fluid provides mechanical protection and immune defense against pathogens to the fetus. Indeed, components of the innate and adaptive immunity, including B cells, have been described in the amniotic fluid. However, limited information concerning phenotype and functionality of amniotic fluid B cells is available. Hence, we aimed to perform a full phenotypical and functional characterization of amniotic fluid B cells in normal pregnancy and in a mouse model of preterm birth. Phenotypic analysis depicted the presence of two populations of amniotic fluid B cells: an immature population, resembling B1 progenitor cells and a more mature population. Further isolation and in vitro co-culture with a bone marrow stroma cell line demonstrated the capacity of the immature B cells to mature. This was further supported by spontaneous production of IgM, a feature of the B1 B cell sub-population. An additional in vitro stimulation with lipopolysaccharide induced the activation of amniotic fluid B cells as well as the production of pro and anti-inflammatory cytokines. Furthermore, amniotic fluid B cells were expanded in the acute phase of LPS-induced preterm birth. Overall our data add new insight not only on the phenotype and developmental stage of the amniotic fluid B1 B cells but especially on their functionality. This provides important information for a better understanding of their role within the amniotic fluid as immunological protective barrier, especially with regard to intraamniotic infection and preterm birth.

 

    Society for Reproduction and Fertility

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    Isolation and characterization of AF cells. (A) Murine feto–maternal unit after removal from the maternal uterus, showing the placenta and the amniotic sac containing the AF. (B) Collection of the AF with a 0.6 × 25 mm syringe (green). (C) Gating strategy for the flow cytometric analysis of CD19+ AF-B cells. (D) Expression analysis of paternal MHCI molecule (H-2Dd) within CD19+ AF-B cells. (E) Pure magnetically isolated CD19+ AF-B cells were stained with fluorochrome-conjugated antibodies against CD45R (B220) and analyzed by flow cytometry. Lymphocytes were identified by their scatter properties (FSC-A × SSC-A plot or FSC-H × SSC-H) and then doublets were excluded by gating on FSC-A × FSC-H. Fluorescence minus one (FMO) was used as control. Data is representative of at least eight independent animals.

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    Phenotypic characterization of isolated AF CD19+ B cells co-cultured with a bone marrow stromal cell line. (A) Magnetically isolated CD19+ AF-B cells were co-cultured with a bone marrow stromal cell line (OP9) for 10 days and then stained with fluorochrome-conjugated antibodies against CD45R (B220). (B) Representative histograms showing gating strategy for the analysis of the expression of B cell activation markers, CD69 and MHCII in AF-isolated CD19+ cells after co-culture with OP9 stromal cell line. Pure isolated CD19+ AF-B cells were co-cultured with OP9 stromal cells for 10 days and then treated with LPS (10 μg/mL) for 48 h. Thereafter, cells were stained with fluorochrome-conjugated antibodies against CD19, CD69 and MHCII and analyzed by flow cytometry using a FACSCanto flow cytometer. Gray histograms represent corresponding unstained controls. (C) Box and whiskers graphs showing mean fluorescence intensity (MFI) of CD69 and MHCII in CD19+ B cells. Data were collected with FACSCanto flow cytometer and analyzed with FlowJo software. Lymphocytes were identified by their scatter properties (FSC-A × SSC-A plot) and then doublets were excluded by gating on FSC-A × FSC-H. Fluorescence minus one (FMO) was used as control. Data were analyzed with PRISM software (version 5.0, GraphPad). Data are shown as mean ± s.e.m. of five mice per group. ***P < 0.001 as analyzed by Mann–Whitney test.

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    Amniotic fluid B cells produce pro as well as anti-inflammatory cytokines. (A) Representative pseudocolor plots showing gating strategy used to analyze intracellular cytokine production by AF-B cells. Pure isolated CD19+ AF-B cells were co-cultured with OP9 stromal cells for 10 days and then treated with LPS (10 μg/mL) for 48 h. Thereafter, cells were stained with fluorochrome-conjugated antibodies against CD19, TNF-α, IFN-γ, IL-17A and IL-10. Data were collected with FACSCanto flow cytometer and analyzed with FlowJo software. Lymphocytes are identified by their scatter properties (FSC-A × SSC-A plot) and then doublets were excluded by gating on FSC-A × FSC-H. Data are shown as mean ± s.e.m. of five mice per group. No statistically significant differences were observed among the groups as analyzed by Student’s t-test.

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    Amniotic fluid B cells spontaneously produce IgM. Pure magnetically isolated CD19+ AF-B cells were co-cultured with OP9 stromal cells for 10 days and then further stimulated with LPS (10 μg/mL) for 48 h. Levels of IgM were quantified in supernatants by ELISA. Data are shown as single dot, whereas means are indicated. No statistically significant differences were observed among the groups as analyzed by Mann–Whitney test.

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    B cells are augmented in the amniotic fluid during acute phase of LPS-induced PTB. Pregnant mice were injected (i.p.) with LPS (10 μg/mice) on day 16 of pregnancy and killed 5 h later. (A) Representative pseudocolor plots showing gating strategy used to analyze different B cell subpopulations in the AF during acute phase of LPS-induced PTB. (B) Bar graphs show percentages and total numbers of CD19+ B cells as well as percentages of CD19-gated B220 and CD19-gated CD5 B1 B cells in the AF during acute phase of LPS-induced PTB. Data are shown as mean ± s.e.m. of six mice per group. ***P < 0.001; ****P < 0.0001 as analyzed by Student’s t-test.

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