CD83 is locally regulated and differentially expressed in disturbed murine pregnancy

in Reproduction
Correspondence should be addressed to D O Muzzio; Email: damian.muzzio@med.uni-greifswald.de
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Alterations in the immunologic balance during pregnancy have been associated with poor pregnancy outcomes. The underlying mechanisms are complex and mouse models delivered valuable information on inflammatory imbalance in disturbed pregnancies and served as model to test potential anti-inflammatory therapies. CD83 is a transmembrane protein (mCD83) with a soluble form (sCD83) which possesses strong anti-inflammatory properties. During murine pregnancy, upregulated mCD83 expression induces sCD83 release after in vitro stimulation with LPS, phorbol myristate acetate (PMA) and ionomycin. The release mechanism of sCD83 and its control are yet to be elucidated. In this study, the expression of mCD83 and sCD83 has been extensively studied in the CBA/J × DBA/2J mouse model of pro-inflammatory-mediated pregnancy disturbances. mCD83 was higher expressed on splenic B cells, uterus-draining lymph nodes T cells and dendritic cells from mice with poor pregnancy outcome (PPOM) compared to mice with good pregnancy outcome (GPOM). PPOM, however, was accompanied by lower sCD83 serum levels. In vitro treatment of splenic B cells with progesterone led to a reduction of TIMP1 expression, mCD83 expression and sCD83 release, while TIMP1 treatment had a positive effect on sCD83 availability. These results suggest that tissue and matrix components are involved in the regulation of CD83 in murine pro-inflammatory pregnancies.

 

    Society for Reproduction and Fertility

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    PPOM express higher mCD83 than GPOM in splenic but not in peripheral B cells in vivo. B cells (B220+), T cells (CD4+) and dendritic cells (DCIR2+) from GPOM (n = 16) were compared to cells from PPOM (n = 22). (A) Representative plots show gating strategies for determination of mCD83 expression. In overlapping histograms (right), the gray area represents the unstained control and the black line the positive staining. Bars show the relative median fluorescence intensity (MFI) of mCD83 (B) or the percentage of mCD83+ cells (C) in live B cells (B220+), T cells (CD4+) and dendritic cells (DCIR2+) from spleen (SPL), thymus (THY), paraaortic (PLN) and inguinal (ILN) lymph nodes. In spleen, B220+ B cells were further subdivided in CD21/35hiCD23low marginal zone B cells (MZ) and CD21/35intCD23hi follicular zone B cells (FO). All data were analyzed using Student’s t-test (n ≥ 16). Significant differences are indicated (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

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    Ex vivo stimulation of splenic B cells reveals no difference in mCD83 expression between GPOM and PPOM. (A) Splenic lymphocytes were stimulated with LPS for 48 h with PMA and ionomycin for the last 5 h (LPI). Plots show a representative gating strategy for determination of mCD83 expression on CD19+ B cells (left). The overlapping histograms display mCD83 expression of untreated (filled line) and LPI-stimulated B cells (dashed line). Gray area represents the FMO for CD83. Bars show the corresponding median fluorescence intensities (MFI) of mCD83 (bottom-right) on LPI-stimulated B cells. (B) Supernatants of the LPI-stimulated lymphocytes were analyzed by ELISA to determine sCD83 levels. All data were analyzed using Student’s t-test (A: n = 10, B: n = 9).

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    Progesterone reduces mCD83 and sCD83 availability in vitro. (A) Sera of pregnant mice were analyzed by ELISA to determine sCD83 levels. Data were analyzed using Student’s t-test (GPOM n = 19, PPOM n = 8). Significant differences are indicated (*P ≤ 0.05). (B) Splenic lymphocytes or magnetically isolated CD19+ B cells from the spleen of non-pregnant CBA/J mice were cultured for 48 h with progesterone or with medium alone as control. Graph shows levels of sCD83 of supernatants determined by ELISA. Data were analyzed using paired t-test (n = 5). Significant differences are indicated (*P ≤ 0.05). (C) Splenic lymphocytes from non-pregnant CBA/J mice were cultured for 48 h with LPS alone or with LPS and 500 ng/mL progesterone. PMA and ionomycin were added for the last 5 h. The plots show a representative gating strategy (left). Overlapping histograms (right) display differences in iCD83 between LPS (filled line) and LPS with progesterone (dashed line) stimulated CD19+ B cells. Gray area represents the FMO for CD83. Data represent the fold increase of iCD83 MFI (middle) or Cd83 mRNA (right) in splenic CD19+ B cells over the corresponding untreated controls. Data were analyzed using paired t-test (flow cytometry: n = 7; qPCR: n = 6). (D) Splenic lymphocytes from non-pregnant CBA/J mice were cultured for 48 h with or without the presence of 50 or 500 ng/mL progesterone. The plots show a representative gating strategy (left). Overlapping histogram displays differences between with progesterone-stimulated B cells (dashed line) and untreated control (filled line). Gray area represents the FMO for CD83. Bars (top-middle) show the corresponding median fluorescence intensity (MFI) of mCD83 within CD19+ B cells (right-bottom). Bars (right) display the fold change of either mCD83 (n = 4) or iCD83 (n = 7) MFI after progesterone treatment. Graph (bottom) shows Cd83 mRNA levels of control vs progesterone-treated B cells (n = 6). Data from flow cytometry were analyzed by ANOVA and Dunnett’s Multiple Comparison test to compare treatments against control (top-middle) or unparited t-test (right). Data from qPCR were analyzed by paired t-test (n = 6). Significant differences are indicated (**P ≤ 0.01, ***P ≤ 0.001).

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    The metallopeptidase inhibitor TIMP1 increases mCD83 and sCD83 availability in vitro. (A) Splenic lymphocytes from non-pregnant CBA/J mice were cultured for 48 h with 50 or 500 ng/mL progesterone. Cells cultured in medium alone served as control. Overlapping histograms (top) display differences between progesterone-stimulated B cells (dashed line) and untreated control (filled line). Gray area represents the FMO for TIMP1. Bars show the median fluorescence intensity (MFI) of TIMP1 within CD19+ B cells (bottom). Data were analyzed by ANOVA and Dunnett’s multiple comparison test to compare treatments with controls (n = 4). Significant differences are indicated (**P ≤ 0.01). (B) Splenic lymphocytes were cultured for 48 h with LPS and for the last 5 h with PMA and ionomycin (LPI). Recombinant TIMP1 or anti-TIMP1 blocking antibody were added from the beginning of the experiment. Scatter dot plots show a representative gating strategy to CD19+ B cells and CD4+ T cells (top). Overlapping histograms display differences between TIMP1 (middle-left) or anti-TIMP1 (middle-right) expression on CD19+ B cells (dashed lines) and the respective controls (filled line). Gray curve represents the FMO for mCD83. The bottom-left graph shows the fold increase of the corresponding MFI of mCD83 on CD19+ B cells after stimulation with LPI and TIMP1 (n = 4). The bottom-right graph represents the normalized MFI of mCD83 on CD19+ B cells after stimulation with LPI and anti-TIMP1 (n = 5). Data were analyzed using paired t-test. Significant differences are indicated (*P ≤ 0.05). (C) Graph shows the amount of sCD83 in supernatants of LPI or LPI and TIMP1-treated splenocytes determined by ELISA. Data were analyzed using paired t-test (n = 5). Significant differences are indicated (*P ≤ 0.05).

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    Schematic summary of CD83 shedding in GPOM and PPOM on splenic B cells. In GPOM (A), a balanced shedding of mCD83 provides the source of sCD83 in healthy pregnancies. In contrast, an imbalanced shedding of mCD83 in PPOM (B) results in reduced sCD83 release.

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    Schematic summary of the proposed progesterone-dependent regulation of CD83 availability by TIMP1. (A) TIMP1 inhibition of a proteinase that degrades mCD83 results in a higher mCD83 expression and therefore improved availability for sCD83 release. (B) Progesterone, acting through inhibition of B cell TIMP1 expression, facilitates the degradation of mCD83, reducing mCD83 expression and sCD83 release.

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