Pregnancy status alters IL-21-mediated effects on murine B lymphocytes

in Reproduction

Correspondence should be addressed to D O Muzzio; Email: damian.muzzio@med.uni-greifswald.de
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A favorable outcome of pregnancy depends greatly on an adequate balance of immune protection and fetal tolerance at the fetomaternal interface. IL-21 is a pro-inflammatory cytokine associated with altering immune responses in autoimmune diseases. IL-21 has pleiotropic functions, including induction of Th17 T cells, inhibition of Treg development, and modulation of antibody responses of B lymphocytes. Genetic polymorphisms of IL21 have been associated to poor pregnancy outcomes. However, the mechanism of IL-21 actions needs further evaluation. Here, we postulate that IL-21 affects splenic B cell function during pregnancy and shapes immune responses. We show that splenic B cells from CBA/J × BALB/c mice with favorable pregnancy outcome expressed lower IL21R levels than in CBA/J × DBA/2J mice, a mouse model for immune-induced bad pregnancy outcome. As a consequence, B cells from CBA/J × BALB/c mice reacted less sensitively to IL-21 than B cells from non-pregnant mice (NPM) or from CBA/J × DBA/2J mice. Also, LPS-induced apoptotic rates were altered in NPM and CBA/J × DBA/2J but not in CBA/J × BALB/c mice. This is accompanied by improved survival of B cells that produce the anti-inflammatory cytokine IL-10 upon stimulation with LPS. We also observed lower numbers of CD4+CXCR5+Bcl-6+ follicular T-helper cells (Tfh) in normal pregnant mice, compared to non-pregnant and mice with disturbed pregnancies. Our data indicate that alterations of the Tfh/IL-21/IL-10 axis may have important influence on pregnancy outcome.

Supplementary Materials

    • Supplementary Figure 1 Gating strategies and raw data from Figure 1. (A) Representative plots showing gating strategies to gate dead cells. (B) Bars display percentages of dead cells. (C) Histograms display propidium iodide representative plots and gating strategies for apoptotic cells (left gate) and cells in the cell cycle phase G2/S+M (right gate). (D) Bars show percentage of apoptotic cells (top) or cells in phase G2/S+M (bottom). (E) Histograms showing CFSE staining as gated on live cells. Black lines represent IL-21 treated cells and grey filled histograms represent controls. Dotted line represents the staining at t = 0. (F) Bars display CFSE fluorescence intensity. White, light gray and dark gray bars represent non-pregnant (n = 5), BALB/c paired (n = 5) and DBA/2J paired (n = 9) CBA/J mice, respectively. All experiments were performed in duplicate. Data was statistically analyzed by paired Student’s t-test and displayed as mean ± SEM. Significant differences are indicated (*p ≤ 0.05, ***p ≤ 0.001).
    • Supplementary Figure 2 IL-21 treatment alters antigen presentation capacity of splenic B cells depending on the type of stimulation. The expression of CD69 (A), MHCII (C), CD80 (E) and CD86 (G) on live CD19+ B cells was assessed after treatment with LPS (left), anti-CD40 (middle) or anti-IgM (right) antibodies in the presence or absence of IL-21. Histograms (A, C, E and G) display fluorescence intensities of each marker with (gray filled histogram) or without (black line) the presence of IL-21 in the culture media. Dotted lines represent unstained controls. Bars (B, D, F and H) display percentage of the fluorescence intensity of each marker normalized to respective controls. White, light gray and dark gray bars represent non-pregnant (n = 5), BALB/c paired (n = 5) and DBA/2J paired CBA/J mice, respectively (n = 9). Data was statistically analyzed by paired Student’s t-test and displayed as mean ± SEM. All experiments were performed in duplicate. Significant differences are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
    • Supplementary Figure 3 Raw data from Figure 1. Immunoglobulin and IL-10 production of magnetically isolated splenic CD19+ B cells after treatment with LPS and the presence or absence of IL-21. Bars display immunoglobulin levels on supernatants of stimulated B cells. White, light gray and dark gray bars represent non-pregnant (n = 5), BALB/c paired (n = 5) and DBA/2J paired (n = 9) CBA/J mice, respectively. Data was statistically analyzed by paired Student’s t-test and displayed as mean ± SEM. All experiments were performed in duplicate. Significant differences are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
    • Supplementary Figure 4 Gating strategies for Figure 3. (A) Gating strategy for assessment of IL-21R in follicular zone (FO), marginal zone (MZ), transitional (TN) and total B cells (top) within splenic lymphocytes of CBA/J female mice. Overlapping histograms (bottom) represent IL-21R expression on each corresponding subset. (B) Gating strategy for analysis of CD4+CXCR5+Bcl-6+ follicular T helper cells (Tfh) within splenic lymphocytes of CBA/J female mice. (C) Gating strategy for analysis of peripheral blood CD4+ICOS+CXCR5+PD-1+ Tfh within CD3+ lymphocytes from non-pregnant women (NP), women on the first (1st) or third (3rd) trimester of pregnancy.

 

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