HIF-1α-Deficiency in Myeloid Cells Leads to a Disturbed Accumulation of Myeloid Derived Suppressor Cells (MDSC) During Pregnancy and to an Increased Abortion Rate in Mice

Abstract

Abortions are the most important reason for unintentional childlessness. During pregnancy, maternal immune cells are in close contact to cells of the semi-allogeneic fetus. Dysregulation of the maternal immune system leading to defective adaptation to pregnancy often plays a role in pathogenesis of abortions. Myeloid-derived suppressor cells (MDSC) are myeloid cells that suppress functions of other immune cells, especially T-cells, thereby negatively affecting diseases such as cancer, sepsis or trauma. They seem, however, also necessary for maintenance of maternal-fetal tolerance. Mechanisms regulating MDSC expansion and function during pregnancy are only incompletely understood. In tumor environment, hypoxia is crucial for MDSC accumulation and activation. Hypoxia is also important for early placenta and embryo development. Effects of hypoxia are mediated through hypoxia-inducible factor 1α (HIF-1α). In the present study we aimed to examine the role of HIF-1α in myeloid cells for MDSC accumulation and MDSC function during pregnancy and for pregnancy outcome. We therefore used a mouse model with targeted deletion of HIF-1α in myeloid cells (myeloid HIF-KO) and analyzed blood, spleens and uteri of pregnant mice at gestational day E 10.5 in comparison to non-pregnant animals and wildtype (WT) animals. Further we analyzed pregnancy success by determining rates of failed implantation and abortion in WT and myeloid HIF-KO animals. We found that myeloid HIF-KO in mice led to an abrogated MDSC accumulation in the pregnant uterus and to impaired suppressive activity of MDSC. While expression of chemokine receptors and integrins on MDSC was not affected by HIF-1α, myeloid HIF-KO led to increased apoptosis rates of MDSC in the uterus. Myeloid-HIF-KO resulted in increased proportions of non-pregnant animals after positive vaginal plug and increased abortion rates, suggesting that activation of HIF-1α dependent pathways in MDSC are important for maintenance of pregnancy.

Keywords: HIF; MDSC; abortion; apoptosis; pregnancy.

Figure 1

Quantification of MDSC in blood, spleens and uteri of wildtype and myeloid HIF-KO mice. Non-pregnant (np) and E10.5 pregnant (p) wildtype (WT) and myeloid HIF-KO (HIF-KO) mice were euthanized and blood, spleens and uteri were collected. Tissues were homogenized and filtered to obtain single cell suspensions. Cells were then analyzed by flow cytometry. (A–I) Scatter diagrams with bars showing percentages of total MDSC (A–C) GR-MDSC (D–F), and MO-MDSC (G-I) from total CD45⁺ leucocytes in peripheral blood (A,D,G), spleens (B,E,H) and uteri (C,E,I). Each symbol represents an individual sample and the median is indicated. n = 10–16, *p < 0.05; **p < 0.01; ns not significant; Kruskal-Wallis test and Dunn's multiple comparison test. (J,K) Representative density plots showing the gating strategy for total MDSC (CD11b⁺/Gr-1⁺), GR-MDSC (CD11b⁺/Ly6C^low/Ly6G⁺) and MO-MDSC (CD11b⁺/Ly6C^high/Ly6G⁻) in spleens (J) and uteri (K).

Figure 2

Inhibition of CD4⁺ T-cell proliferation by MDSC generated from wildtype and myeloid HIF-KO mice. Non-pregnant wildtype (WT) mice and myeloid HIF-KO mice (HIF-KO) were euthanized and bone marrow cells were collected. Cells were cultured for 4 days with G-CSF and GM-CSF. After 4 days non-adherent cells were removed and adherent MDSC were detached with Trypsin/EDTA. MDSC were then added to CD4⁺ T-cells, freshly isolated from spleens of non-pregnant wildtype mice by MACS, stained with CFSE and stimulated with anti-CD3/CD28 microbeads. After 4 days, proliferation of CD4⁺ T-cells was assessed by CFSE dye dilution. Proliferation index was determined as ratio of T-cell proliferation with and without addition of MDSC. (A) Representative histogram plots showing proliferation of CD4⁺ T-cells without (white histogram, w/o MDSC) addition of MDSC and with (gray histograms, w MDSC) addition of MDSC generated from WT mice (left side) and from HIF-KO mice (right side) in T-cell:MDSC ratios of 4:1, 2:1 and 1:1. (B) Inhibitory effect of MDSC from WT mice (white bars) and HIF-KO mice (gray bars) on proliferation of CD4⁺ T-cells. Dashed line shows proliferation of target CD4⁺ T-cells without addition of MDSC. Inhibition of T-cell proliferation by MDSC was measured at the indicated ratios by CFSE dye dilution. Bars show mean and standard deviation of 5 samples pooled from 5 independent experiments. *p < 0.05; **p < 0.01 compared with target cells alone; Wilcoxon matched-pairs signed-rank test.

Figure 3

Expression of chemokine receptors and integrins on uterine MDSC from wildtype and myeloid HIF-KO mice. (A–I) E10.5 pregnant wildtype (WT) and myeloid HIF-KO (HIF-KO) mice were euthanized and uteri were collected. Tissues were homogenized and filtered to obtain single cell suspensions. Cells were then analyzed by flow cytometry. Scatter diagrams with bars showing MFI for indicated chemokine receptor and integrin expression on CD11b⁺/Gr-1⁺ MDSC from wildtype (white bars) and myeloid HIF-KO (HIF-KO) mice. Each symbol represents an individual sample and the median is indicated. n = 5–6, *p < 0.05; **p < 0.01; ns, not significant; Mann-Whitney test.

Figure 4

Apoptosis of MDSC from spleens and uteri from wildtype and myeloid HIF-KO mice. E10.5 pregnant wildtype (WT) and myeloid HIF-KO (HIF-KO) mice were euthanized and spleens and uteri were collected. Tissues were homogenized and filtered to obtain single cell suspensions. Cells were then analyzed for apoptosis annexin V staining and flow cytometry. (A) Representative density plots from spleens (upper plots) and uteri (lower plots) of Wildtype (WT) and myeloid HIF-KO (HIF-KO) mice for GR-1 vs. annexin V. (B,C) Scatter diagrams with bars showing percentages of annexin V⁺ MDSC from total MDSC in spleens (B) and uteri (C) from wildtype (white bars) and myeloid HIF-KO (HIF-KO) mice. Each symbol represents an individual sample and the median is indicated. n = 5–6, *p < 0.05; **p < 0.01; Mann-Whitney test.

Figure 5

Pregnancy success in wildtype and myeloid HIF-KO mice. Wildtype and myeloid HIF-KO mice were term-bred and the day when a vaginal plug was detected was defined as day E0.5. At day E10.5 mice were euthanized and the abdominal cavity was opened. Uteri containing foeto-placental units were removed and inspected. Total implantation sides and resorbing units were counted. (A) Bar graph showing the percentages of non-pregnant (black part) and pregnant (gray part) animals among wildtype (WT) and myeloid HIF-KO (HIF-KO) mice after positive evaluation for vaginal plug. n = 13–20, *p < 0.05, Fishers exact test. (B) Bar graph shows the percentages of individuals without (black part) and with (gray part) at least one abortion among wildtype (WT) and myeloid HIF-KO (HIF-KO) mice. n = 12–16, *p < 0.05, Fishers exact test. (C) Representative pictures of uteri from a pregnant wildtype (WT) and a pregnant myeloid HIF-KO mouse (HIF-KO). Arrows indicate resorbing units. (D) Scatter diagram with bars showing abortion rates of pregnant wildtype (white bar) and pregnant myeloid HIF-KO mice (gray bar). Abortion rates were defined as ratio between resorbing units and total implantation sites. Each symbol represents an individual animal and the median is indicated. n = 12–16, *p < 0.05; Mann-Whitney test.