Impact of the adenosine receptor A2BR expressed on myeloid cells on immune regulation during pregnancy

Abstract

During pregnancy, the maternal immune system must carefully balance protection against pathogens with tolerance toward the semiallogeneic fetus. Dysfunctions of the immune system can lead to severe complications such as preeclampsia, fetal growth restriction, or pregnancy loss. Adenosine plays a role in physiological processes and plasma-level increase during pregnancy. The adenosine receptor A2B (A2BR), which is expressed on both, immune and nonimmune cells, is activated by high adenosine concentrations, achieved during pregnancy. We investigated the impact of A2BR expressed on myeloid cells on immune regulation during pregnancy using a mouse model with myeloid deficiency for A2BR. We demonstrate systemic changes in myeloid and lymphoid cell populations during pregnancy in A2BR-KO (Adora2B923^f/f-LysM^Cre) mice with increased monocytes, neutrophils, and T cells but decreased B cells as well as altered T-cell subpopulations with decreased Th1 cells and Tregs and increased Th17 cells. Lack of A2BR on myeloid cells caused an increased systemic expression of IL-6 but decreased systemic accumulation and function of MDSC and reduced numbers of uterine natural killer cells. The pregnancy outcome was only marginally affected. Our results demonstrate that A2BR on myeloid cells plays a role in immune regulation during pregnancy, but the clinical impact on pregnancy remains unclear.

Keywords: Adenosine receptor; Immune regulation; Myeloid A2BR knockout; Pregnancy.

Figure 1

Immune cell populations in spleens of Adora2B923^f/f‐LysM^Cre and wildtype mice. Wildtype (WT) and Adora2B923^f/f‐LysM^Cre (A2BR‐KO) mice were time mated and the day when a vaginal plug was detected was defined as day E0.5. Mice were euthanized at E10.5, spleens were removed, homogenized, and analyzed by flow cytometry. (A) Proportions of the different immune cell populations in spleens of pregnant WT (left diagram, n = 17) and A2BR‐KO mice (right diagram, n = 17) at mid‐pregnancy (E10.5). (B–N) Percentages of monocytes (B), macrophages (C), neutrophils (D), MDSC (E), dendritic cells (F), CD11b⁺ dendritic cells (G), CD103⁺ dendritic cells (H), T cells (I), T helper cells (J), cytotoxic T cells (K), B cells (L), NK cells (M) and innate lymphoid cells 3 (N) from all spleen leukocytes in WT (n = 17) and A2BR‐KO mice (n = 17). Each symbol represents an individual animal and the mean is indicated. Light grey bars represent WT animals and dark grey bars represent A2BR‐KO animals. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, Mann–Whitney test.

Figure 2

T‐cell subpopulations in spleens of Adora2B923^f/f‐LysM^Cre and wildtype mice. Wildtype (WT) and Adora2B923^f/f‐LysM^Cre (A2BR‐KO) mice were time mated and the day when a vaginal plug was detected was defined as day E0.5. Spleens were analyzed by flow cytometry. (A–D) Percentages of Th1 cells (A), Th2 cells (B), Th17 cells (C), and Tregs (D) from all CD4⁺ T cells, (E–J) percentages of naïve CD4⁺/CD8⁺ T cells (E, H), effector memory CD4⁺/CD8⁺ T cells (F, I) and central memory CD4⁺/CD8⁺ T cells (H, J) from all CD4⁺/CD8⁺ T cells and (K) mean fluorescence intensity of the expression of the activation marker CD44 on all T cells in WT (n = 9) and A2BR‐KO mice (n = 9). Each symbol represents an individual animal and the mean is indicated. Light blue bars represent WT animals and dark blue bars represent A2BR‐KO animals. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, Mann–Whitney test.

Figure 3

Immune cell populations in uteri of Adora2B923^f/f‐LysM^Cre and wildtype mice. Wildtype (WT) and Adora2B923^f/f‐LysM^Cre (A2BR‐KO) mice were time mated and the day when a vaginal plug was detected was defined as day E0.5. Mice were euthanized at E10.5, uteri were removed, homogenized, and analyzed by flow cytometry. (A) Proportions of the different immune cell populations in uteri of pregnant WT (left diagram, n = 17) and A2BR‐KO mice (right diagram, n = 17) at mid‐pregnancy (E10.5). (B–J) Percentages of myeloid cells (B), monocytes (C), macrophages (D), neutrophils (E), MDSC (F), dendritic cells (G), B cells (H), T cells (I), and NK cells (J) from all living leukocytes in uteri of WT (n = 17) and A2BR‐KO mice (n = 17). Each symbol represents an individual animal and the mean is indicated. Light grey bars represent WT animals and dark grey bars represent A2BR‐KO animals. ****p < 0.0001, **p < 0.01, ns = not significant, Mann–Whitney test. (K) Representative histogram plots showing the expression of the uNK cell markers CD122 and CD49b in uteri leukocytes of A2BR‐KO (blue) and WT mice (orange). (L, M) Bar graphs showing the proportion of CD122 expressing (L) and CD49b expressing NK cells from all NK cells in A2BR‐KO (dark grey) and WT mice (light grey). **p < 0.01, Mann–Whitney test.

Figure 4

The functionality of in vitro generated MDSC from Adora2B923^f/f‐LysM^Cre mice. MDSC were in vitro generated from bone marrow cells from nonpregnant wildtype (WT) and Adora2B923^f/f‐LysM^Cre (A2BR‐KO) mice and added to CFSE‐stained and anti‐CD3/CD28 stimulated CD4⁺ T cells isolated from the spleens of nonpregnant mice. (A) Representative histogram plots showing proliferation of CD4⁺ T cells without (orange) and with the addition of in vitro generated MDSC from WT (blue) and (A2BR‐KO) (red) in a 2:1 ratio (T cell:MDSC). (B) Suppressive effect of in vitro generated MDSC from WT (light grey) and A2BR‐KO mice (dark grey) on T‐cell proliferation (n = 9). The dashed line shows the proliferation of target CD4⁺ T cells without the addition of MDSC. The proliferation index was determined as the ratio of T‐cell proliferation with and without the addition of MDSC. *p < 0.05, Wilcoxon matched‐pairs signed rank test. (C–E) Intracellular staining of effector enzymes iNOS (C) and IDO (D) on in vitro generated MDSC from WT and A2BR‐KO mice. Bars represent data from 5–9 independent experiments. Mean and standard deviation are indicated, ns = not significant, Mann–Whitney test.

Figure 5

Concentrations of proinflammatory cytokines in the serum of Adora2B923^f/f‐LysM^Cre and wildtype mice. Adora2B923^f/f‐LysM^Cre (A2BR‐KO) and wildtype (WT) mice were time mated and the day when a vaginal plug was detected was defined as day E0.5. Mice were euthanized at day E10.5 and blood was collected. Blood was centrifuged, and serum was collected and analyzed for expression of IL‐6 and TNF‐α by ELISA. (A, B) Protein concentrations of IL‐6 (A) and TNF‐α (B) in serum of A2BR‐KO and WT mice at E10.5. Each symbol represents an individual animal and the mean is indicated (n = 6–8), ***p < 0.001, ns = not significant, Mann–Whitney test.

Figure 6

Pregnancy outcome in Adora2B923^f/f‐LysM^Cre and wildtype mice. Adora2B923^f/f‐LysM^Cre (A2BR‐KO) and wildtype (WT) mice were time mated and the day when a vaginal plug was detected was defined as day E0.5. Mice were euthanized at mid‐pregnancy (E10.5) or late pregnancy (E16.5) and uteri‐containing fetoplacental units were removed. Total implantation sides and resorbing units were counted and at E16.5 fetuses were weighed. (A, B) Abortion rate (percentage of resorbed fetuses per litter) of WT and A2BR‐KO mice at E10.5 (A, n = 16) and E16.5 (B, n = 8). (C) Proportion of WT and A2BR‐KO mice with and without abortions (n = 24). (D–F) Number of viable fetuses at E10.5 (D, n = 16) and E16.5 (E, n = 8) and weight of fetuses at E16.5 (F). Each symbol represents an individual animal and the mean is indicated. Light grey bars represent WT animals and dark grey bars represent A2BR‐KO animals. ns = not significant, Mann–Whitney test.