Chemokine C-X-C receptor 4 mediates recruitment of bone marrow-derived nonhematopoietic and immune cells to the pregnant uterus†

Abstract

Bone marrow-derived progenitor cells (BMDPCs) are mobilized to the circulation in pregnancy and get recruited to the pregnant decidua where they contribute functionally to decidualization and successful implantation. However, the molecular mechanisms underlying BMDPCs recruitment to the decidua are unknown. CXCL12 ligand and its CXCR4 receptor play crucial roles in the mobilization and homing of stem/progenitor cells to various tissues. To investigate the role of CXCL12-CXCR4 axis in BMDPCs recruitment to decidua, we created transgenic GFP mice harboring CXCR4 gene susceptible to tamoxifen-inducible Cre-mediated ablation. These mice served as BM donors into wild-type C57BL/6 J female recipients using a 5-fluorouracil-based nongonadotoxic submyeloablation to achieve BM-specific CXCR4 knockout (CXCR4KO). Successful CXCR4 ablation was confirmed by RT-PCR and in vitro cell migration assays. Flow cytometry and immunohistochemistry showed a significant increase in GFP+ BM-derived cells (BMDCs) in the implantation site as compared to the nonpregnant uterus of control (2.7-fold) and CXCR4KO (1.8-fold) mice. This increase was uterus-specific and was not observed in other organs. This pregnancy-induced increase occurred in both hematopoietic (CD45+) and nonhematopoietic (CD45-) uterine BMDCs in control mice. In contrast, in CXCR4KO mice there was no increase in nonhematopoietic BMDCs in the pregnant uterus. Moreover, decidual recruitment of myeloid cells but not NK cells was diminished by BM CXCR4 deletion. Immunofluorescence showed the presence of nonhematopoietic GFP+ cells that were negative for CD45 (panleukocyte) and DBA (NK) markers in control but not CXCR4KO decidua. In conclusion, we report that CXCR4 expression in nonhematopoietic BMDPCs is essential for their recruitment to the pregnant decidua.

Keywords: CXCR4; bone marrow; implantation; pregnancy; stem cells; uterus.

Graphical Abstract

Figure 1

(A) A schematic describing the Cre-Lox system utilized in our model. In the CAG-Cre-Esr1 mice, the Cre recombinase is fused with a mutated LBD of the ER, which has very high affinity to the synthetic ER ligand tamoxifen while lacking sensitivity to endogenous estrogen. Upon tamoxifen administration, Cre recombinase translocates to the nucleus where it excises exon 2 of the CXCR4 gene flanked by the LoxP sites. (B) A schematic of the experimental model. WT C57BL/6 J recipient female mice received nongonadotoxic submyeloablative BMT regimen using 5-FU injections on day −6 and day −1 before BMT and SCF injections at −21 and −9 h prior to and +3 h after the second 5FU dose. Cre⁺CXCR4^flox/flox/GFP⁺ (CXCR4KO) or Cre⁻CXCR4^flox/flox/GFP⁺ (WT) mice served as BM donors. BM engraftment was assayed by flow cytometry of peripheral blood on day 21 post-BMT. Subsequently, engrafted female mice received intraperitoneal injection of Tamoxifen at 75 mg/kg body weight for five consecutive days. BMT recipients were mated with fertile males on day 14 post-tamoxifen injections and timed pregnancies were established. Pregnant mice were sacrificed on E9.5 or as nonpregnant (controls). Blood, BM, spleen, and uterus were collected from pregnant and nonpregnant controls and analyzed using multicolor flow cytometry, IHC, and IF. (C and D) Genotyping PCR results for detection of (C) Cre + and (D) CXCR4^f/f and CXCR4 WT genes. (E) Genotyping PCR results of Cre + CXCR4^f/f and Cre-CXCR4^f/f mice after tamoxifen injection demonstrating the recombined CXCR4 deleted gene construct in Cre + CXCR4f/f mouse but not in Cre-CXCR4f/f control. (F and G) Relative CXCR4 mRNA expression levels normalized to GAPDH in control mice and CXCR4KO mice in (F) peripheral blood and (G) bone marrow cells. n = 8 mice/group. (H) In vitro transwell migration assay of BM cells showing representative images of BM cells from control mice and CXCR4KO mice migrated towards CXCL12 ligand at various concentrations (0, 30, or 100 ng/mL). (I) Quantitative summary of transwell migration assay showing the chemotactic index of BM cells of control mice and CXCR4KO mice towards CXCL12 ligand at various concentrations. In vitro data in H-I are representative of three independent experiments. Data in bar graphs are shown as mean ± SEM. ^*P < 0.01, ^**P < 0.001.

Figure 2

(A) Multicolor flow cytometry analysis of cultured BM cells from CXCR4KO and control mice. Live cells were gated on Sca-1+ and CD45– to exclude hematopoietic cells, and were further gated on CD29+ and CD44+ to identify BM MSCs. Representative flow cytometry plots of cultured BM cells are shown. (B) Quantitative summary of percentage of CD29+/CD44+/Sca1+/CD45– BM MSCs in CXCR4KO and control mice. Data shown in (A) and (B) are the mean of n = 4 mice/group. (C) CFU assay showing representative images of colony development in cultured BM cells of CXCR4KO and control mice. (D) Quantitative summary of CFU numbers per well in CXCR4KO and control mice. ^*P < 0.01, n = 4 mice/group. (E) Quantitative summary of mRNA expression of Cxcl12, Nanog, Oct4, Sox2, and Hoxa11 in cultured MSCs of CXCR4KO and control mice. CFU assays and quantitative real-time polymerase chain reaction assays were performed in duplicate and data are the averages of two independent experiments. Data in bar graphs are shown as mean ± SEM.

Figure 3

(A) Flow cytometry analysis showing GFP+ cells in bone marrow, peripheral blood, spleen, nonpregnant uterus, and decidua (E9.5) of CXCR4KO and WT control mice. Live cells were gated on GFP to calculate the percentage of BMDCs (GFP+). Representative flow cytometry plots are shown. (B and C) Quantitative summaries of percentage of GFP+ cells present in bone marrow, spleen, blood, and uterus in (B) WT control and (C) CXCR4KO mice in both pregnant (E9.5) and nonpregnant samples. (D) Quantitative summary of the ratio of GFP+ cell frequency in bone marrow, spleen, and uterus relative to peripheral blood in control and CXCR4KO mice in both pregnant (E9.5) and nonpregnant samples. n = 6 mice/group, ^*P < 0.05, ^**P < 0.01. Data in bar graphs are shown as mean ± SEM.

Figure 4

Immunohistochemistry images of uterine tissue sections from CXCR4KO and control mice. (A) Immunostaining images of nonpregnant uterine tissue sections of control and CXCR4KO stained with GFP antibody (brown). The bottom panel (20×) is a magnification of the dashed area in the upper panel (4×). Scale bar, 200 μm. (B) Immunostaining images of implantation site (E8.5) sections of control and CXCR4KO stained with GFP antibody (brown). The bottom panel (10x) is a magnification of the dashed area in the upper panel (2.5×). Note the concentration of GFP+ BMDCs in the mesometrial (M) area compared to the scant presence in the antimesometrial (AM) area. L, lumen; GC, giant cell layer; E, embryo. Scale bar, 400 μm.

Figure 5

(A) Flow cytometry analysis of nonpregnant uterus and decidua (E9.5) from control and CXCR4KO mice. Live cells were gated on GFP and CD45 to identify GFP⁺CD45⁺ hematopoietic BMDCs and GFP⁺CD45⁻ nonhematopoietic BMDCs. Representative flow cytometry plots are shown. (B and C) Quantitative summary of percentage of GFP⁺CD45⁺ hematopoietic BMDCs and (C) GFP⁺ CD45^– nonhematopoietic BMDCs in nonpregnant uterus and implantation site (E9.5) of (B) control and (C) CXCR4KO mice. n = 6 mice/group, ^*P < 0.05, ^**P < 0.01. Data in bar graphs are shown as mean ± SEM.

Figure 6

(A) Immunofluorescence of E9.5 decidua tissue sections (mesometrial) showing colocalization of CD45 pan-leukocyte marker (white), GFP marker of transplanted BMDCs (green), and DBA marker of NK cells (red) in control and CXCR4KO mice. Sections were counterstained with DAPI for nuclear staining (blue). White arrows point to nonhematopoietic GFP+ BMDCs that are negative for either CD45 or DBA markers. Yellow arrows point to GFP + CD45 + DBA+ BM-derived NK cells. Purple arrows point to GFP + CD45 + DBA– BM-derived non-NK immune cells. Note that nonhematopoietic GFP+ BMDCs (GFP + CD4 – DBA–) are found in control mice (white arrows) but not in CXCR4KO mice, where all GFP+ BMDCs are hematopoietic (CD45+ and/or DBA+). Low magnification picture scale bar, 200 μm. High magnification picture scale bar, 50 μm. (B) Quantitative summary of percentage of DBA+ (NK), DBA– CD45+ (immune non-NK) and DBA-CD45- (nonhematopoietic) BMDCs out of total GFP+ cells in the implantation site (E9.5) of control and CXCR4KO mice. n = 4 mice/group, ^**P < 0.01. Data in bar graphs are shown as mean ± SEM.

Figure 7

Multicolor flow cytometry analysis of GFP+ BM-derived hematopoietic cells in nonpregnant and pregnant (E9.5) mice tissues. (A) Gating strategy for flow cytometry analysis of GFP+ cells in implantation site (E9.5) of control and CXCR4KO mice. Live cells were gated on CD45+ and GFP+ to analyze the hematopoietic BMDC population. NK cells were positive for Nk1.1 (NK) but negative for CD3 (T cell) markers, while T cells were CD3+ NK1.1–. Cells were further gated as NK1.1–CD3– to analyze myeloid cells. The markers CD11b, Ly6G, and F4/80 were used to identify granulocytes (Ly6G+ CD11b+ F4/80–), macrophages (F4/80+ CD11b+ Ly6G–), and monocytes (CD11b+ F4/80– Ly6G–). Representative flow cytometry plots of single cells from E9.5 decidual tissue are shown. (B–E) Quantitative summary of the distribution of various immune cell subsets (neutrophils, macrophages, monocytes, T cells, and NK cells) in GFP⁺ CD45⁺ hematopoietic BMDCs of control and CXCR4KO mice in (B) bone marrow, (C) spleen, (D) implantation site, and (E) peripheral blood. (F) Quantitative summary of ratio of immune cell frequency (NK cells, T cells, monocytes, and granulocytes) in tissue (decidua, BM, or spleen) relative to peripheral blood in control and CXCR4KO mice. Data shown are the mean of n = 6 mice/group; ^*P < 0.05, ^**P < 0.01. Data in bar graphs are shown as mean ± SEM.