Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single-cell RNA sequencing (scRNA-seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue-resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function-related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA-seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal-foetal interface by regulating the activity of NK cells.

Cytochrome P450 26A1 modulates natural killer cells in mouse early pregnancy.

Cytochrome P450 26A1 (CYP26A1) has a spatiotemporal expression pattern in the uterus, with a significant increase in mRNA and protein levels during peri-implantation. Inhibiting the function or expression of CYP26A1 can cause pregnancy failure, suggesting an important regulatory role of CYP26A1 in the maintenance of pregnancy. However, little is known about the exact mechanism involved. In this study, using a pCR3.1-cyp26a1 plasmid immunization mouse model and a Cyp26a1-MO (Cyp26a1-specific antisense oligos) knockdown mouse model, we report that the number of Dolichos biflorus agglutinin (DBA) lectin-positive uterine natural killer (uNK) cells was reduced in pCR3.1-cyp26a1 plasmid immunized and Cyp26a1-MO-treated mice. In contrast, the percentage of CD3- CD49b+ NK cells in the uteri from the treatment group was significantly higher than that of the control group in both models. Similarly, significantly up-regulated expression of CD49b (a pan-NK cell marker), interferon gamma, CCL2, CCR2 (CCL2 receptor) and CCL3 were detected in the uteri of pCR3.1-cyp26a1- and Cyp26a1-MO-treated mice. Transcriptome analysis suggested that CYP26A1 might regulate NK cells through chemokines. In conclusion, the present data suggest that silencing CYP26A1 expression/function can decrease the number of uNK cells and significantly increase the percentage of CD3- CD49b+ NK cells in the uteri of pregnant mice. These findings provide a new line of evidence correlating the deleterious effects of blocking CYP26A1 in pregnancy with the aberrant regulation of NK cells in the uterus.

The Balance between Conventional DCs and Plasmacytoid DCs Is Pivotal for Immunological Tolerance during Pregnancy in the Mouse.

Dendritic cells (DCs), which can shape their functions depending on the microenvironment, are crucial for the delicate balance of immunity and tolerance during pregnancy. However, the mechanism underlying the microenvironment-educated plasticity of DC differentiation during pregnancy remains largely unclear. Here, we found that the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) is regulated in a tissue-specific manner during pregnancy. The ratio of cDCs and pDCs remained constant in the spleen. However, the ratio changed in the para-aortic lymph nodes (LNs), where cDC percentages were significantly reduced concurrent with an increase in pDCs from E8.5 to E16.5. Moreover, the expansion of pDCs and T regulatory (Treg) cells was correlated in the para-aortic LNs, and pDCs had more potential to induce regulatory T cells (Tregs) compared with cDCs (independent of IDO expression). Notably, the balance between cDCs and pDCs is disrupted in IFN-γ-induced abnormal pregnancy, accompanied by lower Treg percentages in the para-aortic LNs and decidua. To further identify the underlying mechanism, we found that elevated IFN-γ can increase the levels of GM-CSF to alter the differentiation of pDCs into cDCs in vivo. Therefore, we provide a novel regulatory mechanism underlying pregnancy-related immune tolerance that involves the balance of DC subsets, which may offer a new target for the prevention of human spontaneous abortion.

Seminal plasma induces inflammation in the uterus through the γδ T/IL-17 pathway.

After insemination, a large number of leukocytes migrate into the uterus, which is accompanied by intense inflammation. However, the details of how seminal plasma interacts with the uterus are still not very clear. Here, we present that neutrophils migrate and accumulate around the uterine epithelium following insemination, which is accompanied by an increase in interleukin (IL) 17A levels. Additionally, we find that γδ T cells are the major source of IL-17A, and the seminal plasma could induce the γδ T cells to secret IL-17A. Blocking IL-17A could reduce the number of neutrophils in the uterus and prevent them from migrating to the epithelium by decreasing the chemokines CXCL1, CXCL2 and CXCL5. Blocking IL-17A did not affect the Th1/Th2 balance but actually diminished the inflammation in the uterus by reducing the expression of IL-1ß and TNF-α. In summary, we found a new mechanism by which seminal plasma could influence the inflammation in the uterus through the γδ T/IL-17 pathway to regulate the expression of various chemokines and cytokines.

Insulin-like growth factor binding protein 7 modulates estrogen-induced trophoblast proliferation and invasion in HTR-8 and JEG-3 cells.

Previous research has reported that IGFBP7 functions as a tumor suppressor gene in different tumors, but its role in the trophoblast has not been elucidated. In this research, we studied the regulation mechanism of IGFBP7 in trophoblast proliferation and invasion in HTR-8 and JEG-3 cell lines. We found that IGFBP7 was abundantly expressed in normal human syncytiotrophoblast tissue samples but that this was lacking in hydatidiform moles. The proliferation and invasion capacities of HTR-8 and JEG-3 cells were significantly inhibited by recombinant IGFBP7. Estrogen (E2) stimulated the expression of IGFBP7 at a concentration of 5-10 ng/mL. This stimulation was inhibited by the estrogen receptor antagonist Fulvestrant (ICI182.780) and a TGFß-neutralizing antibody. In conclusion, our data reveals that estrogen stimulates the expression of IGFBP7 through estrogen receptors and TGFß. The expression of IGFBP7 could be stimulated by TGFß in a dose-dependent manner and inhibited by IFNγ in HTR-8 and JEG-3 cells. IGFBP7 could also inhibit the phosphorylation of ERK and the expression of PCNA, MMP2 and MMP9 in HTR-8 and JEG-3 cells. These findings suggest that IGFBP7 is a key regulator of E2-induced trophoblast proliferation and invasion.

Evidence for the inhibition of fertilization in vitro by anti-ZP3 antisera derived from DNA vaccine.

Previously we have found that DNA vaccine, pCMV4-rZPC' can generate specific antibodies against rabbit ZPC (amino acid 263-415, rZPC'), which binds to ovarian ZP and leads to a significant reduction of fertility in vivo. The purpose of this study was to evaluate the effect of antisera from pCMV4-rZPC(')-immunized mice on sperm-oocyte interaction in vitro. The effect of antisera from DNA vaccine-immunized mice on fertilization and early embryonic development was studied using an in vitro fertilization system. The results showed that the antisera supplemented in fertilization medium (10%, v/v) significantly decreased the rate of fertilization compared to that of control groups (P<0.05); whereas the antisera showed no significant effect on the rate of fertilization when ZP-free eggs were used. Moreover, the antisera pre-neutralized with mouse soluble zona pellucida lost the capacity to inhibit fertilization when compared with that of control groups. In addition, the antisera showed no detrimental effect on early developmental potential of mouse embryos in vitro. Taken together, our study provided herein direct evidence showing that antisera generated by DNA vaccine can block sperm-egg recognition during fertilization via targeting the oocyte ZP proteins.