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.

A novel Cytochrome P450 26A1 expressing NK cell subset at the mouse maternal-foetal interface.

Cyp26a1 had important roles in mouse embryo implantation and was highly expressed in some of NK cells at the human maternal-foetal interface in early pregnancy. However, the regulatory effect of Cyp26a1 on NK cells remains poorly understood. Through qPCR and flow cytometric assays, we found that Cyp26a1 was expressed by mouse uterine NK cells but not spleen NK cells during the peri-implantation period and there was a group of NK cells that highly expressed Cyp26a1, that is Cyp26a1+ NK cell subset. single cell-population transcriptome sequencing on Cyp26a1+ NK and Cyp26a1- NK cell subsets was performed. We found that there were 3957 differentially expressed genes in the Cyp26a1+ NK cell subset with a cut-off of fold change ≥2 and FDR < 0.01, 2509 genes were up-regulated and 1448 genes were down-regulated in Cyp26a1+ NK cell subset. Moreover, cytokine-cytokine receptor interaction signalling pathway and natural killer cell-mediated cytotoxicity signalling pathway were enriched according to KEGG pathway enrichment analysis. We further found that the expression of Gzma and Klrg1 was significantly increased and Fcgr4 was significantly decreased when inhibiting Cyp26a1. Our experimental results show that there is a novel NK cell subset of Cyp26a1+ NK cells in mouse uterus and Cyp26a1 can regulate the gene expression of Gzma, Klrg1 and Fcgr4 in the Cyp26a1+ NK cells.

Cytochrome P450 26A1 modulates uterine dendritic cells in mice early pregnancy.

Cytochrome P450 26A1 (CYP26A1) plays important roles in the mice peri-implantation period. Inhibiting its expression or function leads to pregnancy failure. However, little is known about the underlying mechanisms involved, especially the relationship between CYP26A1 and immune cells. In this study, using Cyp26a1-specific antisense morpholigos (Cyp26a1-MO) knockdown mice model and pCR3.1-Cyp26a1 vaccine mice model, we found that the number of uterine CD45+ CD11c+ MHCIIlo-hi F4/80- dendritic cells (DCs) was significantly decreased in the treated mice. The percentage of mature DCs (CD86hi ) was obviously lower and the percentage of immature DCs (CD86lo ) was remarkably higher in uterine DCs in the treatment group than that of the control group. Further experiments found that ID2, a transcription factor associated with DCs development, and CD86, a DC mature marker molecule, were both significantly reduced in mice uteri in the treated group. In vitro, ID2 and CD86 also decreased in bone marrow-derived DCs under Cyp26a1-MO treatment. These findings provide novel information that CYP26A1 might affect the embryo implantation via modulating the differentiation and maturation of uterine DCs.

Cytochrome P450 26A1 Modulates the Polarization of Uterine Macrophages During the Peri-Implantation Period.

Uterine M1/M2 macrophages activation states undergo dynamic changes throughout pregnancy, and inappropriate macrophages polarization can cause adverse pregnancy outcomes, especially during the peri-implantation period. Our previous studies have confirmed that Cytochrome P450 26A1 (CYP26A1) can affect embryo implantation by regulating uterine NK cells and DCs. The aim of this study was to investigate whether CYP26A1 regulates the polarization of uterine macrophages in early pregnancy. Here, we observed that Cyp26a1 was significantly upregulated in M1 as compared with M2 of uterine macrophages, Raw264.7 and iBMDM. Knockdown of CYP26A1 in mice uterine significantly decreased the number of embryo implantation sites and the proportion of CD45+F4/80+CD206 - M1-like uterine macrophages. Primary uterine macrophages treated with anti-CYP26A1 antibody expressed significantly lower levels of M1 markers Nos2, Il1b, Il6 and Tnf-a. In CYP26A1 knockout Raw264.7 cells, the protein levels of M1 markers TNF-α, IL-6 and CD86 were significantly decreased as compared with the wild type cells. Moreover, CYP26A1 deficiency decreased the ability to produce nitric oxide and increased the phagocytosis capacity of Raw264.7 cells under M1 stimulation state. The re-introduction of CYP26A1 partially reversed the polarization levels of M1 in CYP26A1 knockout Raw264.7 cells. CYP26A1 may regulate the polarization of uterine macrophages to M1 through Stap1 and Slc7a2. In summary, these results indicate that CYP26A1 plays a significant role in macrophage polarization, and knockdown of CYP26A1 can cause insufficient M1 polarization during the peri-implantation period, which has adverse effects on blastocyst implantation.

A Cu(2+) ion-F center complex view on the photoluminescence quenching and correlating ferrimagnetism in (Cu2(+)/Cu1(2+))(0.044)Zn(0.956)O electrospun nanobelts.

Unlike to the most previous reports, mixed-cation Cu(+)/Cu(2+) doping-induced novel nanoscale phenomena, including photoluminescence quenching and a correlating ferrimagnetism with Néel temperature ≈ 14 K, were found in the as-calcined (Cu2(+)/Cu1(2+))0.044Zn0.956O electrospun nanobelts (NBs). There is also high strain (up to 1.98%) and shrunk lattice distortion (ΔV/V0 ∼ 0.127%) in the (Cu2(+)/Cu1(2+))0.044Zn0.956O NBs, leading to broken lattice symmetry in conjunction with nonstoichiometry (i.e., oxygen vacancies or accurate F centers), which could be possible origins of ferrimagnetism in the Cu-doped ZnO NBs. Electron paramagnetic resonance spectra reveal that there are giant and anisotropic g factors, suggesting that there is strong anisotropic spin-orbit interaction between the Cu(2+) ion and F center (i.e., forming Cu(2+)-F(+) complexes) in the (Cu2(+)/Cu1(2+))0.044Zn0.956O NBs. The above correlation enables the potential application of tuning of the optical and ferrimagnetic properties through strain and F-center engineering.