Jingfang Di

CXCL12 enhances exogenous CD4+CD25+ T cell migration and prevents embryo loss in non-obese diabetic mice

OBJECTIVE To investigate the possible role of CXCL12 in the migration of regulatory T (Treg) cells. DESIGN Animal model-based study. SETTING Academic. ANIMAL(S) Pregnant non-obese diabetic (NOD) mice were compared with non-immunodeficient mice. INTERVENTION(S) In vivo and in vitro CXCL12 induction. MAIN OUTCOME MEASURE(S) Flow cytometric analysis and Treg cell migratory assay. RESULT(S) A significantly high percentage of spontaneous embryo resorption was observed in both syngeneic and allogeneic pregnant NOD mice. The percentage of embryo loss in allogeneic pregnant NOD mice was significantly decreased by treatment with Treg cells and CXCL12 injection; however, no such effect was observed in syngeneic pregnant NOD mice. In addition, the migration of Treg cells induced by CXCL12 was confirmed by both in vitro and in vivo migratory assays. CXCR4, the specific receptor for CXCL12, was expressed more intensively on Treg cells than on non-Treg CD3(+) T cells, whereas CXCL12 was dominantly expressed in cytokeratin 7(+) trophoblast cells at an early stage of gestation, and its expression reduced gradually during pregnancy. CONCLUSION(S) The higher level of embryo loss in allogeneic pregnant NOD mice may be due to the lack of Treg cells. CXCL12 can cause CXCR4(+) Treg cells to migrate into the pregnant uterus and establish a beneficial microenvironment for the fetus.

Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy

OBJECTIVE To characterize uterine natural killer (uNK) cells in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice and investigate the potential role of these cells in pregnancy tolerance. DESIGN An animal model-based study. SETTING Academic research center in a university. ANIMAL(S) Syngeneic pregnant NOD/SCID mice were compared with non-immunodeficient BALB/c mice. INTERVENTION(S) Induction of Toll-like receptor (TLR) agonists. MAIN OUTCOME MEASURE(S) Flow cytometric analysis was performed to detect the percentage of cell subsets, and standard (51)Cr release assay was performed to determine cytotoxicity. RESULT(S) The dominant subset of uNK cells in NOD/SCID mice is DX5 (CD49b)(+), asialo ganglio-N-tetraosylceramide(+), CD25(+), CD122(+), Thy-1 (CD90)(hi), c-kit (CD117)(hi), and interleukin-10(+). In addition, the percentage of interferon-gamma(+) subset was slightly increased in response to selected TLR agonists in the NOD/SCID mice, whereas the corresponding percentage in BALB/c mice could be increased dramatically. Such an effect could be abrogated by inhibitors, including LY294002, SP600125, and PD98059. The significant increase of interferon-gamma(+) NK cell percentage in BALB/c mice was concomitant with the increase of the embryo resorption rate. In contrast, the resorption rate in NOD/SCID mice was not significantly increased upon the induction of polyinosinic polycytidylic acid or lipopolysaccharide. As expected, the NK cells from NOD/SCID mice display a detectable but lower cytotoxicity than BALB/c, as determined by standard (51)Cr release assay. In addition, the uNK cells from NOD/SCID mice also display a hyposensitivity to lipopolysaccharide-induced production of inducible nitric oxide synthase. CONCLUSION(S) A considerable percentage of immature NK cells were detected at the fetomaternal interface in NOD/SCID mice. These cells were hyposensitive to the stimulation of selected TLR agonists. Such a status seemed to be beneficial for the maintenance of pregnancy.

Effect of TLR3 and TLR7 activation in uterine NK cells from non-obese diabetic (NOD) mice.

Toll-like receptor (TLR)-TLR cross talk is thought to be important in TLR signaling. Herein, we investigated the effect of specific TLR3 and TLR7 agonists, poly (I:C) and R837, individually and in combination, on uterine immune cell function and their subsequent effects on pregnancy outcome. Allogeneic pregnancies in the non-obese diabetic (NOD) mousexC57BL/6 and wild-type BALB/cxC57BL/6 model were used. An additive increase in embryo resorption was observed after induction with both poly (I:C) and R837, and was associated with elevated numbers of both TNF-alpha- and IFN-gamma-producing CD45(+) cells in the uterus. Further examination showed that while cytokine expression was detected in both CD3(+) cells and CD49b(+) cells in BALB/c mice, NOD mouse cells behaved differently. In NOD mice, elevated cytokine expression was attributed to CD3(+) T cells, with no response detected in the CD49b(+) NK cells. The additive effect of combined agonists was partially inhibited by the Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) inhibitor SP600125 and almost completely abrogated by the extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. These results suggest that increased TLR3 and TLR7 signals are transmitted via Th1-type T cells, rather than NK cells, in NOD mice. Furthermore, the ERK MAPK pathway may be critical in TLR3 and TLR7 signaling.

FTY720‐Induced Conversion of Conventional Foxp3−CD4+ T Cells to Foxp3+ Regulatory T Cells in NOD Mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3−CD4+ T cells to Foxp3+ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3+ and CD49b+ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009.

Insufficient peroxiredoxin‐2 expression in uterine NK cells obtained from a murine model of abortion

The CBA/J × DBA/2 mouse mating combination is prone to spontaneous embryo loss, in contrast to the MHC‐identical CBA/J × BALB/c mating combination, which yields successful pregnancies. The underlying mechanisms for these observations are unclear. In this study, multi‐vision immunohistochemical staining (IHC), flow cytometry and Western blot analysis were used to detect peroxiredoxin‐2 (PRX‐2) expression in the uterine natural killer (uNK) cells from CBA/J × DBA/2 and CBA/J × BALB/c mice. In IHC analysis, co‐localization of PRX‐2 and lectin from Dolichos biflorus agglutinin (DBA‐lectin) was confirmed and the frequency of PRX‐2+DBA‐lectin+ cells was significantly lower in CBA/J × DBA/2 than CBA/J × BALB/c. In flow cytometry and Western blotting, PRX‐2 was found expressed at a significantly lower level in CBA/J × DBA/2 mice. PRX‐2 inhibition with a neutralizing antibody significantly decreased PRX‐2 expression, increased the cytotoxicity of uNK cells, and increased the percentage of embryo loss in CBA/J × DBA/2J mice. Our data suggest that PRX‐2 may be involved in the modulation of maternal–fetal tolerance and that insufficient expression of this protein may correlate with increased embryo loss in CBA/J × DBA/2J mice. J. Cell. Biochem. 112: 773–781, 2011.

Comparison of murine thymic stromal lymphopoietin- and polyinosinic polycytidylic acid-mediated placental dendritic cell activation.

We confirmed previously the existence of thymic stromal lymphopoietin (TSLP)-positive cells in murine placenta by flow cytometry. To compare the characteristics of Toll-like receptor 3 (TLR3)- and TSLP-mediated placental dendritic cell (DC) activation, pregnant BALB/c mouse mated with C57BL/6 male were used as a model of allogenic gestation. Placental CD11c(+) DCs were potently activated by the TLR3-agonist polyinosinic polycytidylic acid [poly (I:C)], subsequently causing increased expression of co-stimulatory molecules. Accordingly, increased intracellular production of interleukin (IL)-12 and interferon (IFN)-gamma, but not IL-4 or IL-10, were detected after stimulation by poly (I:C). In the case of TSLP-stimulation, although increased expression of co-stimulatory molecules was also detected, there was no substantial increase of intracellular production of IL-12, IFN-gamma, IL-4 or IL-10. In contrast, the expression of the Th2 cell-attracting chemokine, the thymus and activation-regulated chemokine (TARC) or CCL17, was significantly boosted in response to TSLP induction, whereas no significant increase of CCL17 was observed when triggering TLR3 with its specific agonist poly (I:C). The data were further supported by a CD4(+)IL-10(+) cell migratory assay. These results suggest that TSLP-TSLP receptor interaction may result in a Th2-type microenvironment at the feto-maternal interface by inducing the production of Th2 cell-attracting chemokine and modulating the immigration of Th2-type cells.

Reduced stathmin-1 expression in natural killer cells associated with spontaneous abortion.

Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to spontaneous abortion, although the reason for this is unclear. In this study, the stathmin-1 expression pattern was evaluated in uterine natural killer (uNK) cells purified from CBA/J×DBA/2J matings. Results were compared with those in a CBA/J×BALB/c control group that yields successful pregnancies. The mean ± SD percentage of stathmin-1(+) cells in the CD49b(+) uNK cell population was lower in CBA/J×DBA/2J mice (0.7% ± 0.4%) than in control CBA/J×BALB/c mice (4.9% ± 1.5%, P < 0.01) using flow cytometry, and the intracellular stathmin-1 level in uNK cells was lower in CBA/J×DBA/2J mice than in control mice using Western blot analysis. Co-localization of lectin from Dolichos biflorus agglutinin (DBA-lectin) and stathmin-1 was confirmed using multivision immunohistochemical analysis. The frequency of stathmin-1(+)DBA-lectin(+) cells was lower in CBA/J×DBA/2J mice than in CBA/J×BALB/c mice. A similar trend in the frequency of stathmin-1(+)CD56(+) cells was seen in patients with unexplained spontaneous abortion compared with normal early pregnancy. A neutralizing antibody against stathmin-1 further increased the percentage of embryo loss in CBA/J×DBA/2J matings. These results provide evidence that stathmin-1 expression in uNK cells at the maternal-fetal interface may help modulate uNK cell function and may be beneficial for a successful pregnancy.