Age-Related Changes in Female Murine Reproductive Mucosa with respect to γδ T Cell Presence.

Many studies have demonstrated a general decline and dysregulation in immune functions with age. It is not clear, however, how the aging affects the immune surveillance of the female reproductive tract (FRT) by γδ T cells, a unique population of T lymphocytes that was shown to regulate homeostasis of epithelial barriers. First, we analyzed γδ T cell presence in FRT in young (2 months) and old (18 months) wild-type (WT) C57BL/6 mice. We did not detect any changes in γδ T cell number nor distribution in the vaginas between the age groups, while in uteri, there was a twofold increase in γδ T cell number in aged mice. To check if γδ T lymphocytes regulate a metabolic and immune status of aging vaginal tissue, we compared the expression of 84 aging-associated genes in young and old WT and γδ T-cell-deficient (Tcrd -/-) mice. We discovered that only the Ltf (lactotransferrin) gene was downregulated in old Tcrd -/- mice. In both mouse strains, we found similar age-dependent changes in cytokine production upon vaginal inflammation due to Toll-like receptor 9 (TLR9) stimulation with CpG. With age in the vaginas, IL-1α and IL-17A levels increased while IL-6, IL-10, MCP-1, and IFNγ levels were diminished in response to CpG. Similar trends were observed in uteri. Interestingly, under the inflammatory state, the lack of γδ T cells in young individuals enhanced MCP-1 production in the vagina and decreased MCP-1 level in the uterus in old females. Our gene expression data point to an antimicrobial role of γδ T lymphocytes. The profile of secreted inflammatory cytokines shifted during aging toward the proinflammatory type, and γδ T cells played a modest fine-tuning role in immunoregulation in aged FRT. We believe this work expands our understanding of γδ T cell functions and the inflammaging in the murine reproductive epithelia.

Peripheral blood subpopulations of Bregs producing IL-35 in women with endometriosis.

PROBLEM: Interleukin 35 (IL-35) is involved in the pathogenesis of endometriosis by suppressing immunoreaction and promoting endometrial cell proliferation. It may also be an essential cytokine in forming the immunosuppressive functions of regulatory B lymphocytes (Bregs). The involvement of Bregs in the pathogenesis of endometriosis has not been previously investigated. In this study, we determined the frequencies of different Breg subpopulations, namely, B10, immature B-cells, and plasmablasts, and their abilities to produce IL-35 in women with endometriosis compared to healthy women. METHODS: The frequencies of different subpopulations of Bregs producing IL-35 were measured in the peripheral blood of women with endometriosis (total pool), women with deep infiltration endometriosis (DIE), women with ovarian endometriosis, and healthy women as a control by flow cytometry. RESULTS: We observed a decrease in the percentage of B10 cells and plasmablasts in women with endometriosis and an increase in the percentage of these Breg populations producing IL-35 in the same experimental group. Interestingly, we also revealed that women with DIE had increased percentages of B10 cells and plasmablasts producing IL-35. CONCLUSION: Taken together, our findings are the first to reveal the frequencies of different subpopulations of Bregs producing IL-35 in women with endometriosis. The results suggest that IL-35 expression in B lymphocytes could be used as a peripheral marker of endometriosis; however, further studies are needed.

CD91 Derived Treg Epitope Modulates Regulatory T Lymphocyte Response, Regulates Expression of Costimulatory Molecules on Antigen-Presenting Cells, and Rescues Pregnancy in Mouse Pregnancy Loss Model.

The loss of immune tolerance to fetal antigens may result in reproductive failure. The downregulated number and activity of T regulatory lymphocytes, which are critical for the establishment of immune tolerance to fetal antigens, during pregnancy may lead to miscarriage. The adoptive transfer of Tregs prevents fetal loss in abortion-prone mice. Recently, we demonstrated that the administration of tregitopes, which are short peptides found in human and mouse immunoglobulins (IgGs), decreased the incidence of abortions in female CBA/J mice mated with DBA/2J mice. Here, two non-IgG source peptides (SGS and LKD) that can potentially bind to the major histocompatibility complex II (MHC II) with high affinity and induce Treg expansion were designed in silico. The immune dysregulation-induced pregnancy failure mouse model was used to evaluate the effect of SGS and LKD on immune response and pregnancy outcome. The fetal death rate in the SGS-treated group was lower than that in the phosphate-buffered saline-treated group. SGS and LKD upregulated the splenic pool of Tregs and modulated the T-helper cell (Th1)/Th2-related cytokine response at the preimplantation stage. Additionally, SGS and LKD downregulated the expression of CD80 and MHC class II molecules in splenic CD11c+ antigen-presenting cells. Thus, SGS treatment can result in beneficial pregnancy outcomes. Additionally, SGS peptide-mediated immunomodulation can be a potential therapeutic strategy for immune dysregulation-induced pregnancy failure.

Differential Signals From TNFα-Treated and Untreated Embryos in Uterine Tissues and Splenic CD4+ T Lymphocytes During Preimplantation Pregnancy in Mice.

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT2 Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4+ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4+ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings.

The imbalance in immune tolerance may cause the variety of reproductive failures. An intravenous immunoglobulin infusion (IVIg) therapy is used to improve the live birth rate in women suffering from recurrent pregnancy loss, recurrent spontaneous abortions and recurrent implantation failures. However, the results of IVIg studies are still inconclusive as IVIg infusion in women suffering from pregnancy loss is sometimes ineffective. One of the mechanisms of action of this treatment is inhibition of B cells differentiation and expansion of Tregs and secretion of interleukin 10. It was proposed that immunomodulatory effects of IVIg may be attributed to tregitopes - self-IgG-derived epitopes present in the structure of immunoglobulins. Similarly to IVIg, tregitopes cause the expansion of Tregs and secretion of antigen-specific effector cytokine response. Here, we studied whether the administration of mouse tregitope 167 and/or 289 can prevent abortions in mouse abortion-prone mouse matings. We revealed that tregitopes reduce the foetal death rate. This may be driven by observed higher pool of peripheral Tregs, increased production of IL-10 by Tregs and Bregs and/or maintaining the tolerogenic phenotype of antigen-presenting cells. We believe that our findings may indicate a potential alternative to IVIg for therapeutic intervention in case of pregnancy failures.

Pre-Growth Culture Conditions Affect Type 1 Fimbriae-Dependent Adhesion of Salmonella.

Among various fimbrial structures used by Salmonella enterica to colonize host tissues, type 1 fimbriae (T1F) are among the most extensively studied. Although some experiments have shown the importance of T1F in the initial stages of Salmonella infection, their exact role in the infection process is not fully known. We suggested that different outcomes of T1F investigations were due to the use of different pre-infection growth conditions for the induction of the T1F. We utilized qPCR, flow cytometry, and a wide range of adhesion assays to investigate Salmonella Choleraesuis and Salmonella Typhimurium adhesion in the context of T1F expression. We demonstrated that T1F expression was highly dependent on the pre-infection growth conditions. These growth conditions yielded T1F+ and T1F- populations of Salmonella and, therefore, could be a factor influencing Salmonella-host cell interactions. We supported this conclusion by showing that increased levels of T1F expression directly correlated with higher levels of Salmonella adherence to the intestinal epithelial IPEC-J2 cell line.

Regulatory B cells with IL-35 and IL-10 expression in a normal and abortion-prone murine pregnancy model.

PROBLEM: Interleukin 35 is a relatively newly discovered cytokine that is produced by regulatory B cells (Bregs) and contributes to their suppressive function, which may contribute to fetal tolerance development and pregnancy maintenance. Therefore, the aim of this study was to determine the frequency of Bregs and expression of IL-35 and IL-10 in these cells in a normal and abortion-prone murine pregnancy model. METHODS OF STUDY: The frequency of Bregs and expression level of IL-35 and IL-10 in these cells were measured in peripheral blood, uterine draining lymph nodes, uterus, and decidua using flow cytometry. The analysis was performed on days 3 and 14 of pregnancy in normal mice (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy model. RESULTS: A decreased percentage of Breg cells expressing IL-35 on day 3 of pregnancy in the uterine draining lymph nodes and in peripheral blood in mice from the abortion group compared with the normal pregnancy group was observed. A similar decrease was also observed in the Breg cells population producing IL-10 in peripheral blood. In the uterus (3 dpc) and decidua (14 dpc), a lower percentage of CD19+ IL-35+ was also noted in the abortion-prone model. CONCLUSION: We indicated that the early stages of abortion-prone pregnancy (3 dpc) in mice were characterized by diminished frequency of B cells producing IL-35 at both local and peripheral levels. These results and the observed lower level of IL-35 in women suffering from recurrent spontaneous abortion suggest that IL-35 may be involved in the maintenance of pregnancy.

Expression of Toll-like receptors and costimulatory molecules in splenic B cells in a normal and abortion-prone murine pregnancy model.

PROBLEM: The regulatory role of B lymphocytes in the pregnancy-induced maternal immune response is not well recognized. B lymphocytes function as antigen-presenting cells (APCs) and regulate Toll-like receptors and costimulatory molecule expression in response to intrinsic and extrinsic signals. Therefore, the aim of this study was to determine the expression of TLR2, TLR4, TLR9, and MHC class II and the costimulatory molecules CD80, CD86, and CD40 in splenic B cells in a normal and abortion-prone murine pregnancy model. METHODS OF STUDY: The expression level of these molecules on female splenic B cells was investigated using real-time PCR and flow cytometry. The analysis was performed on the 3rd and 14th day of normal (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy. RESULTS: The expression of Tlr9, Cd86, and H2-Ab1 in splenic B cells on the 3rd day after mating was upregulated, whereas Tlr2 was downregulated in abortion-prone females. On day 14, we observed lower expression levels of Tlr4 and Cd80 and higher expression levels of Cd86 in CBA/J females mated with DBA/2J males. At the protein level, the differences were observed only on day 3 of pregnancy. TLR4 and CD40 molecules were upregulated in splenic B cells, while TLR9 and CD86 were downregulated in abortion-prone mice. CONCLUSION: Differential expression of TLRs and costimulatory molecules in splenic B cells in abortion-prone and normal pregnancies suggests the involvement of these cells in the regulation of the immune response at the periphery in pregnant females.

Global decrease in the expression of signalling pathways' genes in murine uterus during preimplantation pregnancy.

Early preimplantation embryo-maternal communication is crucial for the establishment and development of pregnancy. Though the involvement of several candidate genes and proteins in this complex event has been described, the hierarchy of molecular networks governing this communication remains unknown. The primary objective of this study was to determine whether the presence of embryos in the uterine lumen stimulated or inhibited gene expression in the uterine tissue on day 3.5 post coitum. To answer this question, we investigated the gene expression of dedicated signal transduction pathways in the uterus of CD-1 mice during the preimplantation stage of pregnancy and compared this expression to mice with induced pseudopregnancy. The expression levels of 84 genes assigned to nine intracellular signalling pathways were investigated by real-time PCR. The results demonstrated down-regulation of the uterine gene expression in the majority of pathways. Among target genes, 27 were significantly (p<0.05) down-regulated, and only three were significantly up-regulated. A majority of the down-regulated genes were found to be regulated by the TGFB and NFKB pathways, which suggests that the presence of the embryo selectively regulates signalling within signal transduction pathways. One of the up-regulated genes crucial for early pregnancy was Ptgs2 (p<0.05). The increased amount of both Ptgs2 gene and protein products indicates that Ptgs2 expression may be the earliest positive embryo signal for implantation and pregnancy recognition in mice. In conclusion, our results not only underline which signalling pathways are regulated in embryo-maternal communication before implantation but also support "the quiet state hypothesis" of silencing gene expression.