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.

CD80 and CD86 costimulatory molecules differentially regulate OT-II CD4⁺ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice.

Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs) derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c(+) dendritic cells and macrophages F4/80(+) and CD11b(+) presenting ovalbumin (OVA) were cocultured with CD4(+) T cells derived from OT-II mice's (C57BL6/J-Tg(TcraTcrb)1100Mjb/J) spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU), activation of these cells (flow cytometry), cytokine profile (ELISA), and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4(+) T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear.

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.

Antigen presenting cells costimulatory signaling during pre-implantation pregnancy.

 Success of pregnancy depends on many factors. Three phenomena inducing immune tolerance against semi-allogeneic conceptus may play a crucial role in the pre-implantation period of pregnancy: influence of sex hormones in sex cycle, presence of oocyte or embryo and the presence of semen in the female reproductive tract. On the other hand dendritic cells are the most effective antigen-presenting cells in regulation of immune phenomena and also are considered as potent participants in inducing immune tolerance in the pregnancy. They communicate with T cells in cell contact-dependent manner or via cytokines. During cell-cell contacts, costimulatory molecules play a key role and their expression is often dependent on cytokines milieu. Both costimulatory molecules and cytokines influence generation of T regulatory cells. Interactions of these molecules are closely related. In this paper we would like to pay attention to the importance of antigen presenting cells costimulatory potency in immune regulation during a pre-implantation period of pregnancy.

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.

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.

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.

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.

CD40, CD80, and CD86 costimulatory molecules are differentially expressed on murine splenic antigen-presenting cells during the pre-implantation period of pregnancy, and they modulate regulatory T-cell abundance, peripheral cytokine response, and pregnancy outcome.

PROBLEM: The object of the study was to investigate the costimulatory phenotype of spleen antigen-presenting cells (APCs) in mice after mating and the influence of costimulatory signal blocking on pregnancy outcome, cytokine production, and Treg cell concentration. METHOD OF STUDY: The levels of CD40, CD80, and CD86 on spleen APCs at day 0.5 and 3.5 after mating (C57BL/6Jx DBA/2J and C57BL/6JxBalb/c) were assessed by flow cytometry and RT-PCR. Blocking antibodies against costimulatory molecules were given i.p. at day 3.5 after mating. Pregnancy outcomes, blood cytokine (ELISA), and spleen Treg (flow cytometry) concentrations were examined at day 10.5 after mating. RESULTS: Differential expression of costimulatory molecules on spleen APCs of mated v. pseudopregnant mice was observed mainly at day 3.5 after conception. Administration of anti-CD86 antibody lowered pregnancy outcome. Cytokine expression was modulated after administration of anti-CD86, anti-CD80 and anti-CD40 antibodies, whereas only anti-CD40 antibody changed the concentration of Treg lymphocytes and the level of Foxp3 protein expression. CONCLUSION: Costimulatory phenotype of female spleen APCs is distinctly modulated after mating. Alteration of costimulatory signal derived from APCs during pre-implantation period of pregnancy may have an adverse effect on pregnancy outcome and the tolerogenic immune response.

Influence of bacteriophage preparations on migration of HL-60 leukemia cells in vitro.

BACKGROUND: Bacteriophage therapy is considered one of the most attractive alternatives to antibiotic treatment, which may be significant due to the rising number of antibiotic-resistant bacterial strains. Patients with cancer frequently suffer bacterial infections resulting from immunosuppression caused by anticancer treatment; thus they constitute a considerable group of patients subjected to phage therapy. In this study, we investigated the influence of bacteriophages on the migration of human leukemia (HL-60) cells. Results of these studies provide data regarding phage treatment of patients with cancer, especially with this type of leukemia. MATERIALS AND METHODS: The influence of phage preparation on migration of HL-60 leukemia cells was evaluated with BD Bioscience Migration Chambers. RESULTS: Bacteriophages have no influence on migration of HL-60 cells. The only phage preparation which stimulated migration of HL-60 cells was Staph.liz, specific to S. aureus, however, the molecular basis of these interactions cannot be currently explained. CONCLUSION: Results of our studies may be in line with previous data indicating that phage therapy is safe for patients with cancer.

Influence of bacteriophage preparations on intracellular killing of bacteria by human phagocytes in vitro.

Bacteriophages are viruses that infect bacteria. It was shown that bacteriophage therapy is an effective method of combating bacterial infections, including infections caused by antibiotic-resistant bacterial strains. One of the main obstacles to widespread use of phage preparations is limited knowledge regarding the influence of bacteriophages on human organisms. In our study, we evaluated whether application of phage preparations impair bactericidal activities of human phagocytes (granulocytes and monocytes). In our study, we used preparations of phages T2 and T4 specific to Escherichia coli and A3 phage specific to Staphylococcus aureus. We found that bacteriophage preparations do not influence intracellular killing of bacteria by human phagocytes. The effect is irrespective of phage preparation type (lysate, purified phage preparation), phage titer of the preparation, and whether bacteria phagocytosed by phagocyte cells are sensitive or insensitive to phage (bacteriophages homologous and heterologous to bacteria). Although the results of our study are preliminary, they support previous data indicating safety of therapeutic application of phages.

Effects of tamoxifen on estrogen receptor-α level in immune cells and humoral specific response after immunization of C3H/He male mice with syngeneic testicular germ cells (TGC).

Estrogens and estrogen receptors (ERs) are potent regulators of the immune response. Disruption of ERα or modulation of its function by selective ligands during experimental autoimmune conditions changes the course of disease by influencing specific humoral and cellular responses. However, it is not known whether fluctuation in the ERα level and the variable accessibility to its ligands in immune cells influence the development of specific immune responses against auto-antigens. This study was designed to evaluate the expression level of ERα in splenic immune cells and the specific humoral immune response in male C3H/He/W mice immunized with syngeneic testicular germ cells (TGC) in the presence of tamoxifen. Levels of ERα protein in immune cell subpopulations of immunized mice (assessed by flow cytometry) increased in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and CD3(+)CD4(+) T cells. Addition of tamoxifen decreased the level of ERα in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and the CD19(+)CD3(- ) cell subpopulation of immunized mice. Therefore, immunization with syngeneic antigen and tamoxifen treatment evoked cell-type specific changes in the level of ERα. Irrespective of tamoxifen treatment the humoral response in immunized animals toward TGCs was similar, suggesting that modulation of the level of ERα in immune cells is not directly related to specific auto-antibody production.