Elsebeth Staun-Ram

Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion

BackgroundThe aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line.MethodsFirst trimester trophoblasts were divided into two groups: 6–8 weeks (early) and 9–12 w (late) of gestation. The two trophoblast groups and JAR cells were cultured in medium, with various concentrations of forskolin and Epidermal Growth Factor (EGF). Proteolytic activity was detected by zymography and invasiveness was assessed by Matrigel invasion assay. Students T-test was used for statistical analysis.ResultsIn 6–8 w trophoblast, proMMP-2 was only slightly dominant over proMMP-9 (53.2% vs. 46.8% respectively), whereas in 9–12 w, proMMP-9 was clearly dominant over proMMP-2 (61.7% vs.38.3% respectively). In JAR cells proMMP-2 was strongly dominant (90.2% vs.9.8% respectively). In JAR cells forskolin significantly increased proMMP-2 and -9 secretion (128.5% +/- 12 and 183.2% +/- 27.9 of control, respectively). EGF had a dual effect on JAR cells: at 8 ng/ml both proMMP-2 and -9 were increased (133.5% +/-15 and 223.9% +/- 32.4 of control, respectively) while at 80 ng/ml both proMMP-2 and -9 were decreased (65.1% +/- 18.3 and 66.6% +/- 37 of control, respectively). Forskolin significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts (125.9% +/- 6.3,128.4% +/- 6.4; 169.7% +/- 20.3, 120.3% +/- 4.5 of control, respectively). EGF also significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts (141.22% +/- 14.8, 138.8% +/- 10.3; 168.3% +/- 18.2, 117.3 +/- 3.8 of control, respectively). Both forskolin and EGF increased trophoblast cells invasiveness in all groups. The invasive ability of trophoblast cells, induced by forskolin, was reduced by MMP-2 antibody in: JAR cells, 6–8 w and 9–12 w trophoblasts. Likewise trophoblast invasion induced by EGF was reduced by MMP-2 antibody in all groups. However the invasive ability induced by forskolin or EGF was inhibited by MMP-9 antibody only in trophoblasts from 9–12 w.ConclusionsFirst trimester trophoblasts express differential gelatinase secretion profile according to the gestational week. In JAR and early trophoblasts (6–8 w) MMP-2 is the main gelatinase and the key enzyme in trophoblast invasion. Thereafter in late first trimester trophoblasts (9–12 w), both MMP-2 and -9 participate in trophoblast invasion.

Human trophoblast function during the implantation process

The implantation process involves complex and synchronized molecular and cellular events between the uterus and the implanting embryo. These events are regulated by paracrine and autocrine factors. Trophoblast invasion and migration through the uterine wall is mediated by molecular and cellular interactions, controlled by the trophoblast and the maternal microenvironment. This review is focused on the molecular constituents of the human trophoblast, their actions and interactions, including interrelations with the uterine endometrium.

Laquinimod modulates B cells and their regulatory effects on T cells in multiple sclerosis.

Laquinimod is an orally administered drug under development for the treatment of Multiple Sclerosis (MS), lacking a fully elucidated mode of action. We assessed the immunomodulatory effects of laquinimod in vitro on human B cells from healthy or MS patients, cultured alone or with CD4(+) T cells. Laquinimod modulated B cell markers, mainly by increasing the regulatory ones CD25, IL10 and CD86, and decreased IL4, while increasing IL10 and TGFβ in both B and T cells, in a B cell-mediated manner. These findings shed additional light on the mechanisms underlying the effects of laquinimod in MS and potentially other immune-mediated diseases.

p53 Mediates Epidermal Growth Factor (EGF) Induction of MMP-2 Transcription and Trophoblast Invasion

Trophoblast invasion is a highly restricted process, regulated by growth factors, hormones and cytokines. Trophoblast invasion is attainable due to proteolytic degradation of the epithelial basement membrane and the extracellular matrix by proteolytic enzymes like the matrix metalloproteinases (MMPs) particularly MMP-2. Epidermal growth factor (EGF), a major mediator of implantation, has been documented to induce MMP-2 and trophoblast invasion. The aim of this study was to investigate the transcriptional regulation of MMP-2 in EGF- stimulated invasive trophoblast cells, using JAR choriocarcinoma cell line and 6-8w 1st trimester trophoblasts. MMP-2 expression was induced by EGF within 1 hour. Gelshift and supershift assay were used to explore transcription factors involved in MMP-2 regulation. EGF induced binding activity and expression of phophorylated p53, AP-2alpha and -gamma, C/EBPepsilon and -lambda to their responding sequences, found in the MMP-2 promoter. Additionally EGF induced binding activity to SP-1, but reduced the expression of SP-1 and SP-4. Inhibition of p53 by antisense reduced both basic and EGF- induced MMP-2 expression. In summary: MMP-2 transcriptional regulation by EGF in invasive trophoblasts is mediated through several binding sites and transcription factors including p53, AP-2alpha and -gamma, C/EBPepsilon and -lambda and SP-1. p53 mediates both basic and EGF-induced MMP-2 transcription.

Cathepsins (S and B) and their inhibitor Cystatin C in immune cells: Modulation by interferon-β and role played in cell migration

We investigated the modulation of Cathepsin S, Cathepsin B and Cystatin C expression in immune cells by interferon (IFN)-β, and their role in cell migration. Cathepsin levels were increased in monocytic and T line cells upon activation. IFN-β abolished this increase of Cathepsin B in monocytes and of Cathepsin S in T cells, while increased Cystatin C. Neutralizing Cystatin C increased cell migration, while migration of monocytes and T cells was inhibited by IFN-β, by Cystatin C, and T cell migration was suppressed by Cathepsin S and B inhibitors. These findings support further studies in the importance of Cathepsins and Cystatins in immunomodulation.

Interferon-beta induces distinct gene expression response patterns in human monocytes versus T cells.

Background Monocytes, which are key players in innate immunity, are outnumbered by neutrophils and lymphocytes among peripheral white blood cells. The cytokine interferon-β (IFN-β) is widely used as an immunomodulatory drug for multiple sclerosis and its functional pathways in peripheral blood mononuclear cells (PBMCs) have been previously described. The aim of the present study was to identify novel, cell-specific IFN-β functions and pathways in tumor necrosis factor (TNF)-α-activated monocytes that may have been missed in studies using PBMCs. Methodology/Principal Findings Whole genome gene expression profiles of human monocytes and T cells were compared following in vitro priming to TNF-α and overnight exposure to IFN-β. Statistical analyses of the gene expression data revealed a cell-type-specific change of 699 transcripts, 667 monocyte-specific transcripts, 21 T cell-specific transcripts and 11 transcripts with either a difference in the response direction or a difference in the magnitude of response. RT-PCR revealed a set of differentially expressed genes (DEGs), exhibiting responses to IFN-β that are modulated by TNF-α in monocytes, such as RIPK2 and CD83, but not in T cells or PBMCs. Known IFN-β promoter response elements, such as ISRE, were enriched in T cell DEGs but not in monocyte DEGs. The overall directionality of the gene expression regulation by IFN-β was different in T cells and monocytes, with up-regulation more prevalent in T cells, and a similar extent of up and down-regulation recorded in monocytes. Conclusions By focusing on the response of distinct cell types and by evaluating the combined effects of two cytokines with pro and anti-inflammatory activities, we were able to present two new findings First, new IFN-β response pathways and genes, some of which were monocytes specific; second, a cell-specific modulation of the IFN-β response transcriptome by TNF-α.

The influence of vitamin D supplementation on melatonin status in patients with multiple sclerosis

BACKGROUND Multiple sclerosis (MS) incidence is higher in geographic regions with less sunlight exposure. Both vitamin D and melatonin are essential mediators of the effect of sunlight in health, and as such are candidates to play a key role in MS. We hypothesized that vitamin D and melatonin may have related influences in patients with MS. METHODS In a randomized, double blind study of 40 IFN-β treated MS patients, 21 patients were assigned to 800 IU of vitamin D3 per day (low dose), while 19 patients received 4,370 IU vitamin D3 per day (high dose) for one year. Serum 25-hydroxy-vitamin-D (25-OH-D) and nighttime urine melatonin metabolite, 6-sulphatoxy-melatonin (6-SMT), were measured at baseline, 3 months and 1 year from enrolment. RESULTS After 3 months supplementation, 25-OH-D levels increased and nighttime melatonin secretion decreased significantly in the high dose group, but not in the low dose group. After 1 year, a decrease in 25-OH-D levels, accompanied by an increase of urine nighttime 6-SMT were observed in the high dose group. Percent change in serum 25-OH-D was significantly and negatively correlated with percent change in urine 6-SMT after 3 months and between 3 months to 1 year. 25-OH-D levels by the end of the study were significantly and negatively correlated to BMI. CONCLUSIONS Melatonin secretion is negatively correlated with alterations in serum 25-OH-D in IFN-β treated patients with MS. The finding suggests that melatonin should be considered as a potential mediator of vitamin D neuro-immunomodulatory effects in patients with MS.

Ets-2 and p53 mediate cAMP-induced MMP-2 expression, activity and trophoblast invasion

BackgroundWe have previously shown that Matrix metalloproteinase (MMP) -2 is a key-enzyme in early trophoblast invasion and that Protein Kinase A (PKA) increases MMP-2 expression and trophoblast invasion. The aim of this study was to examine MMP -2 regulation by PKA in invasive trophoblasts: JAR choriocarcinoma cell-line and 6-8 w first trimester trophoblasts.MethodsThe effect of Forskolin (PKA) on MMP-2 expression was assessed by Northern Blot and RT-PCR. Possible transcription factors binding to consensus MMP-2 promoter sequences in response to Forskolin, were detected by EMSA binding assay and their expression assessed by western blot analysis. Antisense transfection of relevant transcription factors was performed and the inhibitory effect assessed on MMP-2 expression (RT-PCR), secretion (zymography) and trophoblast invasiveness (transwell migration assay).ResultsWe found that Forskolin increased MMP-2 mRNA in JAR cells within 24 hours, and induced binding to p53, Ets, C/EBP and AP-2. Transcription factors Ets-2, phospho- p53, C/EBP epsilon, C/EBP lambda and AP-2 alpha bound to their respective binding sequences in response to Forskolin and the expressions of these transcription factors were all elevated in Forskolin- treated cells. Inhibition of Ets-2 and p53 reduced MMP-2 expression, secretion and invasiveness of Forskolin treated cells.ConclusionMMP-2 is regulated by PKA through several binding sites and transcription factors including Ets-2, p53, C/EBP, C/EBP lambda and AP-2 alpha. Ets-2 and p53 mediate cAMP- induced trophoblast invasiveness, through regulation of MMP-2.

Integrative analysis of methylome and transcriptome in human blood identifies extensive sex- and immune cell-specific differentially methylated regions

The relationship between DNA methylation and gene expression is complex and elusive. To further elucidate these relations, we performed an integrative analysis of the methylome and transcriptome of 4 circulating immune cell subsets (B cells, monocytes, CD4+, and CD8+ T cells) from healthy females. Additionally, in light of the known sex bias in the prevalence of several immune-mediated diseases, the female datasets were compared with similar public available male data sets. Immune cell-specific differentially methylated regions (DMRs) were found to be highly similar between sexes, with an average correlation coefficient of 0.82; however, numerous sex-specific DMRs, shared by the cell subsets, were identified, mainly on autosomal chromosomes. This provides a list of highly interesting candidate genes to be studied in disorders with sexual dimorphism, such as autoimmune diseases. Immune cell-specific DMRs were mainly located in the gene body and intergenic region, distant from CpG islands but overlapping with enhancer elements, indicating that distal regulatory elements are important in immune cell specificity. In contrast, sex-specific DMRs were overrepresented in CpG islands, suggesting that the epigenetic regulatory mechanisms of sex and immune cell specificity may differ. Both positive and, more frequently, negative correlations between subset-specific expression and methylation were observed, and cell-specific DMRs of both interactions were associated with similar biological pathways, while sex-specific DMRs were linked to networks of early development or estrogen receptor and immune-related molecules. Our findings of immune cell- and sex-specific methylome and transcriptome profiles provide novel insight on their complex regulatory interactions and may particularly contribute to research of immune-mediated diseases.

Effector and regulatory B cells in Multiple Sclerosis

The role of B cells in the pathogenesis of Multiple Sclerosis (MS), an autoimmune neurodegenerative disease, is becoming eminent in recent years, but the specific contribution of the distinct B cell subsets remains to be elucidated. Several B cell subsets have shown regulatory, anti-inflammatory capacities in response to stimuli in vitro, as well as in the animal model of MS: Experimental Autoimmune Encephalomyelitis (EAE). However, the functional role of the B regulatory cells (Bregs) in vivo and specifically in the human disease is yet to be clarified. In the present review, we have summarized the updated information on the roles of effector and regulatory B cells in MS and the immune-modulatory effects of MS therapeutic agents on their phenotype and function.