This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.PILOT PROJECT II: Genetic Mouse Model of PreeclampsiaPrincipal Investigator: Zhongbin Lai, M.D., Ph.D.A.
Specific Aims I n this proposal, the specific aims are designed to address the following questions:
Specific Aim #1. Will IL-10 knockout mice (IL-10 KO) exhibit compromised pregnancy outcome (fetal resorption, premature birth, preeclampsia, or intrauterine growth retardation) in response to gestational age-dependent treatment with inflammatory signals such as lipopolysaccharide (LPS), peptidoglycan, and hypoxia?Specific Aim #2. Is the maternal inflammatory programming required for preeclampsia-associated signature features of pro-inflammatory cytokine network, leukocyte invasion of the placenta, hypertension, and proteinurea? Specific Aim #3. Will administration of serum from preeclampsia patients cause placental abnormalities (apoptosis, poor trophoblast invasion of the decidua, and leukocyte invasion) and hypertension in pregnant IL-10 KO mice?B. Studies and ResultsThis report covers the period from 7/04 to 5/05. Our lab is interested in establishing a mouse model for pregnancy-associated hypertension disease preeclampsia. Our published observations point to a temporal role for IL-10 in normal and abnormal pregnancy outcomes. Below, we present our unpublished preliminary results to highlight the in vivo role of IL-10 and uterine NK cells in fetal survival. In addition, we present data to suggest that IL-10 is capable of inhibiting COX-2 expression and prostaglandin release. We also demonstrate that maternal serum from preeclamptic women causes cell death in trophoblasts.Uterine NK cells mediate inflammation-induced fetal demise in IL-10-null mice (Manuscript Suibmitted to Nature Immunology)Fetal demise is a common complication of early pregnancy, and dysregulated local immune responses may underlie its etiology. In this context, it is important to demonstrate a novel regulatory relationship between deficiency in cytokines such as IL-10, inflammation, enhanced uterine NK (uNK) cell cytotoxicity, and pregnancy loss. We show here that mating of IL-10-/- mice resulted in fetal resorption or intrauterine growth restriction (IUGR) in response to very low doses of lipopolysaccharide (LPS). Pregnancy in wild type mice was normal even at 10 fold higher LPS doses (Figure 1). Fetal resorption in IL-10-/- mice was associated with significant increase in uterine natural killer (uNK) cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells or IL-10 administration rescued pregnancy in LPS-treated IL-10-/-animals (Figure 2). Our results identify a two-hit mechanism of fetal demise or IUGR involving IL-10 deficiency and inflammation. Our results may provide insights into adverse pregnancy outcomes in humans. Evidence that maternal serum from preeclamptic women induces cell death in trophoblast cellsInsufficient uteroplacental circulation and endothelial dysfunction are the hallmark features of spontaneous abortions and preeclampsia (13, 14). It has been reported that placental degeneration due to inflammation may result in leakage of cytotoxic cytokines, fragmented nucleic acids, and angiotensinogens into the perpheral circulation. Thus, efforts should be focused on using such observations to detect cytototoxic factors in serum samples from preeclamptic and normotensive patients. If successful, such findings will be of great significance in establishing in vitro assays to predict pregnancy-associated disorders based on the factors present in the sera. To initiate these studies, we have performed apoptosis and propidium exclusion assays on the human extravillous trophoblast cell line TCL1 exposed to serum samples from normotensive pregnant and preeclamptic women.
Our aim was to show that preeclamptic serum contains soluble components that induce cell death. In order to delineate the potential cytotoxic effect of factors in the serum of pre-eclamptic women, extravillous trophoblasts (TCL-1 cell line) were treated with 20% serum samples from preeclamptic patients (n=15) as well as from women experiencing normal pregnancy (n=8). In addition, cells were grown in 10% FCS or 10% FCS + actnomycin D (20 ?g/ml) for normal growth or to induce apoptosis, respectively. Cell injury and apoptosis in these treated cells were measured using propidium iodide exclusion and caspase 3 activation assays. Representative experiments are shown in Figures 3 and 4. In Figure 3, we show that preeclamptic serum, not normal pregnancy serum, induces uptake of propidium iodide as early as two hrs of treatment. The experimental details are provided in the figure legend. This activity is heat sensitive and can be titrated down to 5% of serum (data not shown). Similarly, we find that serum from patients with preeclampsia, but not serum from women with normal pregnancy causes significant apoptosis in TCL-1 cells as measured by FACS analysis of caspase 3-positive population (Figure 4). It is noteworthy that there were interindividual differences in serum samples from preeclamptic patients, underscoring the heterogeneous nature of preeclampsia (data not shown). At this juncture, we have used only a limited number of samples. We intend to use this approach to screen a sizeable number of samples from both preeclamptic patients and normal pregnancy individuals The long term goal is to identify the cytotoxic molecule(s) in maternal serum and to evaluate preeclampsia serum for its in vivo activity in pregnant IL-10 KO mice.Future Directions 1) Our data clearly suggest that IL-10-/- mice experience pregnancy complications in response to inflammatory signals, we plan to exploit this model to study pathophysiologic parameters associated with preeclampsia. We plan to use hypoxic conditions, LPS, and poly I-C as the possible causative factors for preeclampsia. Furthermore, we will attempt to establish a link between preeclamptic symptoms and the cytotoxic activity of uterine NK cells and trophoblast invasion. 2) Our results suggest that a trophoblast cytotoxic activity is present in serum samples from preeclampsia patients. Using IL-10-/- pregnant mice, we will attempt to evaluate the in vivo effect of the serum activity.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
5P20RR018728-05
Application #
7610529
Study Section
Special Emphasis Panel (ZRR1-RI-8 (01))
Project Start
2007-07-01
Project End
2008-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
5
Fiscal Year
2007
Total Cost
$120,648
Indirect Cost
Name
Women and Infants Hospital-Rhode Island
Department
Type
DUNS #
069851913
City
Providence
State
RI
Country
United States
Zip Code
02905
Wang, Ailin; Conicella, Alexander E; Schmidt, Hermann Broder et al. (2018) A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. EMBO J 37:
Chen, Xiaodi; Hovanesian, Virginia; Naqvi, Syed et al. (2018) Systemic infusions of anti-interleukin-1? neutralizing antibodies reduce short-term brain injury after cerebral ischemia in the ovine fetus. Brain Behav Immun 67:24-35
Janke, Abigail M; Seo, Da Hee; Rahmanian, Vahid et al. (2018) Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment. Biochemistry 57:2549-2563
Lange, P T; Schorl, C; Sahoo, D et al. (2018) Liver X Receptors Suppress Activity of Cholesterol and Fatty Acid Synthesis Pathways To Oppose Gammaherpesvirus Replication. MBio 9:
Patra, Aparna; Chen, Xiaodi; Sadowska, Grazyna B et al. (2017) Neutralizing anti-interleukin-1? antibodies reduce ischemia-related interleukin-1? transport across the blood-brain barrier in fetal sheep. Neuroscience 346:113-125
Kalkunte, Satyan; Huang, Zheping; Lippe, Eliana et al. (2017) Polychlorinated biphenyls target Notch/Dll and VEGF R2 in the mouse placenta and human trophoblast cell lines for their anti-angiogenic effects. Sci Rep 7:39885
Taggart, Allison J; Lin, Chien-Ling; Shrestha, Barsha et al. (2017) Large-scale analysis of branchpoint usage across species and cell lines. Genome Res 27:639-649
Spasova, Mariya S; Chen, Xiaodi; Sadowska, Grazyna B et al. (2017) Ischemia reduces inter-alpha inhibitor proteins in the brain of the ovine fetus. Dev Neurobiol 77:726-737
Monahan, Zachary; Ryan, Veronica H; Janke, Abigail M et al. (2017) Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J 36:2951-2967
Ribeiro, Jennifer R; Gaudet, Hilary M; Khan, Mehreen et al. (2017) Human Epididymis Protein 4 Promotes Events Associated with Metastatic Ovarian Cancer via Regulation of the Extracelluar Matrix. Front Oncol 7:332

Showing the most recent 10 out of 170 publications