Grantee Research Project Results
2022 Progress Report: Instrumenting phenotypic immunological responses to toxicants that threaten human reproduction
EPA Grant Number: R839501Title: Instrumenting phenotypic immunological responses to toxicants that threaten human reproduction
Investigators: Osteen, Kevin G , Cliffel, David , McLean, John , Bruner-Tran, Kaylon L.
Institution: Vanderbilt University Medical Center , Vanderbilt University
EPA Project Officer: Callan, Richard
Project Period: August 1, 2019 through July 31, 2022 (Extended to July 31, 2024)
Project Period Covered by this Report: August 1, 2021 through July 31,2022
Project Amount: $848,923
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Our goals are to 1) examine whether a pre-existing inflammatory condition impacts the endometrium’s acute responses to an environmental toxicant known to impact fertility and 2) develop in vitro models that are appropriate alternatives to animal studies for accurate reproductive endpoint assessment.
Progress Summary:
Specific Aim 1: To utilize the EndoChip model to examine how a pre-existing inflammatory disease affects cellular response(s) to an environmental toxicant.
Covid restrictions continue to hamper our ability to obtain primary endometrial biopsies. Although we will continue our efforts to solve this issue, we have now obtained commercially available human endometrial stromal and epithelial cell lines. These cells have been expanded in vitro using the organoid model as we previously used to expand previously obtained primary cells (as detailed in our NCE request). These cells have been cultured in our hands and verified to exhibit normal response to estradiol and progesterone. Using these cells and the primary cells we previously obtained, we are continuing our efforts to examine the impact of TCDD exposure on inducing the endometriosis phenotype as well as promoting secondary inflammatory changes. To date, our studies indicate a synergistic effect of either disease (endometriosis) or TCDD exposure and infection-mediated loss of P4 action. Following co-culture of stromal and epithelial cells within the hydrogel matrix, exposure of cells to the inflammatory cytokine induces expansion and morphological changes with the epithelial cells in conjunction with loss of stromal cell PGR expression (data not shown), mimicking the endometriosis phenotype. Ongoing studies are examining the cellular response to TCDD following the induction of the endometriosis phenotype in both stromal and epithelial cells, including assessment of culture media by raman spectroscopy as described below.
Specific Aim 2: To Examine Disease-Toxicant Interactions in EndoChips by utilizing cell-type specific biosensors combined with metabolomic analysis via IM-MS.
Our separately funded studies using a chimeric mouse model bearing human endometrial tissues revealed that the innate immune cell phenotype becomes modified by the presence of endometriosis such that disease progression is enhanced. Studies herein are examining TCDD/disease alterations in immune cells specifically focusing on innate immune cell “training” responses (a new concept in immunology) that have not yet been examined relative to either endometriosis or toxicant exposure. Immune cell over-training can occur in response to common life-stage infections and subsequently drive inflammatory diseases. In our studies, we found that immune cells acquired from women with endometriosis, compared to control cells, exhibited increased expression of IL-1 and IL-6 in response to either TCDD or the infection mimic lipopolysaccharide (LPS). In turn exposure of endometrial stromal cells to these “activated” immune cell cytokines reduced progesterone sensitivity and elevated MMP-3 secretion while decreasing prolactin secretion. Thus, both epithelial cells and immune cells can act to disrupt endometrial progesterone responses in adjacent stromal cells when exposed to TCDD, a response that is amplified by both the presence of disease and an infection mimic.
Given the complexity and expense of IM-MS, our most recent studies have begun to explore the use of Raman Spectroscopy to reveal the biochemical footprint of endometrial cells in order to identify changes associated with either toxicant exposure or disease processes. Raman spectroscopy is a label-free, non-destructive method that can be used, in vivo or ex vivo, to rapidly assess tissue specimens or biofluids. It is a favorable analytical tool for clinical translation as it allows for rapid biochemical and biomolecular assessment of a specimen with minimum to no sample preparation. We have now demonstrated that SERS was successfully able to discriminate the biochemical footprint of endometrial cells in the presence and absence of TCDD.
Specific Aim 3: To perform Case Studies to assess the EndoChip as an alternative to vertebrate animal testing to identify reproductive toxicants.
The restrictions on access to human tissue due to the Covid-19 pandemic has forced us to approach our studies from multiple, alternative angles. Since an important goal of our studies is to understand the impact of toxicant exposure and/or endometriosis on progesterone response relative to fertility, we have acquired a human trophoblast cell line in order to utilize a modified version of the EndoChip that mimics many aspects of early pregnancy. Within the EndoChip-MFI (Maternal-Fetal Interface), we have replaced the porous membrane with a biological matrix that will allow interaction with trophoblast cells to mimic implantation. This design allowed trophoblast cells to be seeded in the upper chamber while stromal and epithelial cells are grown together in the lower chamber. The center matrix enables us to better visualize and biochemically characterize the initial invasion of the trophoblasts into the endometrial culture, mimicking implantation. This model is currently in use and has been shown to be useful for additional studies of cell behavior at the maternal-fetal interface, including the use of SERS to detect metabolic changes as well as secretory products.
Future Activities:
We will continue to advance our studies using previously obtained cells and/or cell lines. We will continue to examine these cultures in the presence and absence of an inflammatory challenge. The next step will be to introduce immune cells into the cultures. We will measure established biomarkers of P4-mediated stromal-epithelial communication as well MMP3. Together these studies will allow us to further validate the EndoChip as a screening tool for identifying disease-toxicant interactions.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 19 publications | 3 publications in selected types | All 3 journal articles |
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Gnecco JS, Ding T, Smith C, Lu J, Bruner-Tran KL, Osteen KG. Hemodynamic forces enhance decidualization via endothelial-derived prostaglandin E2 and prostacyclin in a microfluidic model of the human endometrium. Human Reproduction 2019;43(4):702-714 |
R839501 (2022) R835736C003 (2018) |
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Stephens VR, Rumph JT, Ameli S, Bruner-Tran KL, Osteen KG. The potential relationship between environmental endocrine disruptor exposure and the development of endometriosis and adenomyosis. Frontiers in Physiology 2022;12:807685. |
R839501 (2022) |
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Supplemental Keywords:
Biology, environment, contaminant, toxicsRelevant Websites:
Department of Obstetrics and Gynecology Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.