Grantee Research Project Results
2023 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, 2022 through July 31,2023
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.
Using a combination of our endometrium on a chip model (the EndoChip), organoid technology, human endometrial cell lines, and peripheral blood immune cells we continue to assess the role of TCDD exposure as a driver of infertility and poor pregnancy outcomes. Using these systems, alone and in combination, has allowed us to identify disease and cellular phenotypes that increase the secondary impact of TCDD exposure and further drive development of the endometriosis phenotype. For example, differential expression of CD55 by epithelia cells related to P4-resistance in endometriosis was also observed within the EndoChip following TCDD exposure. The negative impact of this toxicant was enhanced by the perfusion of immune cells into the device.
Specific Aim 2: To Examine Disease-Toxicant Interactions in EndoChips by utilizing cell-type specific biosensors combined with metabolomic analysis via IM-MS.
Numerous studies have demonstrated that innate immune cell phenotypes in women with endometriosis exhibit dysfunction, but whether these changes are due to the presence of disease or are a consequence of it is not known. Our studies in mice with an induced endometriosis-like uterine phenotype due to developmental TCDD exposure, demonstrated similar altered innate immune cell phenotypes and strongly suggest the immune cell changes precede the reproductive disease. The shift in phenotype we observed in immune cells acquired from women with endometriosis, compared to control cells, included an increased expression of pro-inflammatory cytokines, including IL-1 and IL-6, in response to the infection mimic lipopolysaccharide (LPS). Since our murine model of early life TCDD exposure demonstrated a similar alteration in functional responses we currently hypothesize that TCDD may alter the phenomenon of life-stage immune cell training, an immune system function that has largely not been studied in the context of historical environmental toxicant/infection interactions. As a concept, trained immunity has emerged over the last decade to explain the ability of several types of innate immune cells to acquire and perpetuate phenotypes based on certain past exposures. Interestingly, epithelial cells, including reproductive tract epithelial cells that are a component of mucosal immunity can also be “trained;” thus, our systems are ideal to test the impact of altered training of immune and epithelial cell training due to either disease or disease/toxicant.
Over the last funding period, we have utilized Ramen Spectroscopy (RS) to determine whether TCDD-altered cell training may impact the behavior of reproductive tract cells in our model systems. SERS was successfully able to discriminate the biochemical footprint of endometrial cells in the presence and absence of TCDD. Additionally, while we need to further analyze the results of our most recent SERS analysis, we find that the analytical spectrum following stimulation of immune cell phenotypes acquired from the bone marrow of control versus TCDD exposed mice or cells acquired from healthy versus endometriosis patients equally demonstrate altered spectra. Most notably, control mice mimic the spectra observed from a woman without endometriosis while cells from a toxicant exposed mouse exhibits a Raman spectrum similar to that of an endometriosis patient. While further processing will be necessary to identify specific amino acid or protein expression patterns, our results indicate that Raman Spectroscopy holds promise to predict alterations in immune cell function associated with the disease or toxicant exposure.
Specific Aim 3: To perform Case Studies to assess the EndoChip as an alternative to vertebrate animal testing to identify reproductive toxicants.
The overarching goal of our original grant was to determine if a pre-existing inflammatory disease would increase the health risks associated with a toxicant exposure. Our studies focused on the known endometriosis-related phenotype of reduced uterine response to P4. Since women with endometriosis are known to exhibit reduced fertility, we have now shifted some of our studies to include pregnancy-associated cells. We obtained de-identified, non-laboring human placental tissue according to our IRB approved protocol and isolated placental macrophages. We found that multiple aspects of placental macrophage response to infection were blunted by in vitro exposure to TCDD. These data support the role of environmental toxicant exposure in promoting poor pregnancy outcomes (Stephens et al, manuscript in press). We have also examined the impact of TCDD on macrophage behaviors that are known to impact pregnancy success in response to common infections, such as Group B Streptococcus (GBS). Approximately 10-40% of pregnant patients will test positive for GBS, a bacteria that can be passed to the infant and threaten health. Additional studies are underway to culture placental macrophages within our MFI-Chip model for SERS and biosensor analysis. Finally, we acquired a human trophoblast cell line in order to build a modified version of the EndoChip that mimics many aspects of P4 action during early pregnancy. We denote this model as the EndoChip-MFI-Chip (Maternal-Fetal Interface). We are also developing an organoid-based MicroFormulator MFI model and have begun to examine placental immune cell reactions to TCDD in vitro. The organoid matrix will allow us to better visualize and biochemically characterize the interactions of trophoblast cells with the maternal-derived stromal-decidual cells. This model is currently being analyzed with the same SERS and biosensor approach as noted above.
Additionally, the use of targeted biosensors (IL-1/MMP-3) can give real-time assessment of the impact of toxicants within our MicroFormulator. We are focusing on the MMP-3 and IL-1β sensors based on our findings to date as discussed above. We expect the use of SERS and biosensors allow us to monitor the impact of TCDD on endometrial maturation within the MicroFormulator. As necessary, we can use classic analytical techniques to monitor protein secretion (Prolaction TGF-β, CD55 etc) and molecular epigenetic characterization of the progesterone receptor to support the newer approaches.
EPA site visits have not occurred over the course of our studies. Nevertheless, we engaged in routine phone discussions with EPA administration and communicated our laboratory successes and any delay issues through our regular progress reports. Our financial data and use of funds was readily available. Covid-19 delays in regard to expenditure of funds was communicated directly to the EPA and noted within our progress reports.
We maintain the highest standard of research and meet or exceed all rules and regulations. There have been no occurrences of research misconduct during this or preceding reporting periods.
Future Activities:
Planned activity: We will continue to advance our studies using previously obtained primary 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 : 1 Displayed | Download in RIS Format
Other project views: | All 19 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Gnecco JS, Missmer S, Hawkins S, Osteen KG, Greaves K, Bruner-Tran KL, WERF EPHect-EM-Organoids Consortium. World Endometriosis Research Foundation (WERF) Experimental Models for Endometriosis (EPHect-EM-Organoids):Emerging organoid systems for endometriosis research. World Congress on Endometriosis, Edinburgh, Scotland. May 2023. |
R839501 (2023) |
not available |
Supplemental Keywords:
Biology, environment, contaminant, toxicRelevant 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.