Date of Award

1-1-2022

Embargo Period

4-22-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Melissa Cunningham

Second Advisor

Beth Wolf

Third Advisor

Chenthamarakshan Vasu

Fourth Advisor

Wei Jiang

Abstract

Estrogen can be anti- or pro-inflammatory depending on milieu and has a role in increased incidence of lupus in reproductive age women versus men. Estrogen’s pleiotropic effects are in part due to Estrogen receptorα (ERα) and its variants that localize to cytoplasmic pools, nuclear regions and to the membrane. To understand the role of sex hormone receptors in immune responses, we investigated the effects of altered ERα isoform overexpression in dendritic and B cells. Toll like receptor 7 (TLR7) stimulated endpoints, often dysregulated in lupus were also investigated. Genetically modified MOER (membrane-only ERα) and NOER (nuclear-only ERα) mice were used to investigate the effects of membrane versus nuclear ERα. Transfection experiments were conducted using the Neon Electroporation system. In vitro studies utilized transfected DC2.4 cells (a murine dendritic cell line) as well as a human SLE EBV-transformed B cell line. Transfected cells were stimulated with TLR7 agonist for 1.5 hours and used either for vitality assays or RNA processed for Nanostring analysis. Bone marrow harvested from ovariectomized NOER mice cultured with GM-CSF and IL-4 for 7 days to generate BM-DCs, also treated for 1.5 hrs with TLR7 agonist and stained to observe NFκB p65 translocation. Preliminary results show that cells overexpressing ERα46 compared to ERα66 differentially express multiple genes critical in inflammation and immunity. Top genes altered in DC2.4 include IL1α and IL1β. Interestingly, ERα 46:66 co-expression was similar in DC2.4 while B cells resulted in significant downregulation in genes such as Nfatc3 and Hif1a. Nanostring analysis has started to highlight altered pathways such as NFκB and MAPK. WST-1 assays suggests that ERα66 is more proliferative. NOER BM-DC stains show little to no translocation of NFκB p65 to the nucleus before or after stimulation suggesting membrane ERα is necessary. Continued studies are needed to clarify the role of ERα isoform expression and localization in immune cell function. Results between B cells and DCs highlight the ability for ERα to act on different immune cells. These recent data share similarities in altered genes previously seen in RNAseq of BM-DCs from ERα short mice supporting a connection between ERα short and ERα46.

Rights

All rights reserved. Copyright is held by the author.

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