Assistant Professor University of Maryland, United States
Introduction: Disproportionate disease presentation amongst different races and sexes manifests clinically in heart diseases, cancers, and autoimmune disorders. For example, women make up 80% of autoimmune diagnosis.(1)The cause of these health inequities is not well understood. It is theorized that different environmental exposures and sociocultural trauma may be linked to biological modifications.(2) This may influence sensitivities to immunomodulatory molecules, such as the female sex hormone, estrogen. Estrogens are one of the only female sex hormones that have a direct communication on certain innate immune cell activity. Current models of disease do not consider estrogenic effects on immune cell activity.
Macrophages are pivotal constituents of the innate immune system. Their primary role is phagocytosis of pathogens and cellular debris with subsequent immune system priming. Estrogenic effects are associated with macrophage specialization and immune activation. However, it is not well understood how estrogen impacts macrophages amongst different race/ethnic demographics. Here we investigate how race/ethnicity and sex differences influence immune cell function in response to an estrogen receptor alpha/beta agonist (ER⍺/ERβ), 17-β estradiol (E2). We hypothesize that Black/African American-derived (AA) primary macrophages will be more sensitive to varying E2 treatment compared to European American/White (EA) counterparts. To assess this hypothesis, we will investigate alterations in chemokine/cytokine expression, phagocytosis capability and protease production with E2 treatment.
(1) C. J. Mulligan, American Journal of Physical Anthropology. 175, 399–405 (2021). (2) F. Angum, T. Khan, J. Kaler, etc., Cureus. 12, e8094 (202
Materials and
Methods: We defined two major racial populations in the United States as Black/African American and White/European American in our cohort. Monocytes derived from male and female donor’s peripheral blood mononuclear cells were differentiated into macrophages using M-CSF and subsequently cultured in hormone-free media. Proteomic, phenotypic, and functional assays were then performed to define E2 modulation of donor differentiated macrophages. We implemented a human cytokine and chemokine multiplex array panel (Invitrogen™, Cat#: LHC0009M) to assess 25 proteins secreted by donor cells in response to E2 stimulation. A phagocytosis bead (Vybrant™, Cat#: V6694) and zymography assay were conducted post E2 stimulation of donor groups to determine macrophage phagocytic efficacy and protease production, respectively. Phagocytosis assay was optimized for the lab and E2 dosage outcomes were validated relative to prior literature.[Figure 1B] Immunocytochemistry was used to visualize and quantify ER⍺ and ERβ expression to define ER⍺/ERβ ratios. General summary of workflow is visualized in Figure 1A.
Results, Conclusions, and Discussions: We began investigating immune sensitivities to E2 in different racial groups. Macrophages from AA female donors exhibited greater E2 modulation and baseline expression of certain chemokines/cytokines. Chemokines/cytokines such as IL-1Ra [Figure 1C], IL-7 and MCP1 were present and modulated by E2 amongst AA donors but not EA donors. Inflammation-associated chemokines such as IL-8 and MIP1⍺/β were modulated by E2 for both AA and EA donors. One of the AA donors demonstrated a significant modulatory response to IL-8 between 25 nM (~4119 pg/ml) and 100 nM (~19104 pg/ml) E2 treatment.[Figure 1D] With this preliminary work, we will expand our studies to further investigate IL-1Ra and IL-8 because of their associations with autoimmunity and inflammation. Overall, E2 had bimodal effects in increasing or decreasing chemokine/cytokine production dependent on E2 concentration (high vs low) and given donor. For example, all EA donors experienced IL-8 increase at 25 nM E2 treatment, whereas 2/3 of AA donors experienced decreased IL-8 production at 25 nM treatment. There has been a call to consider the ancestry of human-derived primary cells and cell lines within bioengineering platforms.(2) Most primary samples and cell-lines used in benchtop regenerative engineering studies are of White/European ancestry.(4) Along with race/ethnicity, biological sex is another historically overlooked variable in scientific research. The consideration of sex-based variables in pre-clinical studies has only been mandated by regulatory institutions as a biological variable within the last decade. This historical exclusion has led to detrimental outcomes for women, which are exemplified in the development of drugs such as thalidomide and Ambien. The influence of social ancestry and sex differences on disease outcomes and therapeutic potential has not been thoroughly explored. Considering these gaps in knowledge, we set out to investigate the intersectional impact of race/ethnicity and sex on innate immune cells to build bioengineering platforms that more readily capture the heterogeneity of human disease and biology. (2) E. Moore, J. B. Allen, C. J. Mulligan, et al., Nat Rev Mater. 7, 2–4 (2022). (4) H. Ryan, D. Bl, Regen Eng Transl Med. 8, 499-503 (2022)
