Erika Moore

Ancestry-associated immune differences influence fibrosis risk, however how fibrosis-associated pathways vary across individuals remains poorly understood. Fibroblasts are a main cell type involved in fibrosis. The fibroblast response is shaped by cytokine signaling and macrophage activation. The extent to which these pathways vary across individuals, and how ancestry-associated immune differences influence fibrosis risk, remains poorly understood. Here, a poly(ethylene glycol) (PEG)-based hydrogel microphysiological system was leveraged to model fibroblast-macrophage interactions following oxidative stress and to integrate donor-specific immune signals using matched macrophages and serum. Individuals of self-reported African ancestry exhibited higher monocyte expression of CCL4, lower monocyte expression of OXER1, and increased serum IL-10, compared to individuals of European ancestry. Within the hydrogel, oxidative stress reduced fibroblast prevalence while inducing Ki67 and p16. Exogenous TGF-β1 increased fibroblast prevalence and collagen 3 production but did not independently increase α-SMA. Incorporating donor-specific macrophages and serum revealed that cultures from individuals of European ancestry demonstrated higher fibroblast α-SMA and p16 expression. Pharmacologic inhibition of IL-10 further increased α-SMA expression, particularly in African ancestry-derived cultures, identifying IL-10 as a key protective signal limiting fibroblast activation. This hydrogel system provides a platform for dissecting inter-individual immune variation and identifying mechanisms underlying ancestry-associated fibrosis risk.

Ancestry-associated immune differences influence fibrosis risk, however, how fibrosis-associated pathways vary across individuals remains poorly understood. Fibroblasts are a main cell type involved in fibrosis. The fibroblast response is shaped by cytokine signaling and macrophage activation. The extent to which these pathways vary across individuals, and how ancestry-associated immune differences influence fibrosis risk, remains poorly understood. Here, a poly(ethylene glycol) (PEG)-based hydrogel microphysiological system was leveraged to model fibroblast-macrophage interactions following oxidative stress and to integrate donor-specific immune signals using matched macrophages and serum. Individuals of self-reported African ancestry exhibited higher monocyte expression of CCL4, lower monocyte expression of OXER1, and increased serum IL-10, compared to individuals of European ancestry. Within the hydrogel, oxidative stress reduced fibroblast prevalence while inducing Ki67 and p16. Exogenous TGF-beta increased fibroblast prevalence and collagen 3 production but did not independently increase alpha-SMA. Incorporating donor-specific macrophages and serum revealed that cultures from individuals of European ancestry demonstrated higher fibroblast alpha-SMA and p16 expression. Pharmacologic inhibition of IL-10 further increased alpha-SMA, particularly in African ancestry-derived cultures, identifying IL-10 as a key protective signal limiting fibroblast activation. This hydrogel system provides a platform for dissecting inter-individual immune variation and identifying mechanisms underlying ancestry-associated fibrosis risk.

Background: RAW 264.7 (male-derived) and J774A.1 (female-derived) cell lines are widely used in immunology research and are considered preferred models for studying signaling pathways, yet their responses to gonadal hormones remain poorly understood. Gonadal hormones, particularly estrogen, shape immune cell function and contribute to sex differences in disease outcomes, with macrophages playing a central role through their expression of intracellular estrogen receptors (ERs). Herein, we investigated ER expression and functional responses to 17β-estradiol (E2) in male-derived RAW 264.7 and female-derived J774A.1 macrophages, in 2D culture. Additionally, we looked at sex-matched and mismatched media conditions in a 3D hydrogel system. Our results reveal distinct phenotypic and functional differences between the cell lines, emphasizing the need for sex-aware approaches in immunological research and model design. Methods: RAW 264.7 and J774A.1 macrophages were cultured in basal media for 24 hours, then treated with varying concentrations of 17β-estradiol (5, 25, 100 nM), as well as hormone-free and control media. Post-treatment analyses included viability, estrogen receptor expression, phenotype skewing, matrix metalloprotease 9 (MMP9) activity, and phagocytosis. These macrophages were also used to condition sex-specific media environments and were encapsulated in a hydrogel network containing adhesive and cleavable sites. Encapsulated cells were then exposed to sex-matched or sex-mismatched conditioned media, and proliferation and MMP9 activity were assessed. Results: Our results revealed distinct differences in estrogen receptor gene and protein expression, as well as in core macrophage functions such as proliferation, inflammation, matrix remodeling, and phenotype skewing. Additionally, the sex-derivation of the surrounding molecular environment affected macrophage behavior in a 3D hydrogel system. Female-derived macrophages were more sensitive in terms of proliferation to sex-mismatched environments, while male-derived macrophages exhibited altered enzyme activity when exposed to female-conditioned media. Conclusions: These findings underscore the importance of accounting for both the origin of immune cells as well as the hormonal and environmental context in which they are studied. Without these considerations, experimental models risk missing critical biological differences that shape immune responses and disease outcomes.

Introduction: Ancestry reporting is essential to ensure transparency and proper representation in biomedical studies. However, manually extracting this information from study texts is time-consuming and inefficient. In this paper, we present TRACE (Tool for Researching Ancestry and Cell Extraction), powered by GPT-4 and web-crawling, to automate ancestry identification by detecting cell lines or cultures in texts and tracing their ancestry.Methods: TRACE extracts cell lines and primary cultures from research articles and follows web sources to determine their ancestry. We compared TRACE's outputs to a manually generated database to confirm its performance in identifying and verifying ancestry information.Results: The results reveal an overrepresentation of European/White samples and significant underreporting. TRACE enables large-scale, systematic ancestry analysis—a valuable resource for researchers and agencies assessing biases in sample selection.Conclusions: As an open-source tool, TRACE it facilitates broader use to evaluate and improve ancestry representation in biomedical research.

Uterine fibroids are the most common gynecological tumors, characterized by excessive production of extracellular matrix. Despite their prevalence, the cellular mechanisms governing fibroid growth remain poorly understood. Current in vitro models for fibroids do not replicate the complex 3D tissue mechanics, structure, and extracellular matrix components of fibroids, which may limit our understanding of fibroid pathogenesis. To address this gap, we aimed to develop a 3D in vitro model to mimic aspects of the fibroid microenvironment. By encapsulating human uterine fibroblasts in poly(ethylene glycol) (PEG)-based hydrogels comprising collagen- and fibronectin-derived peptides, this model allows for incorporation of fibroid cellular components, extracellular matrix components, and fibroid or myometrial tissue stiffness. Due to its mechanistic role in fibroblast activation and subsequent extracellular matrix production seen in fibroids, we treated uterine fibroblasts with transforming growth factor beta 3 (TGF-β3) to demonstrate quantification of fibrotic markers observed in fibroids. Here, we establish that human uterine fibroblasts increase α smooth muscle actin, extracellular matrix proteins, and cell elongation, as well as high metabolic activity and matrix remodeling in PEG-based hydrogels in response to TGF-β3. This research represents a physiologically relevant in vitro platform to investigate uterine fibroblast function within a 3D environment that mimics uterine fibroids, with the potential to advance our understanding of the cellular and molecular mechanisms driving fibroid growth and development.

Fibrosis is a dysregulated wound healing response characterized by excessive accumulation of dense scar tissue that inhibits organ function and is estimated to contribute to up to 45% of deaths in the industrialized world. In this work, we sought to uncover new ways to address fibrosis by drawing inspiration from an animal that does not develop fibrosis. The Spiny Mouse (Acomys) has the most extensive regenerative capabilities of any known mammal and can regenerate injuries to the skin, kidney, heart, skeletal muscle, and spine with little to no fibrosis. We hypothesize that the regenerative abilities of Acomys are due, in part, to altered stiffness-mediated fibroblast-to-myofibroblast transition (FMT). In this work, we interrogated stiffness-mediated FMT in Acomys and Mus dermal fibroblasts in vitro by performing RNA Sequencing and found no differential gene expression in Acomys fibroblasts cultured on soft vs stiff substrates. We further investigated the direct impact of stiffness-mediated FMT and species differences on ECM deposition by fabricating cell-derived matrices (CDMs) from Acomys and Mus fibroblasts cultured on varying stiffnesses. After assessing the composition of these CDMs using label-free quantitative proteomics, fibrosis-associated extracellular matrix proteins including fibrillin-1, ADAMTS1, SPARC, and galectin-1 were found to be significantly reduced or absent in Acomys CDMs compared to Mus CDMs. In addition, proteins that have been connected to fibrosis resolution, including Col12a1 and clusterin, were upregulated in Acomys CDMs. When cultured on Acomys CDMs, mouse macrophages downregulated MMP9 mRNA expression and maintained increased expression of iNOS in response to IL-4, a pro-fibrotic cytokine. These results indicate a direct impact of species-specific ECM compositions on macrophage phenotype and suggest that ECM produced by Acomys fibroblasts may impede the development of a pro-fibrotic macrophage phenotype in the presence of pro-fibrotic stimuli.