Cell Line-Specific Estrogen Responses Uncover Functional Sex Differences in Murine Macrophages.

Background: RAW 264.7 (male-derived) and J774A.1 (female-derived), are widely used in immunology research, 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) levels, 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 production 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.