Sex Differences Between Two Major Macrophage Cell Lines in 2D and 3D Cultures Reveal Divergent Functional Responses to 17β-Estradiol (E2)

Introduction: : Gonadal hormones play a critical role in shaping immune cell function, contributing to observed sex differences in disease progression, drug response, and treatment outcomes. Estrogen receptors (ERs) influence key immunological processes, including wound healing, immune activation, and tolerance. Macrophages, a key innate immune cell type, express ERs intracellularly and are implicated in sex-biased immune responses. Macrophages serve as key regulators of inflammatory processes throughout the body. Although widely used in immunology, murine macrophage cell lines RAW 264.7 (male-derived; XY karyotype) and J774A.1 (female-derived; XX karyotype) have uncharacterized responses to gonadal hormones.(1,2) Here, we investigate ER expression and functional responses to 17β-Estradiol (E2) treatment across these two cell lines. We assess proliferation, phagocytosis, extracellular matrix remodeling, and polarization capacity. We also evaluated proliferation and matrix remodeling capabilities within a 3D hydrogel platform under both sex-matched and sex-mismatched media conditions. Our findings reveal significant phenotypic and functional differences between both cell lines in 2D and a 3D environment. These results highlight the importance of considering cellular sex in the context of in vitro models and underscore the need for sex-aware approaches in immunological research and biomedical engineering.

Materials and

Methods: : RAW 264.7 (male) and J774A.1 (female) macrophage cell-lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (basal media) for 24 hours. Cells were then treated with varying concentrations of 17β-estradiol (E2) (5 nM, 25 nM, 100 nM), as well as hormone-free and basal media. The highest dose utilized is implemented in similar macrophage/hormone studies.(3,4) Post-treatment, we assessed viability, estrogen receptor (ER) expression, phenotype response, matrix-protease expression and phagocytosis functional/phenotypic responses. Viability was measured using a resazurin assay and Ki67 immunocytochemistry (ICC). ER gene expression (ESR1/ESR2) was quantified via RT-qPCR; only ESR1 was detected, and ERα protein was confirmed with ICC.
Phagocytic function was measured using pHrodo-labeled bioparticles, and matrix metalloproteinase (MMP) activity was evaluated via zymography. For phenotypic characterization, cells were polarized with IFNγ/LPS or IL-4 for 48 hours, followed by ICC for Ki67, aspect ratio analysis, and gene expression of iNOS and Arg-1.
For 3D culture, the RAW 264.7 (male) and J774A.1 (female) macrophages were encapsulated in respective poly-ethylene-glycol hydrogels containing cell-adhesive and protease-cleavable sites. Hydrogels were initially cultured in basal media for 24 hours, followed by treatment with sex-matched or sex-mismatched conditioned media for 48 hours. Sex-based conditioned media was generated in 2D hormone-free cultures. After treatment, culture supernatants were collected for MMP analysis, and hydrogels were fixed for Ki67 immunostaining.

Results, Conclusions, and Discussions:: Viability studies revealed no differences in metabolic or proliferative capabilities between the two sex-specific cell lines when their hormonal environment was altered (Figure 1.A). Additionally, both cell lines demonstrated a significant increase in proliferation markers in a hormone-stripped environment upon differentiation into an inflammatory macrophage phenotype (Figure 1.B). Both RAW 264.7 and J774A.1 cell lines had an increase in Ki67+/DAPI+ratios in a hormone-free environment compared to basal media environment, with a 5x and 4x increase respectively. An increase in proliferation marker Ki67 trends upwards for J774A.1 with increased 17β-estradiol (E2) dose, but demonstrates a bimodal effect for RAW 264.7 macrophages. Increase and prevalence of an inflammatory macrophage phenotype may allude to the importance of hormones in orchestrating inflammatory processes.
Interestingly, RAW 264.7 and J774A.1 cells displayed opposing Estrogen Receptor-1 (ESR1) gene expression responses to a high dose (100 nM) of E2. RAW 264.7 showed a significant threefold increase in ESR1 expression at a high E2 dose compared to the hormone-depleted condition. On the other hand, J774s exhibited a significant decrease in ESR1 expression at a high E2 dose compared to a hormone free environment.(Figure 1.C) Elevated ESR1 expression has been associated with increased protein expression and receptor sensitivity. In macrophages specifically, heightened ERα expression—at both the genomic and proteomic levels—has been linked to intensified inflammatory responses.(5) There were also divergent responses between the two cell lines in functional phagocytic capabilities. RAW 264.7 had marked increase in %effect phagocytosis at high 100 nM E2 dose (72%) compared to hormone-stripped environment (54%). On the other hand J774A.1 macrophages had a substantial decrease %effect phagocytosis at a high E2 dose (14%), relative to a hormone free environment (62%).(Figure 1.D)
Macrophages are key players of inflammation and tissue repair, and are central to immunological research. However, the influence of sex-based factors on macrophage behavior is often overlooked. In this study we compared male (RAW 264.7) and female (J774A.1) macrophage responses to E2 treatment, revealing significant sex-based differences in proliferation, differentiation, and function.

Acknowledgements and/or References (Optional)::

References:
(1) B. Marquez, et al., PLoS One. 2011
(2) R. Binz, et al., Int. J. Mol. Sci. 2021
(3) A. J. Murphy, et al., PLOS ONE. 2009
(4) L. Campbell et al., J. Invest. Dermatol. 2014
(5) P. Vishnyakova, et al., Biomedicines. 2021