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[Preprint]. 2025 Jun 30:rs.3.rs-6925474.
doi: 10.21203/rs.3.rs-6925474/v1.

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

Alison M Veintimilla  1 , Zoe Turner  1 , Nana Owusu-Boaitey  1 , Varun Deshpande  1 , Margaret McCarthy  2 , Erika Moore  1
Affiliations

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

Alison M Veintimilla et al. Res Sq. .

Update in

Abstract

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.

Keywords: estrogen; gonadal hormones; immunology; macrophage function.

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Conflict of interest statement

Competing interests: The authors have no relevant financial or non-financial interest to disclose.

Figures

Figure 1
Figure 1. There are no major differences in viability after E2 dosing for RAW 264.7 (male-dervived) and J774A.1 (female-derived) macrophage cell lines.
(A) Ki67 to DAPI ratios for RAW 264.7 (left) and J774A.1 (right) based on immunocytochemistry. (B) Resazurin metabolic assay relative fluorescent units (RFU) results for RAW 264.7 (left) and J774A.1 (right) cells. For figure, BM refers to basal media conditions, HF refers to hormone-free conditions and 5, 25,100, refers to E2 treatment in nM. One-way ANOVA and post hoc Tukey statistical analysis conducted.
Figure 2
Figure 2. Differential ER expression at high E2 dose for RAW 264.7 vs J774A.1.
Gene expression of Esr1at high E2 (100 nM) dosage for RAW 264.7 vs J774A.1. J774A.1 cells exhibit significantly higher expression of ERα compared to RAW 264.7 cells under high-dose E2 treatment, as well as relative to other hormonal treatment conditions. (A) Esr1 RT-qPCR quantification for RAW 264.7 (left) and J774A.1 (right) in response to varying E2 dosages with basal media group as calibrator. (B) ERα to DAPI ratios based on immunocytochemistry (ICC). Double slashes (//) in the figure refers to a value jump in the y-axis. (C) Representative images of ICC staining for ERα (green), and cell-nuclear marker, DAPI (blue) on RAWs 264.7 (top row) and J774A.1s (bottom row). Scale bar at 250 μm For all – BM is referring to basal media conditions, HF is referring to hormone-free conditions and 5, 25,100, refers to E2 treatment in nM. One-way ANOVA and post hoc Tukey statistical analysis conducted (*p<.05,**p<.01,***p<.001, ****p<.0001).
Figure 3
Figure 3. High E2 has divergent effects on RAW 264.7 vs J774A.1s’ functions.
(A) RFU values of pHrodro bioparticle phagocytosis assay for RAW 264.7 (left) and J774A.1 (right). (B) Zymmography analysis for ECM remodeling enzyme MMP9 for RAW 264.7 (left) and J774A.1 (right). The area under the curve (AUC) ratios were done based on an isolated MMP9 control. For figure, BM refers to basal media conditions, HF refers to hormone-free conditions and 5, 25,100, refers to E2 treatment in nM. One-way ANOVA and post hoc Tukey statistical analysis conducted (*p<.05,**p<.01).
Figure 4
Figure 4. E2 enhances elongation and proliferation in +LPS+IFNγ activated macrophages.
RAW 264.7 macrophages pre-treated with increasing concentrations of E2 exhibit a more elongated morphology following +LPS+IFNγ activation. Both RAW 264.7 and J774A.1 macrophages show increased proliferation in response to E2 pre-treatment under inflammatory conditions. Aspect ratio (AR) morphology analysis for RAW 264.7 (A) and J774A.1 (B). Ki67 and DAPI ratios based on immunocytochemistry for RAW 264.7 (C) and J774A.1 (D) cells. For figure, +LPS+IFNγ refers to an inflammatorily skewed macrophage post E2 treatment. +IL-4 refers to a pro-regenerative skewed macrophage post E2 treatment. BM refers to basal media conditions, HF refers to hormone-free conditions and 5, 25,100, refers to E2 treatment in nM. One-way ANOVA and post hoc Tukey statistical analysis conducted (*p<.05,**p<.01,***p<.001).
Figure 5
Figure 5. RAW 264.7 and J774A.1 macrophages differ in gene expression of canonical phenotypic markers and production of phenotypic enzymatic proteins.
(A-B) RT-qPCR quantification of phenotypic markers (iNOS, Arg-1) for RAW 264.7 (left/top) and J774A.1 (right/bottom) post phenotypic stimulation (+LPS+IFNγ or +IL-4 media treatment) in response to varying E2 pre-treatment. (C) Detectable quantification of iNOS production in +LPS+IFNγ RAW 264.7 macrophages. (D) MMP9 Expression via zymography for +IL-4 RAW 264.7 (top) and +IL-4 J774A.1 (bottom) macrophages in response to E2 pre-treatment. For figure, BM refers to basal media conditions, HF refers to hormone-free conditions and 5, 25,100, refers to E2 treatment in nM. One-way ANOVA and post hoc Tukey statistical analysis conducted (*p<.05,**p<.01,***p<.001).
Figure 6
Figure 6. Sex-mismatched secretomic environments differentially influence macrophage behavior in 3D culture.
Exposure to a sex-mismatched secretome is associated with a trend toward increased proliferation in RAW 264.7 macrophages, while showing a negative trend in proliferation for J774A.1 cells. Additionally, sex-mismatched conditions elevate MMP9 expression in RAW 264.7 macrophages. (A) Ki67+ over DAPI+ ratios based on immunocytochemistry imaging for RAW 264.7 (striped) and J774A.1 (dotted) in sex-matched and sex mis-matched environments. (B) MMP9 expression via zymography for RAW 264.7 (striped) and J774A.1 (dotted) in sex-matched and sex mis-matched environments. For figure – BM refers to basal media, RM (green) refers to RAW 264.7 conditioned media, JM (pink) refers to J774A.1 conditioned media. One-way ANOVA and post hoc Tukey statistical analysis conducted (*p<.05,**p<.01,***p<.001).
Figure 7
Figure 7
Summary of findings in investigation of sex-based factors influencing macrophage modulation.
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