Poster BB5 - Differential Macrophage Response to Stiffness in Males and Females

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Natalie Schulz

Student
Georgia Institute of Technology
Roswell, Georgia, United States

Co-Author(s)

DA

Derek Avery

PhD Student
Virginia Commonwealth University, United States

Primary Investigator(s)

RO

Rene Olivares-Navarrete

Principal Investigator
Virginia Commonwealth University, United States

Introduction: Macrophages are innate immune cells that direct the immune response within the body by detecting a range of biological, chemical, and physical cues. Macrophages are present in all tissues, but their functional characteristics and inflammatory response vary based on tissue type and microenvironment. Macrophages are capable of changing their physiology in response to environmental cues. These changes give rise to different phenotypic populations of macrophages with distinct functions. Traditionally, macrophages have been classified as classically activated or M1 macrophages (pro-inflammatory) or alternatively activated or M2 macrophages (anti-inflammatory). The extracellular matrix (ECM) is an essential biophysical environmental property that changes from tissue to tissue, and reports have shown that macrophages are responsive to changes in both ECM composition and elastic modulus. Besides the remarkable capacity of macrophages to detect changes in their environment, some reports have shown that there are sex differences in macrophage response during immunological responses. This study aimed to investigate if there are sex differences in the macrophage response to substrate stiffness and if the reduction in the levels of steroid hormones affects this response.

Materials and

Methods: Primary macrophages were derived from bone marrow of 12-week-old male and female C57BL/6J mice. Naïve macrophages were differentiated from bone marrow cells by culturing for seven days in DMEM supplemented with M-CSF (30 ng/mL) and either 10% fetal bovine serum (FBS) or charcoal-stripped FBS (CS), which removes non-polar and lipophilic components such as steroid hormones. To assess the effects of substrate stiffness on macrophage activation, naïve male and female macrophages cultured in FBS or CS were seeded on gelatin-coated PDMS substrates (8 or 32 kPa stiffness). After 12 hours of incubation, conditioned media was removed and pro-and anti-inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α) analyzed. RNA was extracted from macrophages attached to the PDMS substrates and gene expression for pro-inflammatory (Il1b, Tnf, Nfkb1, Nos2) and anti-inflammatory (Il10, Arg1, Klf4) factors and mechanosensitive molecules (Yap1, Piezo1, Itgb1, Itgb2, Itgb3, and Itgb7) were quantified by qPCR. In a second experiment, naïve male and female macrophages cultured on FBS or CS were seeded on the same gelatin-coated PDMS substrates and incubated for 12 in their corresponding FBS or CS media. After incubation, the macrophage phenotype: pro-inflammatory macrophages (CD45+/CD11b+F4/80+/CD80+CD206-) and anti-inflammatory macrophages (CD45+/CD11b+F4/80+/CD80-/CD206+). was assessed by spectral flow cytometry. Data were first subjected to Shapiro-Wilk normality test. Results from this test indicate that the data were normally distributed. A one-factor, equal analysis of variance (ANOVA) was used to test the null hypothesis that group means were equal at a significance level of α=0.05, with post hoc Tukey’s HSD test for multiple comparisons.

Results, Conclusions, and Discussions: Naïve macrophages cultured on stiffer substrates (32 kPa) in FBS had higher expression (Il1b, Tnf, Nfkb1, Nos2) and protein levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α) in both male- and female-derived macrophages compared to macrophages cultured on softer substrates (8 kPa). However, the response to substrate stiffness was more robust in female-derived macrophages than in male-derived macrophages (Figure 1A). On the other hand, expression of anti-inflammatory genes (Il10, Arg1, Klf4) and secretion of IL-10 was reduced in macrophages cultured on the stiffer substrate (32 kPa) independent of macrophage sex when compared to the softer substrate (8 kPa) (Figure 1B). Interestingly, the reduction of IL-10 in response to substrate stiffness was more pronounced in macrophages isolated from female mice than in male-derived macrophages (Figure 1B).
Macrophages cultured in media supplemented with CS showed a similar response to the stiffer substrate. Levels of IL-1β and IL-6 were higher than on macrophages cultured on 8 kPa substrates independent of sex. Interestingly, the increased gene expression and protein levels observed in female-derived macrophages in response to substrate stiffness in the FBS group were nonexistent when macrophages were cultured on CS, suggesting a synergistic effect between mechanosensitive and possible steroid hormone signaling pathways. Additionally, levels of IL-10 and TNF-α were not affected by substrate stiffness but mainly affected by sex. We observed higher levels of TNF-α and lower levels of IL-10 in both stiffnesses compared to male-derived macrophages (Figure 1C). Our results highlight the interplay between sex and response to mechanical cues on macrophages.

Acknowledgements (Optional):