Innate Immune Reprogramming of Macrophages in the Parous Murine Mammary Gland

Uncomplicated childbirth is associated with an increased risk of developing breast cancer (BC) five-to-ten years postpartum (postpartum breast cancer; PPBC) compared with age-matched nulliparous individuals. PPBC is also associated with a poor prognosis relative to nulliparous individuals with BC. Contrastingly, preeclampsia, an inflammatory pregnancy complication, protects against PPBC development. We hypothesize that differential parity-induced innate immune reprogramming of mammary gland macrophages may be associated with parity-associated increased PPBC risk and preeclampsia-associated protection against PPBC. Female BALB/c mice were mated with male C57Bl/6 mice to establish allogeneic pregnancies. On gestational day (GD) 10.5, dams were injected with saline (controls) or lipopolysaccharide (LPS; 20 μg/kg) to induce inflammatory pregnancy complications. Age-matched nulliparous controls also received LPS or saline. Following birth, parous mice were force-weaned on postnatal day (PD) 7 to induce synchronous involution. Macrophages were isolated from mammary glands upon euthanasia in pregnancy (GD 14.5), lactation (PD 7), involution (PD 10) and regression (PD 28), plated (4x104 cells/well) and exposed to a 23-hour secondary challenge (LPS; 100 ng/ml; Pam3Cys; 10 µg/ml). Cytokine concentrations in cell culture supernatant were assessed using a multiplex assay (Eve Technologies) and data were analyzed by two-way ANOVA with Tukey’s posthoc test. During involution, compared with supernatant from mammary gland macrophages of age-matched nulliparous saline-treated mice, supernatant from macrophages from parous saline-treated mice exhibited significantly increased interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), and significantly decreased IL-6 following secondary challenge. TNF-α, IL-6 and IL-10 in supernatant from macrophages from parous LPS-treated mice was significantly decreased compared with parous saline-treated controls. Analysis of data collected during pregnancy, lactation and regression is ongoing to temporally evaluate reprogramming. These preliminary results demonstrate differential reprogramming in mammary gland macrophages relative to parity and inflammatory pregnancy complication status and rationalize understanding contributions of completion of a prior pregnancy on PPBC risk and progression.