Department of Chemistry, University of Massachusetts Amherst, Amherst, MA, USA.
Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, MA, USA.
Integr Biol (Camb). 2022 Jun 8;14(3):62-75. doi: 10.1093/intbio/zyac007.
Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.
巨噬细胞是白细胞，在维持体内平衡和免疫反应中发挥不同作用。它们可以根据环境将表型重新编程为促炎（M1）或抗炎（M2）状态。巨噬细胞转录组的约 8-15%具有昼夜节律波动，包括与它们的功能密切相关的基因。由于昼夜节律与细胞表型有关，我们假设将巨噬细胞极化到相反的亚型可能会以不同的方式影响它们的昼夜节律。我们使用发光报告基因跟踪 RAW 264.7 巨噬细胞的昼夜节律。细胞稳定转染了 Bmal1:luc 和 Per2:luc 报告基因，分别代表分子钟的正、负成分。使用多种方法评估了节律性、周期和时间序列幅度的强度。M1 极化降低了 Bmal1:luc 和 Per2:luc 的幅度和节律性，但对周期没有显著影响，而 M2 极化增加了周期，但对幅度或节律性没有实质性改变。由于在癌细胞存在的情况下巨噬细胞表型也会发生改变，我们测试了来自小鼠乳腺癌细胞的条件培养基对昼夜节律的影响。来自高度侵袭性 4T1 细胞的培养基导致节律性丧失，而来自侵袭性较弱的 EMT6 细胞的培养基则没有变化。由于巨噬细胞在肿瘤中发挥作用，并且致癌特征与昼夜节律有关，我们测试了巨噬细胞的条件培养基是否可以改变癌细胞的昼夜节律。RAW 264.7 细胞的条件培养基导致人骨肉瘤 U2OS-Per2:luc 细胞的节律性降低和周期延长，但幅度增加。我们表明巨噬细胞表型的变化导致昼夜节律特征的改变，并表明昼夜节律与巨噬细胞极化状态之间存在关联。此外，我们的数据表明，用乳腺癌条件培养基处理的巨噬细胞具有类似于 M1 亚型的昼夜节律表型，而用巨噬细胞条件培养基处理的癌细胞则发生昼夜节律改变，这为巨噬细胞和癌症之间的另一种交叉对话提供了新的见解。
