Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, 110029, India.
BMC Immunol. 2022 Nov 2;23(1):53. doi: 10.1186/s12865-022-00527-y.
Macrophages are mononuclear CD34 antigen-presenting cells of defense mechanism and play dual roles in tumor burden. The immunomodulatory and their antitumor function of β-defensin 2 is still unclear, despite the accumulating evidence of the response in infection. So, the aim of present study is to elucidate the role of β-defensin 2 on the level of ROS, cytokines, chemokine expression in macrophages and antitumor function in breast cancer.
Swiss albino mice were used to harvest PEC macrophages and C127i breast cancer cells line for tumor model was used in this study. Macrophages were harvested and characterized by flow-cytometry using F4/80 and CD11c antibodies. MTT was performed to estimate cytotoxicity and dose optimization of β-defensin 2. Oxidative stress was analyzed by HO and NO estimation followed by iNOS quantified by q-PCR. Cytokines and chemokines estimation was done using q-PCR. Co-culture experiment was performed to study anti-tumor function using PI for cell cycle, Annexin -V and CFSE analysis for cell proliferation.
PEC harvested macrophages were characterized by flow-cytometry using F4/80 and CD11c antibodies with the purity of 8% pure population of macrophages. It was found that 99% of cells viable at the maximum dose of 100 ng/ml of β-defensin 2 in MTT. Levels of NO and HO were found to be decreased in β-defensin 2 as compared to control. Expression of cytokines of IFN-γ, IL-1α, TNF-α, TGF-βwas found to be increased while IL-3 was decreased in β-defensin 2 group as compared to control. Levels of chemokines CXCL-1, CXCL-5 and CCL5 increased in treated macrophages while CCL24 and CXCL-15 expression decreased. Adhesion receptor (CD32) and fusion receptor (CD204) were decreased in the β-defensin 2 group as compared to control. Anti-tumor experiment was performed using co-culture experiment apoptosis (Annexin-V) was induced, cell cycle arrest in phage and cell proliferation of C127i cells was decreased.
This is the first report of β-defensin 2 modulates macrophage immunomodulatory and their antitumor function in breast cancer. β-defensin 2 as a new therapeutic target for immunotherapy as an adjuvant in vaccines.
巨噬细胞是防御机制的单核 CD34 抗原呈递细胞，在肿瘤负担中发挥双重作用。尽管感染反应的证据不断增加，但β-防御素 2 的免疫调节及其抗肿瘤功能仍不清楚。因此，本研究旨在阐明β-防御素 2 在 ROS、细胞因子、趋化因子表达水平对乳腺癌中巨噬细胞的作用及其抗肿瘤功能。
本研究使用瑞士白化小鼠收获 PEC 巨噬细胞和 C127i 乳腺癌细胞系建立肿瘤模型。使用 F4/80 和 CD11c 抗体通过流式细胞术收获和表征巨噬细胞。通过 MTT 评估β-防御素 2 的细胞毒性和剂量优化。通过 HO 和 NO 估计分析氧化应激，然后通过 q-PCR 量化 iNOS。使用 q-PCR 进行细胞因子和趋化因子的估计。通过 PI 进行细胞周期、Annexin-V 和 CFSE 分析进行细胞增殖的共培养实验来研究抗肿瘤功能。
使用 F4/80 和 CD11c 抗体通过流式细胞术对 PEC 收获的巨噬细胞进行特征分析，巨噬细胞的纯度为 8%的纯巨噬细胞群体。结果发现，在 MTT 中，β-防御素 2 的最大剂量为 100ng/ml 时，细胞存活率为 99%。与对照组相比，β-防御素 2 组的 NO 和 HO 水平降低。与对照组相比，β-防御素 2 组 IFN-γ、IL-1α、TNF-α、TGF-β 的细胞因子表达增加，而 IL-3 减少。处理的巨噬细胞中趋化因子 CXCL-1、CXCL-5 和 CCL5 的水平增加，而 CCL24 和 CXCL-15 的表达减少。与对照组相比，β-防御素 2 组的粘附受体（CD32）和融合受体（CD204）减少。通过共培养实验进行抗肿瘤实验诱导细胞凋亡（Annexin-V），C127i 细胞的细胞周期停滞和增殖减少。
这是首次报道β-防御素 2 调节乳腺癌中巨噬细胞的免疫调节及其抗肿瘤功能。β-防御素 2 作为一种新的治疗靶点，可作为疫苗佐剂用于免疫治疗。
