Immunomodulatory effects of β-defensin 2 on tumor associated macrophages induced antitumor function in breast cancer

Abstract

Background

Tumor-associated macrophages (TAMs) express cytokines and chemokines that can suppress antitumor immunity and promote tumor progression. The immunomodulatory and antitumor function of β-defensin 2 is still unclear, despite the evidence of infection response. We previously reported that β-defensin 2 modulates immunomodulatory and their antitumor function of macrophages in breast cancer. We investigate the association between β-defensin 2 and TAMs and determined the role in tumor-promoting attributes of TAMs reversal of phenotype in tumor regression.

Methods

Swiss albino mice and C127i breast cancer cell line was used in this study. C127i conditioned media was prepared and generated macrophage-derived TAMs to study antitumor function. Flow cytometry was performed for phenotype identification of macrophages and TAMs. MTT assay was performed to estimate cytotoxicity and dose optimization of β-defensin 2. Oxidative stress was analyzed by H₂O₂ and NO estimation, and qPCR was performed for iNOS, cytokines and chemokines expression.

Results

PEC harvested macrophages were characterized by flow-cytometry using F4/80, CD11c antibodies with 98% pure population of macrophages and cultured in C127i conditioned media for 7 days. TAMs markers were estimated, and it was found that 98% expression of F4/80, CD-206, and CD-115 expression compared to macrophages. Purified 100 ng/ml of β-defensin 2 was used to stimulate the TAMs population was viable, which was confirmed by cell viability assay. ROS levels decreased in TAMs treated with β-defensin 2 compared to control group. Interleukins (ILs)-6, 10, and 3, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β and chemokine ligand (CXCL)-1, 5 and 15, chemokine ligand (CCL)-24 and 5 decreased drastically compared to control.

Conclusion

This is the first report of β-defensin 2 on TAMs to elucidate the immunomodulatory and anti-tumor function. It was found that the cytokines, chemokines, and reactive oxygen species (ROS) expression pliably changed which facilitates tumor regression. β-defensin 2 must be targets as adjuvant for future cancer immunotherapeutic agent.