1. Human β-defensin-3 induces IL-8 release and apoptosis in airway smooth muscle cells
W Wang, X Qu, X Dang, D Shang, L Yang, Y Li, D Xu, J G Martin, Q Hamid, J Liu, Y Chang Clin Exp Allergy. 2017 Sep;47(9):1138-1149. doi: 10.1111/cea.12943. Epub 2017 May 26.
Background: Human airway smooth muscle cells (ASMCs) may have a pro-inflammatory role through the release of inflammatory mediators. Increasing evidence indicates that human β-defensins (HBDs) are related to pathogenesis of asthma. Objectives: To examine the plasma level of HBD-1, HBD-2 and HBD-3 in asthmatic patients and the expression of their mouse orthologues in the lung tissue of a mouse model of chronic severe asthma. Further to investigate the effect of HBD-3 on the release of the pro-inflammatory cytokine IL-8 and to explore the mechanisms. Methods: The plasma levels of HBD-1, HBD-2 and HBD-3 from 34 healthy controls and 25 asthmatic patients were determined by ELISA. The expression of mouse β-defensins MBD-1, MBD-3 and MBD-14 in the lung tissue of asthmatic mice was detected by Western blot. The ASMCs were cultured with HBD-3 for 24 hour, and then the supernatant level of IL-8 was evaluated by ELISA and the cell viability was examined by WST-1 assay. The signalling pathway was investigated with blocking antibodies or pharmacological inhibitors. Results: The plasma levels of HBD-1 and HBD-3 were elevated in asthmatic patients, and the expression of MBD-14, the mouse orthologue for HBD-3, was increased in asthmatic mice. HBD-3-induced IL-8 production in a CCR6 receptor-specific manner and was dependent on multiple signalling pathways. Moreover, HBD-3-induced cell apoptosis concurrently, which was dependent on the ERK1/2 MAPK pathway. Mitochondrial ROS regulated both HBD-3-induced IL-8 production and cell apoptosis. Conclusions and clinical relevance: These observations provide clear evidence of an important new mechanism for the promotion of airway inflammation and tissue remodelling with potential relevance for the treatment of asthma.
2. Immunomodulatory effects of β-defensin 2 on macrophages induced immuno-upregulation and their antitumor function in breast cancer
Sonam Agarwal, Anita Chauhan, Khushwant Singh, Kunal Kumar, Rupinder Kaur, Marilyn Masih, Pramod Kumar Gautam BMC Immunol. 2022 Nov 2;23(1):53. doi: 10.1186/s12865-022-00527-y.
Background: 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. Method: 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 H2O2 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. Results: 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 H2O2 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. Conclusion: 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.
3. Mouse β-Defensin 3, A Defensin Inhibitor of Both Its Endogenous and Exogenous Potassium Channels
Yaoyun Zhang, Yonghui Zhao, Hongyue Liu, Weiwei Yu, Fan Yang, Wenhua Li, Zhijian Cao, Yingliang Wu Molecules. 2018 Jun 20;23(6):1489. doi: 10.3390/molecules23061489.
The human defensins are recently discovered to inhibit potassium channels, which are classical targets of the animal toxins. Whether other vertebrate defensins are potassium channel inhibitors remains unknown. In this work, we reported that the mouse β-defensin 3 (mBD3) was a novel inhibitor of both endogenous and exogenous potassium channels. The structural analysis showed that mBD3 is the most identical to human Kv1.3 channel-sensitive human β-defensin 2 (hBD2). However, the pharmacological profiles indicated that the recombinant mBD3 (rmBD3) weakly inhibited the mouse and human Kv1.3 channels. Different from the pharmacological features of human β-defensins, mBD3 more selectively inhibited the mouse Kv1.6 and human KCNQ1/KCNE1 channels with IC50 values of 0.6 ± 0.4 μM and 1.2 ± 0.8 μM, respectively. The site directed mutagenesis experiments indicated that the extracellular pore region of mouse Kv1.6 channel was the interaction site of rmBD3. In addition, the minor effect on the channel conductance-voltage relationship curves implied that mBD3 might bind the extracellular transmembrane helices S1-S2 linker and/or S3-S4 linker of mouse Kv1.6 channel. Together, these findings not only revealed mBD3 as a novel inhibitor of both endogenous and exogenous potassium channels, but also provided a clue to investigate the role of mBD3-Kv1.6 channel interaction in the physiological and pathological field in the future.
