Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions

Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD_50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD₅₀ value range of HBD3 was 18.2-35.9μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro.