Human β-defensin-3 alters, but does not inhibit, the binding of Porphyromonas gingivalis haemagglutinin B to the surface of human dendritic cells

Abstract

Human β-defensin-3 (HBD3) is a small, cationic, host defence peptide with broad antimicrobial activities and diverse innate immune functions. HBD3 binds to many microbial antigens and, in this study, we hypothesised that the known binding of HBD3 to Porphyromonas gingivalis recombinant haemagglutinin B (rHagB) alters, but does not inhibit, the binding of rHagB to human dendritic cells. To test this, human myeloid dendritic cells were incubated for 5 min with rHagB, HBD3 + rHagB (10:1 molar ratio), HBD3 or 0.1 M phosphate-buffered saline (PBS) (pH 7.2) and were then rapidly fixed and processed for confocal microscopy and ultramicrotomy. rHagB and HBD3 could be detected with primary monoclonal mouse antibody to rHagB (MoAb 1858) or polyclonal rabbit antibody to HBD3 (P241) and secondary fluorescent-labelled anti-mouse or anti-rabbit antibodies (confocal microscopy) or protein A–colloidal gold (immunoelectron microscopy). In cells incubated with rHagB only, fluorescence and protein A–colloidal gold were seen at the cell surface and throughout the cytoplasm. In cells incubated with HBD3 + rHagB, fluorescence was observed only at the cell surface in a ‘string of pearls’ configuration. Overall, these results suggest that HBD3 binding to rHagB alters, but does not inhibit, the binding of rHagB to human myeloid dendritic cells.

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

“…Van Hemert et al observed HBD3 in the cytoplasm of DCs min after it was added to cells in culture (Van Hemert et al, 2012), and Semple et al showed that HBD3 rapidly enters toll-like receptor (TLR) 4-stimulated macrophages and targets TLR signaling pathways to inhibit the transcription of pro-inflammatory genes involved in the regulation of nuclear factor kappa B (Semple et al, 2011). Lioi et al showed that HBD3 causes membrane perturbations in RAW264.7 macrophages incubated with TAMRA labeled HBD3 (HBD3 TAMRA ), but not T or B cells (Lioi et al, 2012).…”

Defensin DEFB103 bidirectionally regulates chemokine and cytokine responses to a pro-inflammatory stimulus

“…The binding of DEFB103 to HagB1721 may play a partial role by altering the binding of HagB to TLR on the dendritic cell surface partially retarding the activation of underlying MAPK pathways leading to the attenuated HagB-induced response. However, when administered separately and in differing order to dendritic cells, DEFB103 and HagB may synergistically activate similar pathways and exaggerate a HagB-induced response stronger than that seen by HagB alone.…”

“…However, when administered separately and in differing order to dendritic cells, DEFB103 and HagB may synergistically activate similar pathways and exaggerate a HagB-induced response stronger than that seen by HagB alone. Finally, it is also possible that the ability of DEFB103 to rapidly enter cells21 also plays a part. The trend in responses seen after temporal exposure of human and murine JAWS II dendritic cells to DEFB103 and HagB are similar to the responses seen after temporal exposure of JAWS II dendritic cells to surfactant protein-A (SP-A)-derived peptides and E. coli lipopolysaccharide31.…”

“…Influencing events in ‘defensin controlled inflammation’ likely involve extracellular binding of defensins to microbial antigens171819; defensin-altered binding of antigens to cell surfaces2021; or defensin-altered signaling1222.…”

Antimicrobial Peptides in Host Defense: Functions Beyond Antimicrobial Activity

“…It inhibits the interaction of LPS with CD14 on cell surfaces by competing with the LPS-binding protein (Molhoek et al 2009 ), and it blocks DC-SIGN-gp120 interaction and prevents dendritic cellmediated HIV type 1 transmission (Groot et al 2005 ). HBD3 alters the binding of Porphyromonas gingivalis hemagglutinin B (HagB) to the surface of dendritic cells (Van Hemert et al 2012 ) and HNP-1, HBD1, HBD2, HBD3, DEFB104, and LL-37, all inhibit binding of Alexa Fluor 546-labeled P. intermedia and T. forsythia LPS to THP-1 human monocytes (Lee et al 2010 ).…”