Exploring Pleiotropic Functions of Canine ß-Defensin 103: Nasal Cavity Expression, Antimicrobial Activity, and Melanocortin Receptor Activity.

Canine ß-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a ß-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.

Caspase-1 activation and mature interleukin-1ß release are uncoupled events in monocytes.

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1ß (pro-IL-1ß) and extracellular release of mature IL-1ß from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 µg/mL), and/or nigericin (Nig, 30 µmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1ß antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1ß antibodies. RESULTS: We found that pro-IL-1ß was processed to mature IL-1ß in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1ß, but not the processing of pro-IL-1ß to IL-1ß, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1ß, but not the processing of IL-1ß, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1ß processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1ß by caspase-1 and the release of mature IL-1ß from human monocytes are distinct and separable events.