The in Vitro Immune-Modulating Properties of a Sweat Gland-Derived Antimicrobial Peptide Dermcidin

Abstract

The epidermal barriers of the skin serve as the first layer of defense by limiting the access of many pathogens to the blood circulation. In addition, human skin also contains sweat glands that can secrete a wide array of antimicrobial peptides to restrain the growth of various microbes. In the case of microbial infection, macrophages and monocytes constitute the first line of defense by producing a wide array of proinflammatory cytokines and chemokines. This process is triggered either by pathogen-associated molecular pattern molecules (such as bacterial endotoxin) or damage-associated molecular pattern molecules (such as HMGB1). In light of our findings that a sweat gland-derived antimicrobial peptide, dermcidin, affected both pathogen-associated molecular pattern and damage-associated molecular pattern-induced cytokines/chemokines by macrophages/monocytes, we propose that dermcidin may play an important role in the regulation of the innate immune responses to infection and injury. Future investigations are warranted to further test this understudied hypothesis in both preclinical and clinical settings.

Figure 1. Expression and purification of recombinant dermcidin

A). Amino acid sequence of dermcidin precursor and various proteolytic peptides. DCD-1L is a 48 amino acid peptide corresponding to the C-terminal of the full length of dermcidin precursor. B). Expression and purification of recombinant histidine-tag dermcidin precursor (20–110) (DCD). Recombinant dermcidin corresponding to residue 20–110 amino acid with an N-terminal histidine tag was expressed in E. coli BL21 (DE3) pLysS cells (Panel B, left gel), and purified by histidine-affinity chromatography (Panel B, right gel) and Triton X-114 extraction to remove contaminating endotoxins. Note that recombinant dermcidin migrated on SDS-PAGE gel as a 12–14 kDa monomer (DCD M) in the presence of a reducing agent (DTT), but migrated as both a monomer and 24–28 kDa dimer (24–28 kDa) in the absence of DTT, suggesting possible cross-linking between dimers through disulfide bonds. C). Confirmation of the identify of recombinant protein by Western blotting analysis using dermcidin-specific antibodies.

Figure 2. Dermcidin dose-dependently attenuated LPS- and CIRP-induced NO release by murine macrophages

Murine macrophages were stimulated with LPS or CIRP alone or in the presence of recombinant dermcidin (DCD) for 16 hours, and extracellular levels of nitric oxide (NO) were determined by the Griess Reagent. *, P < 0.05 versus “− control”; #, P < 0.05 versus “+ LPS”, or “+CIRP” alone.

Figure 3. Dermcidin modulated LPS- and HMGB1-induced chemokine release by human monocytes

Human peripheral mononuclear cells (huPBMCs) were stimulated with LPS (0.8 µg/ml) or HMGB1 (4.0 µg/ml) alone, or in the presence of DCD (1.0 µg/ml) for 16 hours, and extracellular levels of cytokines and chemokines were determined by Cytokine Antibody Arrays (Panel A, B). A, Representative cytokine antibody arrays. The name of the cytokines and positive controls (“Pos”, or “+Ctrl”) were labeled in the table below. B, Relative cytokine levels. The relative cytokine levels were estimated by measuring the intensity of corresponding signal, and expressed as mean ± SEM [% of positive controls (“+Ctrl”) of respective arrays] of two independent experiments. *, P < 0.05 versus “untreated”; #, P < 0.05 versus “+ LPS” or “+HMGB1” alone.