The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury

Abstract

Epithelia establish a microbial barrier against infection through the production of antimicrobial peptides (AMPs). In this study, we investigated whether catestatin (Cst), a peptide derived from the neuroendocrine protein chromogranin A (CHGA), is a functional AMP and is present in the epidermis. We show that Cst is antimicrobial against relevant skin microbes, including gram-positive and gram-negative bacteria, yeast, and fungi. The antimicrobial mechanism of Cst was found to be similar to other AMPs, as it was dependent on bacterial charge and growth conditions, and induced membrane disruption. The potential relevance of Cst against skin pathogens was supported by the observation that CHGA was expressed in keratinocytes. In human skin, CHGA was found to be proteolytically processed into the antimicrobial fragment Cst, thus enabling its AMP function. Furthermore, Cst expression in murine skin increased in response to injury and infection, providing potential for increased protection against infection. These data demonstrate that a neuroendocrine peptide has antimicrobial function against a wide assortment of skin pathogens and is upregulated upon injury, thus demonstrating a direct link between the neuroendocrine and cutaneous immune systems. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.

Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1. Cst acts as an antimicrobial through membrane disruption

Membrane permeability was determined by measuring the release of β-galactosidase from E. coli ML-35p as detected by optical density (OD) 420 nm. (a) A 1 µg ml−1 portion of polymixin B induces membrane leakage in the presence of 25 mm NaHCO3 (open circles) but not in the absence of NaHCO3 (closed circles). A 25 mm portion of NaHCO3 alone (open squares) had no effect compared to untreated cells (solid squares). (b) LL-37 (4 µm, open circles) or Cst (20 µm, open squares) induces membrane leakage in the presence of 25 mm NaHCO3 but not in the absence of NaHCO3 (solid circles and squares). (c–h) Electron microscopy of E. coli 25922 treated for 10 minutes with (c) 50 µm Cst (original magnification × 40,000), (d) untreated (original magnification × 40,000), or 10 minutes with (e) 32 µm LL-37 (original magnification × 40,000). (f–h) Original magnification × 15,000 of E. coli shown in (c–e), respectively. Arrows in images (f–h) represent areas chosen for high magnification.

Figure 2. Epidermal keratinocytes express Chga

Immunofluorescence analysis of Cst expression in human skin. Aldehyde-fixed sections of human skin were processed for immunohistochemistry using (a) a polyclonal anti-human Cst or (b) rabbit IgG. (c) Relative expression of Chga mRNA in human neuroblastoma SK-N-SH cells, whole human skin, cultured human keratinocytes, and cultured human fibroblasts using quantitative real-time PCR. Data represent the mean of three samples from the same tissue or cells normalized to GAPDH and then normalized to fibroblasts.

Figure 3. CHGA is processed into Cst in skin

(a) HPLC chromatogram of total human skin extract. The inset shows a peak at fraction 16 corresponding to the fraction indicated by the arrow that eluted at the same CH3CN% (28.5%) as human Cst synthetic peptide applied identically in a following run. Electrospray-ionization mass spectrometry identified the mass of this peak as 2,326 Da. (b) Immunoreactivity of HPLC fractions shown in (a) as determined by reactivity to Cst antibody. Highest immunoreactivity is detected in fraction 16 indicated by the arrow.

Figure 4. CHGA is upregulated following barrier disruption and infection

Immunofluorescence analysis of Cst expression in injured mouse skin. Acetone-fixed sections of mouse skin subjected to consecutive tape-stripping +/− occlusion were processed for immunohistochemistry using a polyclonal anti-rodent Cst antibody. Cst staining is shown at (a) baseline, (b) 3 hours post-tape stripping and (c) 3 hours post-tape stripping with occlusion. (d) Rabbit IgG as a negative control. Bar =40 µm. The expression of Camp (e) or Chga (f) was analyzed by quantitative PCR in normal, non-injured skin (control), or in skin 3 hours after tape stripping +/− occlusion. The expression of Camp (g) or Chga (h) was analyzed by quantitative PCR in normal, non-lesional skin from uninfected mice (control), or in lesional skin 24 hours after infection with GAS. Data are shown as the mean ± SEM by Student’s t-test, n = 3–4 per group, repeated twice. *P < 0.05.