Beta-defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin

Abstract

Background: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis.

Methodology/principal findings: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects.

Conclusions/significance: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Figure 1. Analysis of beta-defensin mRNA expression in normal and inflamed human tissues.

Quantitative real-time PCR was performed on RNA of normal human tissues (mostly obtained from one individual), purified epidermis from skin biopsies of healthy controls, psoriasis patients and atopic dermatitis patients, and inflamed synovium from rheumatoid arthritis patients. Expression of target genes was normalized to that of RPLP0. For graphical representation all values were expressed relative to hBD-2 in tongue, which was set at unity . Primer sequences and efficiency of amplification are given in supplemental table S1. For details on normal human tissues see materials and methods. Bars represent mean and SD.

Figure 2. Immunolocalization of hBD-2 in human epithelia.

Immunohistochemical staining of normal human tissues (tongue, plantar skin and trunk skin, A–C) with a polyclonal rabbit antiserum against recombinant hBD-2. Note that protein data largely follow the mRNA data demonstrating the absence in normal skin, low expression in tongue and plantar skin. Bar = 100 µm. Control sections stained with pre-immune serum were negative (not shown).

Figure 3. Correlation between serum hBD-2 protein levels and genomic copy number.

Serum hBD-2 protein levels of 70 healthy controls (determined by ELISA) were plotted against the genomic copy number of the beta-defensin repeat on chromosome 8p23, as determined by MAPH, REDVR and PRT. A significant linear correlation was found. Pearson's R = 0.46 and p<7×10⁻⁵.

Figure 4. Correlation between serum hBD-2 protein and PASI score.

Serum hBD-2 protein levels of 38 psoriasis patients of varying disease severity were plotted against their PASI score. A significant linear correlation was found. Pearson's R = 0.82, p<4×10⁻¹⁰.

Figure 5. Correlation between the change in serum hBD-2 concentration and change in clinical score.

Serum hBD-2 protein levels of 15 patients for which PASI scores and serum was available on two different occasions over a 6–18 week interval, were plotted against the change in PASI score (ΔPASI). A significant linear correlation was found. Pearson R = 0.74, p<0.002. Note that there was a decrease in serum hBD-2 in most patients that showed clinical improvement (negative ΔPASI) and an increase in serum hBD-2 in a few patients that showed exacerbation (positive ΔPASI).

Figure 6. Induction of hBD-2 protein expression in reconstructed skin by proinflammatory cytokines.

Expression of hBD-2 in 3-D reconstructed skin following stimulation with psoriasis-associated cytokines (10 ng/ml IL-1α, 5 ng/ml TNFα and 5 ng/ml IL-6) for 72 hours. Note that without stimulation there is no hBD-2 expression (A), whereas the cytokine mixture induces high expression levels that are secreted into the underlying culture medium (147 ng/ml in 24 h). Part of the hBD-2 protein remains adsorbed to the dermal matrix as witnessed by the staining of structures in the dermis (B). Bar = 100 µm.