Expression of Antimicrobial Peptides in Cutaneous Infections after Skin Surgery

M.R. Kesting; M. Stoeckelhuber; F. Hölzle; T. Mücke; K. Neumann; K. Woermann; F. Jacobsen; L. Steinstraesser; K.-D. Wolff; D.J. Loeffelbein; N.H. Rohleder

Disclosures

The British Journal of Dermatology. 2010;163(1):121-127. 

In This Article

Discussion

We have (i) confirmed and expanded previous knowledge about cutaneous gene expression and protein levels of HDPs, (ii) demonstrated no difference in the expression of DEFB1 (HBD-1) in postoperatively inflamed epithelium compared with healthy epithelium, (iii) revealed the quantitative upregulation of the expression of DEFB4A (HBD-2), DEFB103A (HBD-3) and S100A7 in cutaneous surgical site infections, (iv) shown that the protein levels of hBD-1 do not differ in tissue from wound infections and healthy epithelium, and (v) found that the protein level of psoriasin is strongly elevated in surgical wound infections in comparison with that of healthy epithelium.

These results are in accordance with previous reports concerning the presence of DEFB1 expression in human skin.[14] An interesting issue is whether cutaneous DEFB1 expression is constitutive or is affected by inflammation. An earlier study found that the transcription of DEFB1 is not upregulated by inflammatory stimuli in various epithelial tissues and thus that it seems to be a constitutively expressed defending agent for epithelial and mucosal surfaces.[21] Another study described the elevated expression of DEFB1 in keratinocytes of whole human skin after exposure to lipopolysaccharides, peptidoglycan from Staphylococcus aureus and SpeB, which is a virulence factor of Streptococcus pyogenes.[22] However, our investigations support the assumption that DEFB1 expression does not seem to be affected in inflamed cutaneous wounds.

We found a 40-fold upregulation of DEFB4A expression in surgical wound healing disorders. This is in accordance with previous studies describing the elevated transcription of DEFB4A after incubation with bacterial pathogens in epidermal cultures.[23] As the hBD-2 peptide has also been found to act chemotactically on effector cells of the adaptive immune system,[24] the observed elevation in its synthesis not only seems to be a rapid mechanism of direct defence against bacterial growth, but also appears to be important for the long-term immune response in the case of impaired wound healing.

The results also demonstrated a 10·8-fold upregulation in the expression of DEFB103A in infected wounds. As mentioned above, hBD-3 shows broad antimicrobial activity, and the detected elevation in expression can be assumed to be part of a general defence reaction against bacterial growth. The result also supports the assumption that hBD-3 plays a major role in wound healing, based on the observation that hBD-3 promotes the healing of infected diabetic wounds.[25]

Patients with psoriasis have been found to suffer to a lesser extent from cutaneous infections than might be expected according to the reduced integrity of their skin.[26] An explanation for this phenomenon might be the above-mentioned upregulation of the expression of S100A7 in the skin lesions of these patients. The present study demonstrates that the skin upregulates S100A7 expression and protein levels of psoriasin not only in psoriatic lesions, but also in infected surgical wounds (17·1-fold change). Thus, the elevation of S100A7 expression might also be a fundamental long-term defence mechanism in nonhealing wounds.

Immunohistochemical analysis revealed no differences in hBD-1 staining intensities between cutaneous surgical site infections and healthy epithelium. In both groups, weak-moderate staining was observed in the basal and upper layers, and weaker staining was found in the SC. This result is in accordance with the observed consistency of mRNA expression between the groups and supports the above-mentioned argument that hBD-1 is constitutively produced in human skin and is not affected by pathological stimuli.

We found that the staining intensity of hBD-2 in wound infections is weak in the basal layer and pronounced in the upper epidermis, which confirms previous data.[27] However, the previous study detected only minimal staining of hBD-2 in normal skin at the day of tissue sampling,[27] whereas we saw moderate intensities not only in wound healing disorders, but also in healthy epithelium. We therefore speculate that hBD-2 is also permanently produced in healthy human skin at a baseline level, and that the observed elevation in gene expression might compensate for increased consumption in situations of microbial infestation.

However, the immunoreactivity of both hBD-2 and hBD-3 shows some variability between the samples within each group. This indicates that the increase in the amount and the distribution of these peptides in skin is a dynamic process, which has been suggested previously with respect to hBD-2.[27] Therefore, a thorough immunohistochemical analysis of these AMPs will require multiple sampling intervals according to a precise timing protocol after the initial event causing the lesion.

The staining intensity of psoriasin is weak-moderate in the SB, SS and SG and moderate in the SC of healthy epithelial samples. In contrast, it is strongly elevated in all samples of infected wounds in all epidermal layers except the basal layer, which exhibits weak staining. These observations are in accordance with the observed upregulation of S100A7 gene expression. The consistency of psoriasin staining intensities throughout most of the samples also demonstrates that the methods used in this study adequately measure HDP protein levels. We assume that the higher concentration of psoriasin generates increased antimicrobial activity, especially at the site of microbial invasion.

For all investigated peptides, staining intensities intensify from the SB to the SG. The accumulation of HDPs in the upper layers might be a result of the direction of cell proliferation. However, this pattern of distribution can be assumed to be useful for the host, as the highest levels of HDPs are thus assembled at the entry point of the microbial invaders.

Disruption of the 'shield' mechanism of endogenous peptide antibiotics has been suggested to be a causative factor in recurrent skin infections.[11] Thus, a decrease in DEFB4A expression has been found in burn wounds and is assumed to be a reason for wound infection.[28] However, the results of the present study indicate that there is no deficiency of the tested HDPs in inflamed cutaneous wounds after surgery. Nevertheless, there might be a decrease in the levels of other HDPs not investigated in this study resulting in reduced resistance against bacterial growth.

Attempts have been made to establish the use of (intravenously administered or topically applied) natural and synthetic HDPs for the treatment of infections, as an alternative to conventional antibiotics.[29,30] Higher antimicrobial activity than that for established antibiotics has been verified for some of the HDP derivatives.[31] Moreover, the rates of emerging resistance against HDP-derived agents have been found to be much lower than those for other antibiotics. However, in addition to high production costs and rapid degradation by proteolysis,[19,32] a major problem in the process of development of such treatment is the narrow therapeutic index of the HDPs. Thus, the effect of enhanced antimicrobial activity has often been observed to be accompanied by an increase in cytotoxity.[7,32] In the case of cutaneous surgical site infections, these negative effects might be avoidable by the use of collagen membranes as HDP-derivative carriers for topical application.[32]

In this study, we provide reference values for the development of pharmaceuticals for the treatment of cutaneous surgical site infections with HDPs. With knowledge of the quantities of the HDPs in inflamed surgical wounds, subsequent research should focus on finding differences in gene expression and protein levels between inflamed and regularly healing wounds. As a consequence, a possible deficiency of HDPs in wound infections after skin surgery might be remedied with a resulting improvement in the healing process. On the other hand, we cannot yet exclude that a dysregulation of HDP synthesis in infected wounds might also cause an overexpression, and that an accompanying rise in cytotoxity might be one factor responsible for prolonged inflammation. Our findings are in accordance with a recent study describing the strong induction of psoriasin and hBD-2 in margins of chronic venous ulcers.[33] Therefore, the regulation of these HDPs seems to bear similarities in surgical site infections and chronic venous ulcers.

The knowledge of possible differences in HDP levels between postoperatively inflamed and regularly healing skin should be of great value for prospective research into new treatment strategies. The results of this study should also encourage further investigations into such differences.

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