**Lactoferricin**

184 Antimicrobial Agents

skin was considered as a simple physical barrier, but it is in a process of continual regeneration

Skin is composed of three layers, from inside to outside (Figure 2): i) *hypodermis* or subcutaneous tissue, ii) *dermis*, or *corium*, with a 3 to 5 mm thickness, iii) *epidermis*, with a thickness varying from 0.06 to 0.8 mm. *Epidermis* can be subdivided itself into four layers : *stratum basale*, *stratum spinosum*, *stratum granulosum* and *stratum corneum* (Figure 2). The deeper layers are composed of keratinocytes, melanocytes, Langerhans cells, Merkel cells and malpighian cells (Figure 2). Epidermis is composed as a gradient of differentiated keratinocytes, synthesizing keratine in *stratum granulosum*, and losing nuclei and organelles. Skin, and more specifically *stratum corneum*, acts as a barrier in several ways (Elias, 2007). Corneocytes and extracellular matrix represent a physical barrier ("brick wall" model) (Figure 2).

and has its own immunological, histological and nervous responses to environment.

Fig. 2. Schematic representation of the different skin layers, adapted from different sources. A: Epidermis, dermis and subcutaneous tissues are shown with the different structures

during skin diseases, such as atopic dermatitis or psoriasis.

**Lysozyme** 

composing them. (according to Metz-Boutigue, M.H. et al., Curr Pharm Des. 2010;16(9):1024-39).x

The slightly acidic surface (pH~ 5.0), as well as the low hydration level of the skin represents a hostile area for pathogens, such as *Staphylococcus aureus*. Lipids (ceramides, cholesterol, free fatty acids) and their metabolic products present in *stratum corneum* act also as antimicrobial defense. Last, constitutive (and/or inducible) expression of antimicrobial peptides and proteins helps to maintain skin integrity and to prevent pathogen colonization. On the contrary, the surface of healthy skin is ideal for the growth of the normal cutaneous microflora (*Micrococcacea*, i.e. *Staphylococcus epidermidis* and *Corynebacteriacea*) that colonizes skin, competes with pathogens for nutrients and synthesizes antimicrobial compounds. These evolutionary conserved components of the innate immune system can act as direct antimicrobial agents and exert a role as immunomodulatory molecules in normal skin and

Lysozyme is the first antimicrobial protein found in human skin. It was located in cytoplasm of epidermal cells in granular layers and in malpighian cells present in the *stratum spinosum* layer (Ogawa et al., 1971; Papini et al., 1982). Lysozyme is mainly active against GramLactoferricin is an antimicrobial peptide originally produced by pepsin digestion of lactoferrin. It is active against Gram-positive, Gram-negative bacteria and also against *Candida albicans* (Bellamy et al., 1993). This molecule was also detected in skin wash of adult and newborn children (Walker et al., 2008). Synthesized by melanocytes, cutaneous lactoferrin is an iron-binding protein with antibacterial properties due to its ability to sequester iron in biological fluids or to destabilize bacterial membranes, limiting microorganism proliferation and adhesion. It has also immunomodulatory properties by up and down regulating immune cells involved in inflammatory processes (Legrand et al., 2005). The protective anti-inflammatory role of lactoferrin is due to its ability to bind free ferric ion acting as an anti-oxidant (Walker et al., 2008). It can bind to LPS and their receptors during an infection as well (Legrand et al., 2005). Expression of virulence factors of *S. aureus* is modulated by transferrin and lactoferrin (Kansal et al., 2005), demonstrating that these iron-binding proteins play an important role in the host-pathogen interaction in skin and in mucosal tissue probably by LPS or its receptors binding.

### **Dermcidin and its derived peptides**

Dermcidin, is constituvely and specifically expressed in the eccrine sweat glands within the dermis of human skin, secreted into the sweat and transported *via* sweat to the epidermal surface (Schittek et al., 2001). It is a 47 amino acids peptide produced from hydrolysis of a 9.3 kDa precursor by cathepsin D (Baechle et al., 2006). It possesses antibacterial properties at low concentration against *S. aureus*, *E. faecalis*, *E. coli* and *C. albicans*. The *in vivo* importance of DCD in prevention of infections has been demonstrated by its low expression in patients with atopic dermatitis. It was shown that dermcidin induces the production by SepA of *S. aureus*, a proteolytic virulence factor that cleaves and inactivates dermcidin (Lai et al., 2007). In the eccrine sweat, several proteolytically generated DCD fragments (DCD-1, DCD-1L) have been identified. DCD-1L is the most abundant antimicrobial peptide present in sweat, but other peptides derived from dermcidin by proteolysis are also found (Baechle et al., 2006; Rieg et al., 2006). The distribution of these peptides was found to be different according to the individuals. Most of them have 2 to 4 of the major DCD-derived peptides with the constant presence of at least one of the following peptides: DCD-1L (63-110), LEK-45 (66-110) and SSL-29 (63-91). The authors also showed that the distribution of these peptides is dependent on the body sites, which correlates with the presence of eccrine sweat glands and not with apocrine glands. Body parts in contact with pathogens (arms, face etc.) produce high levels of DCDderived peptides. The molecular analysis of the antimicrobial activity of dermcidin-derived peptides showed that peptides like DCD-1L or SSL-23 do not disrupt bacterial membranes, but kill bacteria by still unknown mechanisms (Steffen et al., 2006).

Recently, by using a proteomic approach, a dermcidin precursor was found in human cervico-vaginal fluid (Shaw et al., 2007), together with haptoglobin, neutrophil defensin,

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

hBD-2 by primary keratinocytes (Schroeder & Harder, 2006).

keratinocyte differentiation *in vitro* (Lichti et al., 2008).

wounds (Sorensen et al., 2006).

proliferation.

**Defensins** 

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 187

concern S100 proteins as substrate for transglutaminase, resulting in an incorporation of S100 in the cornified envelope; a last role could be a response to exogenous agents that modulate S100 proteins distribution and consequently their function (Eckert et al., 2004). Psoriasin has been found to be overexpressed in psoriasis. It is produced in *stratum corneum* by keratinocytes (Martinsson et al., 2005) and its basal expression is influenced by extracellular calcium level. Its expression in normal adult tissue is low, but high expression levels were detected in fetal skin, as for transferrin, suggesting a protective role in innate immunity. Psoriasin was found to be the main *E. coli*-cidal agent in the skin. It is a chemotactic agent for neutrophils and CD4+ T cells (Jinquan et al., 1996). Moreover, psoriasin mediates the production of several inflammatory cytokines and chemokines from neutrophils *via* MAPK p38 and ERK activation. It also induces reactive oxygen species production and the exocytosis of alpha-defensins from neutrophils (Zheng et al., 2008).

To date 4 defensins (hBD-1 to -4) in neutrophils and 2 defensins (hBD-5 and hBD-6) produced by Paneth cells were identified. The first inducible human defensin, hBD-2, was identified in psoriatic lesions as the most abundant AMP. It was found to be expressed in terminally differentiated keratinocytes, in a structure located in *stratum corneum*, lamellar bodies that contain lipid-rich secretory granules. It is probably released with lipid-like content of these lamellar bodies (Oren et al., 2003). hBD-2 is also up-regulated locally by infections (Radek & Gallo, 2007) or wounds (Butmarc et al., 2004). It has preferential bactericidal properties against Gram-negative bacteria (Harder et al., 1997) and like LL-37, its effect is sensitive to the concentration of NaCl. hBD-2 derived from neutrophils, promotes prostaglandins production and histamine release from mast cells, playing a role in allergic response (Bals et al., 1998). hBD-2 has also chemotactic properties for immature dendritic cells and memory T cells; it was described to bind to CCR-6, the receptor for macrophage inflammatory protein 3 alpha. In monocytes, hBD-2 expression is stimulated by several cytokines (Ganz, 2003; Kanda & Watanabe, 2008) and Il-1 seems to be the major inducer of hBD-2 production. Bacteria can also stimulate the expression of hBD-2 by epithelial cells, in a cytokine-independent pathway. *P. aeruginosa* is a powerful inducer of

hBD-1 was considered as a constitutively expressed antimicrobial peptide and in particularly not induced by proinflammatory cytokines. However, its production can be induced by peptidoglycan or LPS exposure (Sorensen et al., 2005). It is expressed in malpighian layer and in *stratum corneum* (Ali et al., 2001) and this expression is induced by increasing concentration of calcium (Harder et al., 2004), condition that provokes

hBD-3 has its expression induced by EGF that provokes keratinocytes proliferation in skin

It has chemotactic properties for monocytes (Garcia et al., 2001). While its expression is not induced by infection, hBD-3 displays a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria, as well as against fungi (Harder et al., 2001). Regarding the adaptive immune system, hBD-2, 3 and 4 stimulate expression of proinflammatory cytokines, IL-10 and MCP-1 (Niyonsaba et al., 2007). They also stimulate the phosphorylation of STAT-1 and STAT-3 that induce keratinocytes migration and

lysozyme and lactoferrin. Dermcidin precursor was also found in human gestational tissue (Lee Motoyama et al., 2007), where it is proposed to play a role in pregnancy by regulating trophoblastic functions.
