**Phospholipase A2**

The hydrophobic layer of phosphatidylcholine (PC) overlies and protects the surface of the gastrointestinal (GI) tract, contributing to barrier integrity. In addition, phospholipase A2 is synthesized by Paneth cells and this enzyme hydrolyses bacterial membrane phospholipids to generate both free fatty acids and lysophospholipids. An important prerequisite for the action of phospholipase A2 is the successful binding to the phospholipids surface. *In vitro* studies utilizing recombinant enzymes and artificial phospholipids substrates have shown that phospholipases act on anionic phospholipids (phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine), but are inactive with phosphatidylcholine due to the lack of high affinity binding (Wu et al., 2010). During critical illness such as sepsis, gut barrier integrity may be compromised, which could be related to degradation of PC. Pretreatment with an orally active sPLA(2) inhibitor blocks the LPS-induced increase in GI permeability, and may suggest a new approach to reinforce the GI mucosal barrier and prevent complications from endotoxin in trauma in other septic conditions (Zayat et al., 2008).

### **Cathelicidins**

Cathelicidin LL-37/hCAP18 is synthesized by neutrophils, where it was first identified (Romeo et al., 1988) and epithelial cells of the colon (Hase et al., 2002). *In vitro*, it has chemotactic properties for monocytes, macrophages and T cells (Koczulla et al., 2003). LL-37 is found in sites of inflammation where it modifies dendritic cells (DCs) differentiation, relying innate and adaptive immunity. *In vitro*, modified DCs had, among others characteristics induced by the peptide, enhanced secretion of Th-1 inducing cytokines and promoted Th1 responses (Davidson et al., 2004). LL-37 acts synergistically with IL-1-beta to increase the production of suppressive cytokines (IL-6 and IL-10) and chemokines MCP-1 and MCP-3 by macrophages (Yu et al., 2007). It acts *via* the transcription factor CREB and the activation of phosphorylation of the kinase Akt. In LPS-stimulated monocytes, LL-37 inhibits the release of TNF-alpha modulating inflammatory response induced by LPS, endotoxins and other agonists of TLRs (Mookherjee et al., 2006).

#### **RNAses**

Angiogenin-4 (Ang-4) is a member of the ribonucleases family. This protein is synthesized by Paneth cells and is similar to RNase 7 found in skin. Its secretion is stimulated by exposure to LPS. Ang-4 kills *E. faecalis* and *L. monocytogenes* at concentrations as low as 1 µM, whereas its concentration in crypts can be 1000 times greater (Hooper et al., 2003). Similarly to defensins, it is sensitive to salt concentration and is potentially cytotoxic to eukaryotic cells (Saxena et al., 1992).

#### **C-type lectins**

C-type lectins HIP/PAP are synthesized in human by enterocytes and Paneth cells. The same protein exists in mouse and is named RegIII gamma. These lectins bind Gram-positive peptidoglycan and act by direct killing. Several members of this family are found in gastrointestinal tissues (Dieckgraefe et al., 2002).

#### **Defensins**

As in skin, defensins have a direct antimicrobial role as well as immunomodulatory function. Alpha-defensins are synthesized by Paneth cell in the gastrointestinal tractus (Porter et al., 2002). Alpha-defensin expression does not require microbe induction since they are synthesized in germ-free conditions (Putsep et al., 2000) and/or prenatally (Mallow et al., 1996). In transfected mouse, it was shown that the alpha-defensin hBD-5 protects efficiently against *Salmonella typhimurium*, demonstrating the direct antimicrobial effect of this peptide. In mouse, alpha-defensins are named cryptdins and several families of peptides related to cryptdins are regrouped under the term CRS (Cryptdin Related Sequences). Interestingly, these CRS can form homo- or heterodimers, thus allowing a combinatorial diversity to struggle against pathogens (Hornef et al., 2004).

Beta-defensins are expressed in enterocytes of the small and large intestines. 28 beta-defensin encoding genes have been identified in human genome, but only 8 were found to be expressed. hBD-1 is constitutively expressed in absence of stimulus or bacterial infection (O'Neil et al., 1999), while some nutrients can stimulate its production in cell lines (Sherman et al., 2006). In mouse, an infection by the *Cryptosporidium* parasite resulted in a down-regulation of mBD-1 (Zaalouk et al., 2004), while *in vitro*, sporozoites are killed by this defensin. Some authors conclude on an unique and important regulation of hBD-1, during small intestine infections (Dann et Eckmann, 2007). hBD-2 is not constitutively expressed, but is induced by an infection or by proinflammatory stimuli (O'Neil et al., 1999). hBD-3 and -4 are inducible and particularly expressed in crypt regions (Fahlgren et al., 2004). Defensins can also act as chemotactic agents for immune cells in a similar way to that described for the skin.

### **Bactericidal Permeability Increasing protein**

Bactericidal/permeability-increasing protein (BPI), a constituent of primary neutrophil granules, is a potent natural antibiotic and anti-BPI antibodies are detected during infectious enteritis. In addition, BPI is a target antigen for anti-neutrophil cytoplasmic antoantibodies in inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (Walmsley et al., 1997).

#### **Neuropeptides**

182 Antimicrobial Agents

The hydrophobic layer of phosphatidylcholine (PC) overlies and protects the surface of the gastrointestinal (GI) tract, contributing to barrier integrity. In addition, phospholipase A2 is synthesized by Paneth cells and this enzyme hydrolyses bacterial membrane phospholipids to generate both free fatty acids and lysophospholipids. An important prerequisite for the action of phospholipase A2 is the successful binding to the phospholipids surface. *In vitro* studies utilizing recombinant enzymes and artificial phospholipids substrates have shown that phospholipases act on anionic phospholipids (phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine), but are inactive with phosphatidylcholine due to the lack of high affinity binding (Wu et al., 2010). During critical illness such as sepsis, gut barrier integrity may be compromised, which could be related to degradation of PC. Pretreatment with an orally active sPLA(2) inhibitor blocks the LPS-induced increase in GI permeability, and may suggest a new approach to reinforce the GI mucosal barrier and prevent complications from endotoxin in trauma in other septic conditions (Zayat et al., 2008).

Cathelicidin LL-37/hCAP18 is synthesized by neutrophils, where it was first identified (Romeo et al., 1988) and epithelial cells of the colon (Hase et al., 2002). *In vitro*, it has chemotactic properties for monocytes, macrophages and T cells (Koczulla et al., 2003). LL-37 is found in sites of inflammation where it modifies dendritic cells (DCs) differentiation, relying innate and adaptive immunity. *In vitro*, modified DCs had, among others characteristics induced by the peptide, enhanced secretion of Th-1 inducing cytokines and promoted Th1 responses (Davidson et al., 2004). LL-37 acts synergistically with IL-1-beta to increase the production of suppressive cytokines (IL-6 and IL-10) and chemokines MCP-1 and MCP-3 by macrophages (Yu et al., 2007). It acts *via* the transcription factor CREB and the activation of phosphorylation of the kinase Akt. In LPS-stimulated monocytes, LL-37 inhibits the release of TNF-alpha modulating inflammatory response induced by LPS,

Angiogenin-4 (Ang-4) is a member of the ribonucleases family. This protein is synthesized by Paneth cells and is similar to RNase 7 found in skin. Its secretion is stimulated by exposure to LPS. Ang-4 kills *E. faecalis* and *L. monocytogenes* at concentrations as low as 1 µM, whereas its concentration in crypts can be 1000 times greater (Hooper et al., 2003). Similarly to defensins, it is sensitive to salt concentration and is potentially cytotoxic to

C-type lectins HIP/PAP are synthesized in human by enterocytes and Paneth cells. The same protein exists in mouse and is named RegIII gamma. These lectins bind Gram-positive peptidoglycan and act by direct killing. Several members of this family are found in

As in skin, defensins have a direct antimicrobial role as well as immunomodulatory function. Alpha-defensins are synthesized by Paneth cell in the gastrointestinal tractus (Porter et al., 2002). Alpha-defensin expression does not require microbe induction since

endotoxins and other agonists of TLRs (Mookherjee et al., 2006).

**Phospholipase A2** 

**Cathelicidins** 

**RNAses** 

**C-type lectins** 

**Defensins** 

eukaryotic cells (Saxena et al., 1992).

gastrointestinal tissues (Dieckgraefe et al., 2002).

Enterochromaffin cells (EC) (Siddique et al., 2009) (Figure 1) are enteroendocrine cells present in the intestine, especially colon (Kuramoto et al., 2007) and containing large amounts of serotonin (5-HT). These cells can sense luminal content before its basolaterally release, and activate afferent neuron endings within *lamina propria*, allowing information exchange between gut and central nervous system (Hansen & Witte et al., 2008). Besides this important role, EC secrete also numerous other products, among which VIP (Zanner et al., 2004), Substance P (Heitz et al., 1976), CgA, CgB and secretogranin II/CgC (Cetin & Grube, 1991) and melatonin (Raikhlin et Kvetnoy, 1976).

Despite the crucial role of these cells, their sparse repartition and their low number did not allowed their extensive study. However, the BON cells were proposed as a model (Kim et al., 2001), that will enhance further research. When EC were stimulated by odors, they released serotonin, showing that these cells can also be stimulated by spices and fragrances (Braun et al., 2007). Moreover, a new method was proposed allowing isolating and purifying EC from biopsies (Modlin et al., 2006).

## **1.4 Skin and antimicrobial peptides**

Mammal skin is an essential defense barrier against external aggressions, such as microbial pathogens, oxidant stress, chemical aggressions, mechanical insults, burns etc. For a long time,

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

and in mucosal tissue probably by LPS or its receptors binding.

kill bacteria by still unknown mechanisms (Steffen et al., 2006).

**Dermcidin and its derived peptides** 

in preventing infections in newborn children.

**Lactoferricin** 

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 185

positive bacteria (*S. aureus*), but is also active against Gram-negative bacteria, acting probably as a control of bacterial growth. Still recently, the contribution of lysozyme to cutaneaous defense was subjected to debate since it was not detected in *stratum corneum* as well as in washing fluid. However, it was recently detected in skin wash of adults, and lysozyme concentration was 5 times higher in newborn skin than in adult (Walker et al., 2008), confirming its status of antimicrobial molecule, as well as giving it an important role

Lactoferricin 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

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

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,

skin was considered as a simple physical barrier, but it is in a process of continual regeneration and has its own immunological, histological and nervous responses to environment.

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).

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 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 during skin diseases, such as atopic dermatitis or psoriasis.

#### **Lysozyme**

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 Grampositive bacteria (*S. aureus*), but is also active against Gram-negative bacteria, acting probably as a control of bacterial growth. Still recently, the contribution of lysozyme to cutaneaous defense was subjected to debate since it was not detected in *stratum corneum* as well as in washing fluid. However, it was recently detected in skin wash of adults, and lysozyme concentration was 5 times higher in newborn skin than in adult (Walker et al., 2008), confirming its status of antimicrobial molecule, as well as giving it an important role in preventing infections in newborn children.
