Antibacterial activity: APs show antimicrobial activity against a broad range of microorganisms including Gram-positive and -negative bacteria [35], and there are several hypotheses describing mechanism function. The initial contact between the AP and the microbe is electrostatic, because of the anionic bacterial surface. Firstly, the positively charged cationic peptides (APs) bind to the negatively charged cellular membrane components from the bacterial cell wall such as lipopolysaccharide. The amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’ or ‘toroidal-pore’ mechanisms (see Figure 1; [36]). For the barrel-stave, the peptides aggregate and insert into the membrane bilayer in order to align the hydrophobic peptide regions with the lipid core region. The hydrophilic peptide regions form the inner core of the pore. For the toroidal, the peptides aggregate and induce the lipid monolayers to bend continuously through the pore so that the water core is lined by both the inserted peptides and the lipid head groups and for the carpet channel, the peptides disrupt the membrane by orienting parallel to the surface of the lipid bilayer and forming an extensive layer or carpet. This results in the formation of a transient channel, micellarization, dissolution of the membrane or translocation across the membranes causing an increase of the membrane permeability. The permeability allows an efflux of essential ions and nutrients leading to rapid cell death [37]. In contrast to prokaryotic membranes, mammalian membranes are enriched with zwitterionic phospholipids (neutral in net charge). Moreover the presence of cholesterol, a major constituent of mammalian membranes, can reduce the activity of APs by stabilizing the lipid bilayer or by directly interacting and neutralizing the APs [38]. The composition of the membranes is likely to provide an important determinant for the AP.

A growing body of evidence indicates that the activity of APs is mediated by intracellular targets. AP molecules pass through and dissociate from the membrane and bind to intracellular targets (Figure 1). They mediate the activation of autolytic enzymes, the inhibition of cell wall biosynthesis and synthesis of DNA, RNA and protein [39]. These intracellular mechanisms can act independently or synergistically with membrane permeabilization. Nevertheless, interaction with the bacterial cell membrane appears to be the killing mechanism of the vast majority of APs [40].

Defensins show microbicidal activity against Gram-positive and -negative bacteria and against various yeast strains such as the opportunistic pathogenic yeast Candida albicans. Interestingly, studies indicate that defensins accumulate at a significantly higher rate and to a greater extent in bacteria- and C. albicans-infected lesions in mice and rabbits compared to non-infected, but inflamed, tissues. These data indicate that peptides distinguish between microorganisms and host tissues, and in doing so, accumulate at sites of infection in vivo [41]. The infection results in a reduced pH, which facilitates the accumulation of the AP.

For cathelicidins, a correlation between their antimicrobial activity and their structure has been observed. A greater extent of α-helical conformation is beneficial for their ability to kill both Gram-negative and -positive bacteria [42]. The cathelicidins from sheep and cows, sheep myeloid antimicrobial peptide (SMAP-29) and bovine myeloid antimicrobial peptide (BMAP) respectively, along with the two-disulfide bridged protegrins from pigs, are among the most rapid and potent APs [15]. Another important feature of these peptides is their capacity to bind and neutralize bacterial lipopolysaccharide in vitro, which may account for the ability of exogenously administered peptides to protect against sepsis in vivo [43,44]. However, several studies indicate that their activity is sensitive to alterations in the assay conditions, including salt, pH and the bacterial growth phase [45]. Cathelicidins have cytotoxic effects on eukaryotic cells but this is neutralized by serum components. It is suggested that serum lipoproteins play an important role in protecting human cells from damage caused by cathelicidins [42].

Among histatins, histatin 5 has the strongest antimicrobial activity focusing most of the research on this peptide. It has potent antifungal activity against the pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus [12]. Like cathelicidins, histatin 5 inhibits the leukotoxic activity of Actinobacillus actinomycetemcomitans [47] and inhibits host and bacterial enzymes implicated in periodontal diseases [48].

Interesting insights into the importance of APs have been shown by several approaches. On the one hand, depletion of AP has been linked to several pathologic disorders. For example patients with specific granular deficiency syndrome lack α-defensins and suffer from severe and frequent infections [49]. A deficiency in cathelicidin peptide LL-37 and neutrophil peptides (HNP1-3) in humans known as Morbus Kostmann causes patients to suffer from frequent oral bacterial infections and severe periodontal diseases [50]. On the other hand, β-defensin-1 and CRAMP (cathelin-related antimicrobial peptide; cnlp) gene depletion in knockout mice results in a higher susceptibility to infections and the failure to clear them [9,51,52].

Antiviral activity: In addition to their antibacterial activity, APs possess antiviral activity. The α-defensin HNP-1 inhibits the replication of human immunodeficiency virus (HIV) and influenza virus following viral entry into target cells [53,54]. Similarly, HNP-1 can inactivate papillomavirus, herpes simplex virus, cytomegalovirus, vesicular stomatitis virus and adenovirus [55,56]. Other APs such as human β- and synthetic θ-defensins (Retrocyclin 2) or cathelicidins also block HIV-1 replication and influenza virus infections [57,58,59]. A recent paper using β-defensin-1 (BD-1) deficient mice showed that BD-1 plays a role in preventing viral replication in immune cells [60]. Besides inhibiting the replication of viruses, defensins inhibit HIV entry into cells by antagonizing the virus proteins that fuse with target cells (glycoprotein gp120/gp41—env) or the cellular receptors involved in virus internalization (chemokine receptor CXCR4) [61,62]. Altogether, several studies show AP potential as broad anti-viral agents. In contrast to antibacterial activity, the antiviral activity seems to depend on an interaction with virus proteins. However, the underlying mechanisms of virus inhibition by APs are far from clear and further investigation needs to be done.

Antifungal activity: Various APs show antifungal activity. The number of identified peptides has been increasing steadily in recent years. The increase in mycoses, the development of resistance to available drugs among fungal pathogens, as well as these drugs’ adverse side effects have led to increased efforts to explore the antifungal activity of APs and to identify new candidates for therapy. The mechanisms of AP antifungal action include cell lysis by binding and disruption of the outer membrane, interference with cell wall synthesis and induction of depolymerization of actin cytoskeleton [63,64]. The suggested mechanisms seem to be similar to antibacterial models of the AP. Interestingly, in contrast to antiviral peptides, where it appears to be nearly impossible to predict antiviral activity based on secondary structures of the peptide, antifungal peptides tend to be relatively rich in polar and neutral amino acids [65]. Recent results showed that the formation of α–helical and/or β sheet secondary structures may increase the amphipathicity of the APs and enable them to act specifically with their targets in the fungal membrane [66]. However, the precise mechanisms are not fully understood. For HNP-1 and -2 as well as rabbit NP (1–3), significant activity against Candida spp. has been shown [67,68]. Furthermore, the histatins and cathelicidins also show a high efficiency against Candida and Cryptococcus spp. [69,70,71]. To date, at least 100 different APs have been investigated for their antifungal activity [72]. As such, APs may represent a new generation of antifungal agents.

Antiparasitic activity: With the identification of the Magainins in the 1980’s, studies have shown antiparasitic activity for this AP [73]. Since then, many APs with antiparasitic activities have been identified. At the same time, studies have shown the efficiency of defensins and cathelicidins against the African trypanosome Trypanosoma brucei, the cause of sleeping sickness, by disrupting their cell membrane integrity [24]. More recently, BMAP-18, a truncated form of the bovine myeloid antimicrobial peptide-27 (BMAP-27), exhibited strong action against several parasites, including trypanosomes and Leishmania spp. [74]. At low concentrations, BMAP-18 disrupts mitochondrial potential without obvious alteration of parasite plasma membranes, whereas at higher concentrations membrane lesions in the parasites are induced [74]. Studies have suggested that several APs exhibit antiparasitic modes of action resembling their antibacterial, antiviral or antifungal modes of action. For example, the cathelicidin-derived AP PMAP-23 (porcine myeloid antimicrobial peptide-23) exerts antinematodal and antifungal activities by disrupting the cell membrane by pore formation [75]. However, structure–activity relationship studies revealed that the antiparasitic activities of APs may be dependent on certain peptide motifs. These motifs differ from those required for antibacterial, -viral and -fungal activities [65].

In addition to their role as antimicrobial agents, APs participate in multiple aspects of immunity. Early in 1989, Territo et al. [76] demonstrated that neutrophil derived α-defensins were chemotactic towards human monocytes for the first time. Over the years, many additional features of APs have been discovered. At present, the hypothesis that immunomodulation is the primary task of the AP is advancing. That the minimal required microbicidal concentrations were rarely found in vivo supports this hypothesis. However, some studies showed local high concentrations of AP at sites of infection and inflammation which were sufficient for a direct killing [77]. Also, the high intracellular AP concentrations in phagocytes, such as neutrophil granulocytes, clearly contribute to direct killing of ingested microbes [34]. Furthermore, several studies have shown the sensitivity of antimicrobial activity to cationic concentrations, serum and anionic macromolecules [78], whereas the immunomodulatory activities appear to be less sensitive [79]. The influence of these functions on innate immunity could represent a novel adjuvant therapy in addition to direct antimicrobial activity. In this context, APs can enhance the potency of existing antibiotics in vivo, probably by facilitating access of antibiotics into the bacterial cell resulting in a synergic effect [80]. Furthermore, APs could act as adjuvants stimulating innate immunity by three basic mechanisms: enhancing recruitment of immune cells to the inflamed site, promoting the activation of those cells and polarizing them to achieve the desired response (T helper cells). Altogether, this results in an increase of innate and also adaptive immunity in response to infections. Figure 2 shows a summary for the induction and potential biological role of AP.

APs are primarily chemotactic for immune and non-immune cells. Defensins including α-defensins (e.g., HNP1-3) and β-defensins (human β-defensin 3 and 4; HBD3 and 4) recruit phagocytes, neutrophil granulocytes and monocytes to the site of inflammation [81]. In addition, HBD1 and HBD3 are chemotactic for immature dendritic cells (iDC) and memory T-cells, whereas human α-defensins selectively induce the migration of human native CD4+, CD45+ and CD8+ cells [81]. The human cathelicidin LL-37 is chemotactic for monocytes, T-cells and neutrophils, but not for dendritic cells. Interestingly, recruitment depends on the G-protein coupled receptor (GPCR) formyl peptide receptor-like 1 (FPRL1; [82]). LL-37 suppresses neutrophil apoptosis via this receptor [83]. Concerning defensins, HBD2 recruit memory T-cells and iDC via GPCR CC-chemokine receptor 6 [84]. In addition to the chemotactic activity, defensins induce mast cell activation, including degranulation, increased prostaglandin D2 production and intracellular Ca²⁺ mobilization [85]. Beside the direct chemotactic effect, it has been shown that both defensins and cathelicidins act indirectly by inducing chemokines such as Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-3α (MIP-3α; CCL20) and interferon-γ inducible protein-10 (IP-10; CXCL10) in human epidermal keratinocytes [86]. Furthermore, recent studies of LL-37 showed an interesting dual role in binding DNA (and RNA) to influence inflammatory response. On the one side, LL-37 is able to bind self-DNA and RNA which activates plasmacytoid dendritic cells (pDCs) in the skin disease psoriasis [87,88]. On the other side, it has been shown that LL-37 interacts and neutralizes psoriasis-associated cytosolic DNA in keratinocytes and blocked AIM2 (DNA sensor interferon-inducible protein absent in melanoma 2) inflammasome activation [89].

APs also modify pro- and anti-inflammatory cytokine expression to modulate immune response. They influence the balance between induction of inflammation, and at the same time protect the organism from the detrimental effects of an excessive inflammatory response. For example, LL-37 induces potent pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in immune and non-immune cells, as well as anti-inflammatory cytokines such as IL-10 in brain glial cells or macrophages [90,91]. Furthermore LL-37 suppresses pro-inflammatory response, including both extracellular neutralization the bacterial cell wall component lipopolysaccharide (LPS) and inhibition of LPS-induced cellular response such as TNF-α, nitric oxide and prostaglandin E2 [92,93]. The cytokines themselves are able to increase or decrease AP expression. While TNF-α, IL-1β and IL-6 lead to an increase in the expression of cathelicidin and defensin, IL-10 depresses AP production [10,20,94,95].

The influence of cytokine production, the recruitment of dendritic cells and monocytes to the site of injury, the enhancement of phagocytosis and the maturation of dendritic cells are mediated by APs to modulate adaptive immune functions [90]. All of these effects augment the uptake, processing and presentation of antigens and stimulate the clonal expansion of T-lymphocytes and B-lymphocytes. Previous studies have shown that co-administering of α-(HNP-1-3) and β-defensins (human BD-1 and 2) enhance ovalbumin-specific immunoglobulin G (IgG) response in mice [96,97]. Along with increased iDC maturation, APs induce more effective antigen presentation and subsequent T-cell activation. At the same time, Mader et al. demonstrated that LL-37 induces apoptosis in cytotoxic and regulatory T-cells [98].

Interestingly, CRAMP-deficient mice were more susceptible to pathogenic skin infection [18]. In addition, LL-37, HBD-2 and 3 are highly expressed in epidermal keratinocytes in response to injury or infections of the skin [99,100]. Therefore, it has been suggested that APs play an important role in maintaining the skin barrier and possibly are involved in its restoration. The hypothesis is supported by several findings: Treatment with HBD-3 induced re-epithelialization of wounds in a porcine model [101]. Growth factors such as insulin-like growth factor I and the transforming growth factor-α have been shown to induce the expression of LL-37 or HBD-3 [99]. Subsequently, the induced APs activate epithelial cells and fibroblasts to form granulation tissue and act chemotatic for macrophages and other immune cells [99]. At the same time APs increase growth factors and cytokine expression in the cells of the skin barrier that induce and control wound healing [8] In addition, Koczulla et al. detected that LL-37 mediates angiogenesis and vasculogenesis [102]. HBD-3 is also involved in tissue remodeling by increasing matrix-metalloproteases protein expression [94]. Altogether, APs appear to be promising candidates for new therapeutic approaches in wound healing. Interestingly, we have been able to show that CRAMP expression in rat glial cells is induced by different neurotrophic factors, for example by neurotrophic growth factor (NGF). CRAMP stimulation also results in an increase in NGF and other neurotrophic factors [103]. We hypothesize that CRAMP is involved in brain protection and promotes neuronal survival following bacterial infection of the brain. Furthermore, recent studies show anticancer activities for APs. For example buforin IIb, a 21-amino acid AP derived from histone H2A, has been shown to induce mitochondrial-dependent apoptosis of tumor cell lines and suppressed growth of tumors implanted in mice [104].