Antimicrobial peptides and their potential application in inflammation and sepsis

Starting treatment early is key to increasing survival in patients with severe sepsis and septic shock. The crucial significance of timing has been demonstrated for the treatment of circulatory failure [1], use of antibiotics [2] and use of activated protein C as adjunctive therapy [3]. Whereas it is of vital importance not only to begin anti-infective therapy as soon as possible but to also choose the adequate anti-infective drug [4], the impending problem is the growing number of multiresistant bacteria [5]. Therefore, there is an increasing interest in the identification and development of new anti-infective agents.


Introduction
Starting treatment early is key to increasing survival in patients with severe sepsis and septic shock. Th e crucial signifi cance of timing has been demonstrated for the treatment of circulatory failure [1], use of antibiotics [2] and use of activated protein C as adjunctive therapy [3]. Whereas it is of vital importance not only to begin antiinfective therapy as soon as possible but to also choose the adequate anti-infective drug [4], the impending problem is the growing number of multi-resistant bacteria [5]. Th erefore, there is an increasing interest in the identifi cation and development of new anti-infective agents.
Antimicrobial peptides (AMPs) are found in species ranging from bacteria and insects to mammals. Th ey were identifi ed over 100 years ago as an important part of innate immunity and can be isolated in body fl uids and on body surfaces either constitutively or after infl ammatory stimulation [6]. Compared to conventional antiinfective agents, some AMP may kill bacteria but also simultaneously neutralize released pathogenic factors, like lipopolysaccharide (LPS) or lipoprotein (LP), thus preventing the devastating consequences of the proinfl ammatory cascades in severe sepsis and septic shock. Th e obstacle in the application of naturally occurring AMPs is their high toxicity, promoting hemolysis, nephrotoxicity and neurotoxicity [7]. Th e challenge is, therefore, to develop synthetic peptide-based drugs on the basis of naturally occurring AMPs in order to eff ectively treat septic patients without causing harm.

Naturally occurring antimicrobial peptides in infl ammation
It has been realized for decades that AMPs have anti-Gram-positive and -negative eff ects [8] as well as antiviral and anti-yeast eff ects. Th ese eff ects are limited by several mechanisms including modulation of the surface charge and the use of active extrusion [9][10][11]. Compared to resistance against AMPs, the bacterial mechanisms to defeat conventional anti-infective agents are more evident [12].
Th e majority of studies have addressed the so-called 'defensins' , consisting of an alpha and beta subgroup. Th ere are six diff erent human alpha-defensins [6]. Th ree, highly homologous human defensins are most important: Th e human neutrophil peptides (HNP)1-3. HNP1-3 are stored in the azurophilic granules of polymorphonuclear leukocytes (PMN). HNP1-3 deliver approximately 5% of total PMN protein and comprise about 99% of the total defensin content of the neutrophils. Th eir antimicrobial activity is directed against bacteria (Gram-positive and Gram-negative) and viruses (herpes simplex virus [HSV], cytomegalovirus [CMV], human immunodefi ciency virus [HIV]-1). HNP1-3 are chemotactic and regulate the release of cytokines and complement [6].
A comparable range of effi cacy with potent antimicrobial activity against bacteria and fungi is described for LL-37. LL-37 is generated by the cleavage of hCAP18, the human cationic antimicrobial peptide of 18 kDa and is isolated by neutrophils and epithelial tissues in respiratory, gastrointestinal and urogenital tracts [6].
Th e human beta-defensins (HBD)1-3 come from a variety of epithelial cells in diff erent organs. HBD1-3 have broad antimicrobial activity. Th ey are directed against bacteria, viruses and fungi, inducing chemokines and cytokines and thus recruiting cells of the adaptive immune system [6]. HBD3 downregulates pro-infl ammatory Antimicrobial peptides and their potential application in infl ammation and sepsis cytokines, like tumor necrosis factor (TNF)-α or interleukin (IL)-6, in human and mouse macrophages after exposure to LPS in vitro and in vivo, suggesting a role for this defensin in the resolution of infl ammatory processes. In contrast to other naturally occurring or synthetic peptides, this eff ect is not mediated through direct LPSpeptide LPS binding [13]. Defi cits in the production of AMP usually expressed by human epithelial cells may lead to increased susceptibility to bacterial or viral infections [14]. Th ese defi cits may be caused by immunosuppressive drugs preventing the induction of AMP, such as HBD2, thus promoting infections [15]. Th e importance of HBD2 for protection against infections was demonstrated by Milner and Ortega who detected normal levels of HBD1 in burn patients, but the burn-associated loss of epithelium led to a decrease in HBD2 [16]. Since HBD2 eff ectively kills Escherichia coli, Staphylococcus aureus and other bacteria, reduced levels of this AMP may result in local bacterial proliferation [17].
Recently, these fi ndings were confi rmed in patients with multiple trauma [18]. Th e authors noted that despite open bone fractures and severe soft tissue trauma in multiple trauma, the rate of bacterial infection is surprisingly low and hypothesized that this may be related to serum concentrations of AMPs. Concentrations of HBD2, HBD3 and LL-37 were elevated after trauma suggesting a higher anti-bacterial eff ect compared to healthy donors thus explaining the relatively low infec tion rates [18].
Moreover, an increase or decrease in susceptibility to infl ammation may be related to the highly variable interindividual composition of AMPs in body fl uids [19]. Surprisingly, the capsular polysaccharide production in multiple Group A streptococci strains is upregulated by LL-37, thus increasing virulence [20]. LL-37 itself has functions other than chemotaxis, as shown by investi gations of the eff ects of LL-37 on human omental arteries and veins [21]. In these studies, LL-37 induced endothelium-dependent relaxation by involvement of nitric oxide (NO) of endothelial origin. A localized increase in LL-37 can be mediated by degranulation of granulocytes following the process of granulocyte 'rolling' and 'sticking' on the endothelium [22]. Despite this possible negative consequence, therapeutic application of AMPs may have positive eff ects in a variety of infl ammatory diseases. In experimental sepsis, the administration of AMP in certain doses was associated with increased survival [23]; the eff ective application of AMPs in animals was not confi rmed in human trials. Iseganan, an analog of protegrin-1, a naturally occurring peptide with broadspectrum microbicidal activity, was not shown to be benefi cial in reducing stomatitis in patients receiving chemotherapy [24].
In recent years, an increasing number of synthetic (cationic) peptides have been developed, but none has been approved for human use. Omiganan penta hydrochloride (synthetic cationic peptide; MBI 226), administered at insertion sites, was superior to povidone iodine in preventing central venous catheter-related bloodstream infections in a phase III trial; however, although the study was completed many years ago, the results have not been published in a peer-reviewed journal.
Local treatment of diabetic ulcer with AMPs had at least an equipotent anti-infl ammatory eff ect compared to antibiotics [12]. In recent years, there have been at least 20 diff erent peptides in several steps of development scheduled for anti-infective trials [23]. Two studies on human lactoferrin 1-11 peptide (hLF1-11) were aborted at Phase I and II stages. Th e purpose of these studies was to establish tolerance to treatment with hLF1-11 adminis tered intravenously as a single daily dose for 10 consecutive days. Th e target population was patients with bacteremia due to Staphylococcus epidermidis (clinicaltrials.gov-identifi er NCT00509847) or patients with proven candidemia (clinicaltrials.gov-identifi er NCT00509834). Th e reasons given for withdrawal were that recruitment was not feasible within the timeframe (bacteremia) or the patient population was not available (candidemia).

Development and anti-LPS eff ects of synthetic antimicrobial peptides
Compounds to neutralize bacterial endotoxins were originally synthesized based on the binding region of the Limulus anti-LPS factor (LALF) [25]. Ried et al. were the fi rst authors to report synthesis of peptides based on this domain. Th ese authors found that the complete binding sequence, a cyclic peptide called cLALF22, had the greatest ability to bind to the lipid A part of LPS, its "endotoxic principle" [26]. Analogs shortened down to cLALF10 were much less active. In biophysical studies by Andrä et al., interaction of the complete LALF (called ENP = endotoxin-neutralizing protein in its recombinant form) and of part structures with endotoxins was studied, and considerable inhibition of the biological activity of LPS was found at protein [ENP]:[LPS] ratios greater than 20 to 200:1 molar [27][28][29].
Data on the supramolecular structure of LPS were indicative of a change of the lipid A cubic aggregate structure into a multilamellar one. Th is is of considerable interest, since it has been shown that the cubic aggregate structure of lipid A is its bioactive form, whereas some non-enterobacterial biologically inactive LPS/lipid A structures with a diff erent acylation pattern adopt multilamellar aggregates [30]. In subsequent studies, LALFderived cyclic peptides were analyzed, starting with cLALF22 and various shortened analogs [27,29]. Consistently in all these investigations, a change in the endotoxin aggregate structure from a preferentially cubic into a multilamellar one was described, as well as a change in the morphologies of the LPS Re aggregates, as evidenced by freeze-fracture electron microscopy, from 'open eggshells' (i.e., spherical particles in the range 100 to 200 nm), into large stacks of some 1000 nm. Th e binding of the peptides to LPS was characterized in all cases as an exothermic process by isothermal titration calorimetry (ITC), driven by the Coulomb interaction of the positive charges of peptides with the negative charges of the endotoxins [27,29].
Pan et al. synthesized the terminal part of the shrimp anti-LPS-factor, a peptide with 24 amino acids, in a cyclic and linear form. Pre-treatment of mice with the cyclic compound led to considerably enhanced survival of mice infected with approximately 10 6 colony-forming units (cfu) of Pseudomonas aeruginosa. Parallel to this, the peptides were able to reduce the bacteria-induced produc tion of TNF-α in the animals. However, already at a concentration of 2 μg/ml the peptides exhibited significant cytotoxicity in HeLa, MCF-7, and HT1080 cell lines, impeding their use as an anti-infective drug [31].
Because of the insuffi cient specifi city of the above described peptides, we used a new approach, constructing a new series of peptides of amino acid lengths in the range 17 to 23. Th e systematic study of various peptides diff ering in their amino acid chain lengths from 9 to 12 and 17 to 19 showed that the basic sequence, as given by Pep9 (FRRLKWKFW), was already able to confer signi ficant inhibition of LPS-induced cytokine produc tion. Relatively high concentrations of the peptides, however, were still necessary to inhibit cytokine production. Th ere fore, longer peptides were constructed to adapt to the physico-chemistry of the lipid A part of endotoxins. For this process, the composition of the N-terminal side of the peptide was preferentially provided by polar and basic amino acids, and the C-terminal by more hydrophobic ones. Furthermore, the exact number and type and sequence of amino acids was shown to be important, as deduced from the comparison of the peptides with diff erent chain lengths [32][33][34][35].
Additional improvements were obtained by constructing the amino acid sequences on the basis of optimal binding to the lipid A moiety of bacterial LPS. Modifi cations of the sequences (for amino acid sequences see Table 1) led to a nearly complete inhibition of LPS-induced cytokine secretion at a [Pep]:[LPS] 3:1 molar ratio [33,34]. Th is was particularly evident in the case of the lead structure, Pep19-2. More experiments were performed in an in vitro assay of human mononuclear cells, studying the peptideinduced inhibition of TNF-α induction by LPS, alone and in the presence of common antibiotics as a model for combination therapy in septic patients. Th e data for the peptide alone ( Figure 1) were indicative of the strong inhibition capacity of Pep19-2.5, whereas the antibiotic alone (streptomycin, see Figure 2) showed nearly no action. Th e combination of streptomycin with Pep19-2.5 at a 1:1 weight ratio, however, was associated with strong inhibition of cytokine production, exceeding that of the peptide alone indicative of a synergistic action (Figure 2, right hand side). Th erefore, from these data, use of a combination of antibiotic and peptide in septic patients seems promising.
Furthermore, for therapeutic use, it is important that inhibition of LPS-induced cytokine release also takes place in the presence of proteases that are known to decompose peptides. Th erefore, the peptides were incuba ted in 20% AB serum for diff erent time periods. As illustrated in Figure 3, the inhibition of TNF-α production decreased with increasing time of serum incubation; however, even after 2 hours incubation there was still considerable inhibition of cytokine release.
Additional experiments were performed with sequence variants of Pep19-2.5, i.e., the duplicated form, Pep19-2.5Dup, the variant with all amino acids in a D-confi guration (Pep19-2.5D-AA), and a variant in which the fi nal sequence at the C-terminus is lacking (for sequences see Table 1). As demonstrated in Figure 4, duplication of the amino acid sequence did not lead to improvement in the inhibition activity of the peptide, and the peptide with Dconfi gured amino acids still had good inhibitory activity. Th is latter information is important with respect to the activity of proteases in blood serum, which rapidly decompose L-confi guration peptides. A further sequence variation, the peptide Pep19-2.5short, lacking the C-terminal hydrophobic sequence FWFWG, led to a near complete abrogation of any inhibitory activity ( Figure 5). Th is fi nding is of importance since the driving force between the peptides and LPS was shown to be the Coulomb interaction between the basic residues of the peptides (R and K), and the negative charges of LPS [33]. In a second step of the interaction, however, intercalation of the C-terminal proximal hydrophobic peptide part into the lipid A hydrophobic moiety was described [34]. As can be deduced from the lack of inhibition of the shortened variant, the latter process has considerable impact on understanding the high binding constant of the Pep19-2.5-LPS interaction [34]. Th erefore, from these and earlier data, the change of lipid A/LPS cubic aggregate into a multilamellar structure is an important step in the ability of the peptides to neutralize endotoxins, associated with an extremely low saturation value of binding at [peptide]:[LPS] = 0.3 M/M, corresponding to the binding saturation of 3 peptides with 10 LPS molecules [33,34]. Th us, human binding proteins, such as LPS binding protein (LBP) and CD14, are impeded from binding to LPS epitopes, the charged head groups, and thus cannot initiate the infl ammatory reaction. It has been found, furthermore, that the peptides still exert their eff ect when they are added in a time-delayed mode, i.e., when they are administered up to 3 hours after LPS addition [36]. Th is observation is indicative of a membrane process of the interaction, in accordance with recent fi ndings that the peptides readily intercalate into membranes made from phosphatidylcholine and phosphatidylserine as characteristic of eukaryotic cells [34]. After membrane incorporation, the peptides apparently act at the site of membrane receptors such as CD14 and the TLR4/MD2-system, thus competitively inhibiting the interaction with LPS. Th e details of these processes are currently under investigation. Detailed descriptions of the newest aspects of the use of AMPs compared to other therapeutic approaches are summarized in a recent review [35].

Naturally occurring and synthetic antimicrobial peptides in human and experimental sepsis
Certain anti-microbial peptides kill bacteria without causing cell disruption. Patients with septic shock, in particular, would benefi t because antibiotics are known to promote the liberation of pro-infl ammatory cell compo nents and thus augment the severity of septic shock [37,38]. A promising therapeutic approach would involve either the combination of AMP analogs with antibiotic therapy or the combination of AMPs with antibiotic drugs. Th e resultant synergism between the two antimicro bial components may be especially important for the treatment of critically ill patient with severe infections.
Studies on AMPs in patients with severe sepsis or septic shock are limited. LBP and bactericidal/permeability increasing (BPI) protein are comparable with respect to a high-affi nity binding domain for the lipid A component of LPS. Th e diff erence is that signal transduction is not hampered by LBP, because it binds to CD14 after CD14 has itself formed complexes with endotoxin. In contrast, BPI prevents endotoxin binding to CD14, hence inhibiting cytokine liberation.
In 49 patients with abscesses, peritonitis or uninfected body fl uids, LBP and neutrophil granular BPI protein were investigated. In abscesses, compared to peritoneal fl uids and non-infected fl uids, the BPI/LBP ratio was signi fi cantly elevated. Moreover, BPI concentration was increased more in abscesses with Gram-positive compared to those with Gram-negative organisms. BPI may weaken local and systemic eff ects in response to an infl ammatory stimulus induced by endotoxin release [37]. A comparison of BPI bound to the leukocyte surface and BPI and LBP in plasma in healthy volunteers after endotoxin challenge and in patients with Gram-negative sepsis was performed several years ago [39]. In both groups, there was an increase in leukocyte-bound BPI and plasma LBP. Plasma BPI was signifi cantly elevated as a sign of inadequate competition compared to the more frequently detected LBP [39]. A further study investigated BPI levels and BPI/ neutrophil ratios in 42 healthy controls and 34 patients with severe sepsis defi ned according to the ACCP/SCCM consensus conference [40]. BPI was elevated in sepsis compared to controls (median 15.3, range < 1.6-205 μg/l vs 5.2; < 1.6-24 μg/l; p< 0.001). Moreover, sepsis due to Gram-negative compared to Gram-positive pathogens was associated with higher levels of BPI (16.8, range < 1.6-205 μg/l vs. 16.0, < 1.6-60 μg/l; p = 0.05). Because of a possible association with decreased mean arterial pressure, the authors concluded that BPI may refl ect the severity of organ dysfunction in sepsis [40].
Th e development of a recombinant fragment of human BPI (rBPI21) triggered a large study on the therapeutic use of AMPs in pediatric meningococcal infection. Th ree hundred and ninety-fi ve children with suspected meningococcal sepsis were randomly assigned to receive rBPI21 within 8 hours of diagnosis [41]. Overall, the study failed to demonstrate a signifi cantly reduced mortality after rBPI21 compared to placebo (7.4% vs. 9.9%; p = 0.48). Adjusted for patients with incomplete infusion of study drug, the mortality was non-signifi cantly lower in the intervention group (rBPI21 2.2% vs. placebo 6.2%; p = 0.07). Furthermore, the rate of amputations decreased non-signifi cantly (rBPI21 3.2% vs. placebo 7.4%; p = 0.067) and signifi cantly more children regained preillness performance when treated with rBPI21 (77.3% vs. 66.3%, odds ratio 1.75 [95% CI 1.08-2.83]; p = 0.019). Th e study may have failed to reach its target to reduce mortality because of lower than expected placebo group mortality (9.9% vs. 25% expected), but the authors hypothesized that an increased benefi t may be possible by using parallel and immediate treatment of rBPI21 and anti-infective agents [41].
A prospective case-control study investigated plasma HBD2 in 16 patients with severe sepsis compared to 18 controls [42]. HBD2 was signifi cantly higher in severe sepsis compared to non-septic critically ill patients and to healthy controls. Additionally, HBD2 gene expression in non-septic patients and healthy controls was signifi cantly higher after induction with LPS compared with sepsis patients. Survival status was independent of the inducibility of HBD2 gene expression. Th is may be understood as a sign of exhaustion after severe infections and may be related to the complex dysfunction of the innate and adaptive immune system in patients suff ering from severe sepsis [43]. Administration of intravenous hydrocortisone as adjunctive therapy in septic shock reduced HBD3 expression in contrast to undisturbed HBD2-inducibility [43].
An additional study in children with sepsis and critically ill control patients revealed that levels of HNP1-3 (α-defensins) were increased with onset of sepsis in plasma of non-neutropenic septic patients (median     [44]. Recently, Berkestedt et al. demonstrated that levels of HNP1-3, lactoferrin, BPI and heparin-binding protein (HBP) were higher in 31 patients with sepsis compared to 25 non-septic controls [45]. Out of these AMP, only BPI was associated with outcome, revealing signifi cantly higher levels in non-survivors compared to survivors. Th e neutrophil granula-derived HBP, BPI and HNP1-3 and lactoferrin were increased in sepsis, thus refl ecting the ongoing battle of the innate immunity against invading organisms [45].
Th e use of polymyxin B and polymyxin E (colistin), cationic polypeptides that neutralize the lethal eff ects of endotoxin, was abandoned, because nephrotoxicity and neurotoxicity were reported. However, these adverse eff ects observed in early clinical studies were most likely due to a limited understanding of the pharmacokinetics, pharmacodynamics, and toxicodynamics of these agents, and the use of incorrect quantities [46]. Use was reinstated in a recent study of hemoperfusion with polymyxin-B loaded cartridges in patients with abdominal septic shock (EUPHAS) [47]. In this Italian multicenter trial, 64 patients were assigned to receive standard of care or standard of care plus 2 sessions of polymyxin-B hemoperfusion within 72 hours after the surgical procedure. In addition to improvements in mean arterial pressure and organ dysfunction, 28-day mortality was lower in the polymyxin B group compared to conven tional therapy alone (32% vs. 53%, unadjusted hazard ratio [HR] 0.43; 95% CI 0.20-0.94; adjusted HR, 0.36; 95% CI 0.16-0.80). In view of the mortality reduction the study was stopped by the ethics committee. To confi rm the fi ndings in other patients and to address concerns raised after publication of the EUPHAS study, two further studies are underway (EUPHRATES: clinicaltrials.gov-identifi er NCT01046669 and EUPHAS 2 [48]).
Recently, it was demonstrated that a single 25 mg/kg dose of a synthetic peptide protected mice from infection with P. aeruginosa and E. coli [49]. Th e application of the AMP led to elimination of bacteria in the blood 18 hours after infection, and the bacterial count was signifi cantly lower in other specimens (peritoneal fl uid, spleen and liver) compared to control animals. After an additional 22 hours all tested samples were free of bacteria [49]. A further synthetic AMP, named s-thanatin, showed activity against Gram-positive and -negative bacteria [50]. After combination with the antimicrobial peptide it was demonstrated that the minimum inhibitory concentration (MIC) of various antibiotics was decreased by a factor of 2 to 8 (1-3 dilution steps). Moreover, survival after intraperitoneal bacterial challenge was increased after treatment with s-thanatin, in a dose-dependent manner [50]. Th e most recent study is investigating talactoferrin alfa in a phase III study in patients with severe sepsis (OASIS; clinicaltrials.gov identifi er NCT01273779).

Conclusion
Investigations of the past decade show increasing interest in antimicrobial peptides as a tool for sepsis diagnosis and furthermore as a possible therapeutic intervention. Newly designed peptides with decreased toxicity and a broader range of effi cacy may have the potential to provide signifi cant improvements in the treatment of infections.