INTRODUCTION
Sepsis is a major challenge in medicine. Despite enhanced antibiotics and the best available supportive care, sepsis remains one of the most common life-threatening complications in intensive care units (1, 2). According to studies from North America, the incidence is about 3 cases per 1,000 population, an annual burden of 750,000 cases in the United States resulting in 215,000 deaths (3, 4). It is estimated that severe sepsis is responsible for 2% to 11% of all admissions to intensive care units (2, 3). The hospital mortality rate associated with severe sepsis and septic shock (30% and more than 60%, respectively) has not changed much during the last decades (2, 3), and deaths are as frequent as from acute myocardial infarction (3).
Sepsis develops when the first moderate inflammation in response to infection becomes systemic and unregulated (2). In cases of severe sepsis and septic shock, this leads to organ dysfunction, perfusion abnormalities, and hypotension (5), which can be associated with multiple organ dysfunction syndrome, the most common cause of death in patients with sepsis (6).
Because of alarmingly high mortality rates, critical care measurements and new therapeutic approaches for patients with sepsis are the focus of research (3). Recently, new consensus guidelines for the management of sepsis have been published (7), detailing the importance of early goal-directed therapy and later measurements such as lung-protective ventilation, broad-spectrum antibiotics, low-dose corticosteroids, and possibly activated protein C (6). Broad-spectrum antibiotic therapy is one of the most important cornerstones in the management of sepsis because it is usually a result of bacterial infections (2). However, common antibiotic therapy often becomes insufficient because infections with resistant germs are more prevalent (8). The prevailing theory of the pathogenesis of sepsis, the development of an unregulated systemic hyperinflammatory response or immunoincompetence (4) that gradually directs against the host's organism and thus leads to massive tissue destruction, indicates that sufficient therapy includes the eradication of responsible germs and control of the host's own immoderate inflammation response (8-10).
Currently, there is a major effort to develop new substances that combine antimicrobial activity similar to common antibiotics with immunomodulating and anti-inflammatory properties such as anticytokine therapies (tumor necrosis factor-α, interleukin-1) and antiendotoxin strategies using antibodies against endotoxins (12,13). Unfortunately, these approaches often have failed clinically (14), and in many cases, the efficacy of these treatments depends on the severity of sepsis. Short cationic amphiphilic peptides present another promising approach.
ANTIMICROBIAL PEPTIDES
Antimicrobial peptides include all oligopeptides or polypeptides that can kill microbes or inhibit their growth (15). Consequently, this definition also includes all non-ribosomally synthesized peptides, which are primarily produced by bacteria, are well known, and are already used for clinical applications such as penicillin, cyclosporine, cephalosporins, and polymyxin B (15). In a more narrow sense, antimicrobial peptides are defined as ribosomally derived (15) and comprising between 12 and 50 amino acids with 2 to 9 positively charged lysine or arginine residues and up to 50% hydrophobic amino acids (16, 17). More than 900 of these cationic peptides have been discovered to date (18). They exist in organisms from insects to plants to mammals and nonmammalian vertebrates. Antimicrobial peptides show an exceptional broad spectrum of activity, ranging from gram-negative and gram-positive bacteria to fungi and viruses (19-21). In humans, two major classes of antimicrobial peptides in mammals have been described: defensins and cathelicidins (22). hCAP-18/LL-37, the only endogenous cathelicidin that is found in humans to date, is one of the best-investigated cationic peptides. It is a major protein of the specific granules in neutrophils, but also occurs in monocytes, testes, human keratinocytes, and airway epithelia (23). Defensins comprise 30% to 50% of the granule proteins in human neutrophils (24) and can structurally be distinguished in α and β defensins (25): α defensins are mainly expressed by neutrophils and intestinal Paneth cells (25), whereas β defensins are primarily produced by epithelial cells of the skin, kidneys, and trachea-bronchial lining (25).
The mammalian antimicrobial peptides are stored as propeptides in cells and released into extracellular space upon stimulation (15, 24). At body sites such as epithelial tissues that are steadily exposed to potential infectious microbes, there is a constitutive production of cationic peptides such as human β defensins (15). Secretion is induced by the contact of cells with microbes and proinflammatory mediators (15). Elevated levels of β defensins could also be detected in human body fluids during inflammatory diseases, such as pneumonia and urinary tract infections (15, 26, 27). Evidence for the key role of cationic antimicrobial peptides includes mutations of Drosophila and genetic inactivation of single murine genes that led to a decrease in secretion of cationic peptides and simultaneously to increased susceptibility to fungal or bacterial infections (23, 28, 29). These findings suggest an important role of cationic antimicrobial peptides in host defense, and exploiting the potential of these molecules has developed in recent years. This review will give an overview about the role of cationic effector molecules of the innate immune system in pathogenesis and development of sepsis. Thus, we discuss the promise and challenges for the use of antimicrobial peptides in sepsis. Finally, this review focuses on host defense peptides as a potential future therapy for sepsis, discussing its perspectives, difficulties, and current limitations.
FROM ANTIMICROBIAL PEPTIDES TO HOST DEFENSE PEPTIDES
The first discovered property of these cationic peptides was their direct antimicrobial activity, which was thought to be decisive for their role in innate immunity and host defense. Virtually all cathelicidins and defensins show direct antimicrobial activity in vitro (16). They possess broad-spectrum antimicrobial effectiveness including direct killing of gram-negative and gram-positive bacteria, fungi, and parasites (17). Cationic antimicrobial peptides have been described as membrane-targeted agents; many of them adopt a secondary α-helical peptide structure (30, 31) and penetrate the outer membrane of gram-negative bacteria by self-promoted uptake mediated by hydrophobic or electrostatic interactions with lipid A, polyanionic surface LPS, or anionic phospholipids (17, 32). Associating with the negatively charged phospholipid membrane, they form transmembrane channels, which cause changes in permeability and therefore may kill bacterial cells (33). In addition, intracellular targets like DNA can be affected, which ultimately leads to cell destruction (33). Although antimicrobial activities of many cationic peptides are well described under in vitro conditions, their activity invivo is less clear (16). One contradiction against the assumption that most peptides support innate immunity mainly by direct antimicrobial activity is that concentrations found at body sites such as mucosa are relatively low compared with those used in successful in vitro studies (34). Moreover, it has been shown that direct killing of microbes in vitro is often hampered by physiological salt concentrations and monovalent and divalent ions such as Mg2+ or Ca2+ and serum (35, 36).
This supports the theory that direct antimicrobial activity may be only one aspect of how cationic peptides support innate immunity. Recently, more attention has focused on investigating alternative properties.
To demonstrate that antimicrobial activity was not necessarily required for protection in vivo, Bowdish et al. (35) synthesized model peptides tested for antimicrobial and immunomodulatory activities. A peptide with no antimicrobial activity was found to be protective in animal models of Staphylococcus aureus and Salmonella infection, implying that host defense peptides can protect the host by exerting immunomodulatory properties. These findings do not prove, but at least indicate, an alternative mode of action in anti-infective immune response (35). A more recent study by Cirioni et al. (37) showed that hCAP-18/LL-37 can protect rats against lethal sepsis caused by gram-negative bacteria. In different rat models of peritoneal sepsis, hCAP-18/LL-37 turns out to be at least as successful as three different commonly used antibiotics in reducing lethality rates. However, it is less potent in lowering bacterial counts in organs and blood (37). These results confirm the hypothesis that hCAP-18/LL-37 seems to act as a successful modulator of the immune response. Regarding these properties, antimicrobial peptides are increasingly labeled host defense peptides.
INNATE IMMUNITY IN SEPSIS
The detection of the presence of microorganisms and the rapid initiation of immune cascades is essential to compete with invading infections efficiently (38). However, excessive inflammatory response can induce sepsis and septic shock and is related with high mortality (9, 39).
Innate immunity is the first line of defense against invading microorganisms; cells like macrophages or monocytes express receptors (toll-like receptors, CD14) on their surface to recognize foreign antigens by specific molecular patterns (1, 39). Binding between microbial pathogens and pattern recognition receptors stimulates an inflammatory response. Several intracellular pathways are activated and result in the stimulation of transcription factors (nuclear factor-κB [NF-κB]), controlling the expression of immune response genes (40). The release of effector molecules such as cytokines induces inflammatory reaction by recruitment of other immune competent cells. However, bacterial patterns can provoke an overwhelming immune reaction, which can cause severe injury in the host organism. According to current thinking, many different factors such as overwhelming production of proinflammatory and anti-inflammatory mediators (hyperinflammatory and hypoinflammatory response) contribute to the pathological processes of sepsis and may lead to organ failure and death (4, 6, 41). Major microbial components causing inflammatory responses include endotoxins of gram-negative and gram-positive bacteria (cell wall components such as peptidoglycans, lipoteichoic acids), and lipoarabinomannan of mycobacteria (39).
GRAM-NEGATIVE SEPSIS PATHWAY
The most common gram-negative microbes isolated from patients with severe sepsis and septic shock are Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa (42).
The LPS is an important component of the outer membrane of gram-negative bacteria and presents one of the major virulence factors of bacteria that stimulate inflammation (1, 39). The LPS-binding protein (LBP), a plasma protein, controls the process leading to activation of innate immune cells (43). Released from dead gram-negative bacteria, LPS is present as an aggregate because of its amphiphilic structure. The LBP binds to LPS, transforms it into monomers, and transfers to CD14, a receptor for LPS on the surface of circulating monocytes. The CD14 interacts with toll-like receptors ([TLRs] such as TLR4) and therefore activates intracellular cascades resulting in the release of proinflammatory mediators (6, 44). Furthermore, LBP was shown to detoxify and neutralize LPS by transferring into high density lipoproteins (HDL) in vitro (45). Injected intraperitoneally into mice, LBP inhibited LPS-induced cytokine release and therefore decreased the mortality rate of mice in septic shock (45).
GRAM-POSITIVE SEPSIS PATHWAY
In the 1960s and early 1970s, most bloodstream infections were caused by gram-negative bacteria (42). This trend changed during the 1980s, when the proportion of gram-positive infections increased (42). Today, gram-positive infections are almost as frequent as gram-negative ones, which has been mirrored by an increase in the prevalence of gram-positive sepsis (5, 42). A variety of gram-positive bacteria cause sepsis, most frequently staphylococci (S. aureus and coagulase-negative staphylococci), enterococci, and pneumococci (5, 42).
Because gram-positive bacteria are very heterogeneous, it is difficult to determine one main agent (such as LPS in the case of gram-negative sepsis) responsible for initiating immune response (5). Principally, gram-positive bacteria are capable of causing sepsis by two different mechanisms: by producing exotoxins that act as superantigens, or by components of their cell walls stimulating immune cells (1).
Gram-positive bacteria lack both an outer membrane and endotoxin (5). However, their cell wall consists of certain major components that can stimulate immune cells, namely peptidoglycan and lipoteichoic acid (LTA). Both have been shown to induce cytokine production in monocytes by a CD14-dependent pathway (1, 39). Similarly to LPS, peptidoglycan and LTA bind to TLRs on the surface of monocytes and induce intracellular signaling, resulting in secretion of nitric oxide (NO) and proinflammatory cytokines as tumor necrosis factor (TNF)-α.
Considering the impact of endotoxin and cell wall components of gram-positive bacteria in the pathogenesis of sepsis, researchers spent much effort finding substances able to neutralize their proinflammatory response. The urgency of new therapeutic approaches is further emphasized by the fact that common antimicrobial substances have been shown to trigger additional release of endotoxin (46). Animal models of gram-negative and gram-positive sepsis indicate that antibiotics can stimulate the release of biologically active cell wall components (46-48). After application of β lactams, cytokine plasma levels are increased because of LPS-mediated influence (46, 48). After sepsis development as described previously, antibiotics may worsen the patient's condition with their effect on endotoxin levels, although there is an ongoing discussion if the stimulation of cytokine secretion and the release of endotoxin correlate among mortality in patients with sepsis (46). However, developing new substances that can neutralize proinflammatory bacterial cell wall components promises the therapeutic ability to alleviate the complications of sepsis caused by uncontrolled inflammation.
ENDOTOXIN BINDING OF HOST DEFENSE PEPTIDES
Research indicates that host defense peptides can neutralize the effect of gram-negative and gram-positive cell wall components. Human CAP18/LL-37 is one of the best-investigated endotoxin-neutralizing host defense peptides. In vitro murine macrophages or human monocytes were incubated with LPS derived from E. coli, Salmonella typhimurium, or P. aeruginosa in the presence or absence of hCAP18/LL-37, and levels of TNF-α and NO were measured by enzyme-linked immunosorbent assay (22, 23, 37). Physiological concentrations of hCAP-18/LL-37 (≤1 μg/mL) could inhibit greater than or equal to80% of LPS-stimulated TNF-α production (22). Despite the presence of serum, treatment with hCAP-18/LL-37 still resulted in a TNF-α reduction by 70% (23). And the production of NO was dose-dependently decreased by 45% and 90% (8 μg/mL and 24 μg/mL, respectively) (37). Furthermore, hCAP-18/LL-37 also showed a similar reduction of TNF-α production when cells were stimulated with LTA of S. aureus instead of LPS (23). Various α-helical cecropin/melittin hybrid peptide (CEME) have also been shown to inhibit LTA-induced cytokine secretion (49).
The property of host defense peptides to inhibit endotoxin-induced cytokine and NO release and therefore proinflammatory stimulation of the immune system has been widely investigated, and different mechanistic steps have been found, ranging from direct neutralization to altered gene transcription. Larrick et al. (50) demonstrated that CAP18/LL37 is expressed in normal human granulocytes and that the C-terminal portion of human CAP18 binds to LPS, neutralizes LPS-mediated activation of monocytes, and protects mice injected with lethal quantities of LPS. This mechanism of host defense peptides neutralizing endotoxin, by direct binding to LPS, was confirmed by various studies (51-54) and specified by Scott et al. (55), showing that structurally different cationic antimicrobial peptides block the interaction between LPS and LBP, inhibiting transport and binding of LPS to the CD14 receptor. This interaction generally correlated with the ability of cationic peptides to decrease LPS-induced TNF-α production by RAW 264.7 macrophage cells (23, 55). Rosenfeld et al. (51) reported that some endotoxin-binding peptides can dissociate LPS aggregates, preventing the binding to the LBP and CD14 receptor. The direct LPS-binding and endotoxin-neutralizing effect of many host defense peptides can be associated with their antimicrobial activity, and a correlation between both properties is proposed (51); the key mechanistic step may be the binding between the polycationic peptides and the negatively charged residues of LPS by electrostatic and hydrophobic interactions (48). However, although most LPS-neutralizing cationic peptides show antibacterial activity simultaneously (particularly against gram-negative bacteria), not every host defense peptide has the property to neutralize endotoxins (9).
Nagaoka et al. (56) reconfirmed the LPS-binding activity of CAP18 and the inhibiting effect on both the binding of LPS to LBP and on the TNF-α expression by murine macrophages. However, the researchers also found that preincubation of cells with CAP18 inhibits the binding capacity of LPS to RAW264.7 macrophages (56). To confirm the assumption that both CAP18 and LPS use similar binding sites on the cells, anti-CD14 antibodies that can recognize the murine CD14 epitope and inhibit the binding of LPS to CD14+ cells were used (57). The binding of anti-CD14 to macrophages was similarly inhibited when cells were preincubated with LPS or CAP18, indicating the competition between peptide and endotoxin on the binding site within the CD14 receptor (56). Recently, Rosenfeld et al. (51) confirmed these findings.
IMMUNOMODULATORY FUNCTIONS OF HOST DEFENSE PEPTIDES
It has become more and more clear that host defense peptides play an important immunomodulatory role in innate immunity (Table 1). As described previously, many of them neutralize endotoxin and therefore inhibit the production of proinflammatory cytokines such as TNF-α and IL-6. In addition to their endotoxin-neutralizing properties, they interact directly with eukaryotic cells (35) and can induce alterations in the transcription of hundreds of genes in cells of the innate immune system (34). In 2002, Scott et al. (23) investigated the transcriptional responses of macrophages to hCAP-18/LL-37. RAW 264.7 cells were incubated for 4 h with medium or 50 μg/mL hCAP-18/LL-37 and analyzed by complementary DNA expression arrays and semiquantitative reverse transcription-polymerase chain reaction. The surface expression of receptors was additionally confirmed by flow cytometry, and chemokine secretion was measured by enzyme-linked immunosorbent assay. The authors reported that hCAP-18/LL-37 treatment upregulated the expressions of at least 30 genes, including receptors, cell surface antigenes, and other genes that encode for cell-cell communication (such as cytokines, chemokines, IL). Simultaneously, at least 20 genes were downregulated by hCAP-18/LL-37 treatment, including several inflammatory mediators like macrophage-inflammatory protein and IL-12. The authors anticipated that cationic peptides can have reverse outcomes, namely proinflammatory and anti-inflammatory effects. On the one hand, they may stimulate the recruitment of additional immune cells and promote leukocytes to combat bacterial infections by upregulating the production of chemokines such as IL-8 and monocyte chemoattractant protein 1 (MCP-1), and chemokine receptors such as CXCR-4 and CCR-2. On the other hand, host defense peptides seem to function as feedback inhibitors of the innate immune response, downregulating proinflammatory mediators like TNF-α. The authors hypothesize that the influence could be determined by different peptide concentrations at sites of bacterial infections (23). In a more recent study by Mookherje et al. (22), the effect of hCAP-18/LL-37 on LPS-induced gene expression profile in monocytes was investigated. THP-1 cells were stimulated with LPS in the presence or absence of 20 μg/mL of hCAP-18/LL-37, and gene expression in response after different time points was calculated using microarray analysis. The authors described more than 160 genes that were upregulated in LPS-stimulated cells and could often be identified as proinflammatory, which were suppressed in the presence of hCAP-18/LL-37. The TLR4-to-NF-κB pathway plays a major role in LPS-induced inflammatory responses and pathogenesis of gram-negative sepsis. They further investigated associated genes and described that LPS generally upregulates genes encoding elements of the TLR4-to-NF-κB-pathway, although hCAP-18/LL-37 generally suppresses this stimulation. Whereas the expression of TNF-α and other proinflammatory genes was substantially reduced, the expression of chemoattractants like IL-8, CCL4, CXCL-1, and certain anti-inflammatory genes (negative regulators of the TLR4-to-NF-κB pathway) were only slightly reduced (22). Thus, the authors concluded that the dumping effect of hCAP-18/LL-37 on LPS-induced gene transcription is selective (22). Scott et al. showed that CEME, a model cationic peptide derived from parts of the silk moth cecropin and bee melittin peptides (58), blocked at least 30% of genes responsible for elevated levels of inflammation mediators after stimulation of RAW macrophages with LPS (17). In addition, CEMA also had a pleiotropic effect on the macrophage gene expression by upregulating genes encoding for cell surface antigens and adhesion proteins (17).
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Table 1: Major biological activities of the human host defense peptides LL37 and the human β defensins 1-3
These studies showed that host defense peptides can alter the gene transcription of immune cells to suppress the production of inflammatory mediators. Simultaneously, they stimulate the secretion of chemokines and chemokine receptors, promoting leukocytes to combat bacterial infections. Furthermore, host defense peptides show chemotactic activity to monocytes and neutrophils (59, 60). Yang et al. identified the receptor that is used by hCAP-18/LL-37 as formyl peptide receptor-like 1 and found that the chemotactic activity of LL37 is not significantly inhibited by the presence of human serum (60). Moreover, the cathelicidin hCAP-18/LL-37 and human β defensin 2 have been shown to induce mast cell chemotaxis, degranulation, and histamine release (61), which is less significant in the pathogenesis of sepsis than in chronic or allergic inflammation. Other studies confirmed that host defense peptides such as defensins and hCAP-18/LL-37 affect adaptive immunity, including chemotaxis of dendritic and T cells (35, 60), modulation of dendritic cell differentiation (35), and reinforcement in the production of specific immunoglobulins (24). These properties, combined with the confirmed stimulation of wound healing (62, 63) and promotion of angiogenesis (64, 65), indicate the versatile role of host defense peptides in the fight against bacterial infections and the regulation of immune responses.
IMPACT OF HOST DEFENSE PEPTIDES IN EXPERIMENTAL MODELS OF SEPSIS
For burn patients in particular, sepsis is a major complication. We showed that topically applied protegrin-1 to burn wounds in rats led to the reduction of multiresistant P. aeruginosa colonization by up to 10,000-fold (66). Using the same animal model, we demonstrated that Histone 2.1 application induced a 3-fold higher reduction in bacterial counts of P. aeruginosa within 4 h compared with controls (67). Furthermore, hCAP18/LL37 proved effective when used in an adenoviral gene transfer approach by reducing bacterial infection significantly and more effectively than the topically applied treatment control (68). Bals et al. (69) investigated whether overexpression of LL37 leads to improved survival in infected mice. C57BL/6 mice were given an adenovirus vector encoding for hCAP-18/LL-37. Mice treated with intratracheal hCAP-18/LL-37 gene transfer showed lower bacterial load and a smaller inflammatory response than did untreated mice after pulmonary challenge with P. aeruginosa. Systemic expression of hCAP-18/LL-37 after i.v. injection of recombinant adenovirus vector resulted in improved survival rates after i.v. injection of LPS with galactosamine or E. coli. Gamelli et al. (70) investigated the effects of the synthetic human defensin D4B in a murine model of burn wounds. Full-thickness scald burn wounds (15% of total body surface area) were inoculated with P. aeruginosa, and subeschar injections of D4B (200 μg/animal) were administered 2 and 24 h after injury. D4B treatment improved long-term survival to day 14 after injury, and the survival rate was nearly 2-fold greater than in the control groups. Moreover, bacterial counts in wound sample cultures were significantly reduced (70).
Cirioni et al. (37) investigated the therapeutic efficacy of hCAP-18/LL-37 in three different rat models of gram-negative sepsis. In the first model, rats were injected intraperitoneally (i.p.) with 1 mg LPS derived from E. coli and received one i.v. dose of 1 mg/kg of hCAP-18/LL-37, which resulted in a marked decrease of endotoxin, IL-6, and TNF-α plasma levels compared with the control group. In another model, rats received an i.p. injection of 2 × 1010 colony forming units (CFU) of E. coli followed by injection of 1 mg/kg of hCAP-18/LL-37. hCAP-18/LL-37 application induced significant reductions in plasma cytokine and endotoxin levels. Moreover, the lethality rate was reduced to 26.6%, whereas the control group showed a lethality of 100% (37). In the third model, animals received cecal ligation and puncture followed by i.v. injection of 1 mg/kg of hCAP-18/LL-37. Again, the lethality rate was lowered to 33.3% in the hCAP-18/LL-37-treated group (37). Other host defense peptides were tested using similar rat models (47, 53, 71-73). One study was designed to investigate the therapeutic efficacy of P-113D, a derivative of human histatin, in different rat models of P. aeruginosa infection and cecal ligation and puncture (72). Histatins are antimicrobial peptides secreted in human saliva and have been shown to have LPS-binding and -neutralizing activity (72). P-113D showed significantly lower cytokine and endotoxin levels and increased the survival rate to 73.4% compared with 100% lethality in the control groups (72). However, animal host defense peptides like buforin II (isolated from an Asian toad) and indolicidin (isolated from bovine neutrophils) (47), sheep myeloid antimicrobial peptide 29 (71), MSI-78 (isolated from an African frog) (73), and amphibian temporin L (53) also were beneficial in rat models of lethal sepsis. All peptides lowered endotoxin and cytokine levels and the lethality rate significantly and were similar or more successful compared with commonly used antibiotics (47, 53, 71-73).
To determine the effect of the amphibian peptide magainin-2, Giacometti et al. (74) induced sepsis in rats by bile duct ligation and injected 2 mg/kg of magainin-2. The amphibian peptide had similar reductions of plasma endotoxin, TNF-α concentrations, and bacterial growth as the tazobactam-piperacillin control. The efficacy of different host defense peptides was also investigated in murine models of sepsis and septic shock. Larrick et al. used galactosamine- and actinomycin D-sensitized mouse model assays to determine whether fragmented human CAP18 could inhibit LPS lethality in mice (50). As the pretreatment with galactosamine or actinomycin D enhanced their sensitivity to LPS, mice received an i.p. injection of actinomycin or galactosamine D, human CAP18 (1.0 or 0.1 μg), and LPS derived from E. coli (1.0 or 0.1 μg). In this study, fragmented human CAP18 reduced the lethality rate significantly (50). Nagaoka et al. (56) further investigated the effect of human CAP18 on D-galactosamine-sensitized mice by sequential i.p. injections of D-galactosamine, CAP18 (10 μg), and LPS (100 ng). In addition to noting increased survival rates when treated with the cathelicidin, the authors described lowered TNF-α levels in serum and cytokine expression of peritoneal macrophages. Motzkus et al. (75) tested the effect of another LPS-neutralizing host defense peptide, DEFB123, which is a synthetic form of human β defensins. Using a D-galactosamine-sensitized murine model, the authors showed that application of 1,000 μg of DEFB123 increased the survival rate to 75% to 100%, depending on application mode (75). In addition to these human peptides, host defense peptides from other mammals like the bovine BMAP-28 (76), isolates from insects (MBI-27, MBI-28, or MP-1) (52, 58), and from plants (ginsan) (77) were beneficial in murine models of sepsis by lowering cytokine and NO levels, and the lethality rates.
OBSTACLES FOR THE TRANSITION TO THE BEDSIDE
Despite the seemingly successful application of host defense peptides in animal models of sepsis and septic shock, cytotoxicity could be a serious limitation to clinical studies.
We demonstrated that application of protegrin-1, a mammalian host defense peptide, could increase bacterial clearance but decrease survival in a sepsis model in mice. Overall, this study suggests that the host defense peptide protegrin-1 overwhelms the host with endotoxin and elicits large alterations in host innate immune responses to infection (78). Fukumoto et al. (79) treated septic neonatal rats with hCAP-18/LL-37 in different concentrations ranging from 50 to 3,000 μg/kg. All rats that received more than 300 μg/kg of hCAP-18/LL-37 after injection with LPS died within 5 to 8 h. However, mortality was significantly reduced in groups that received lower doses of hCAP-18/LL-37 (79). In fact, hCAP-18/LL-37 has shown to have nonselective cell toxicity and to be hemolytic (31). At doses ranging from 13 to 25 μM of hCAP-18/LL-37, cytotoxicity to human leukocytes and the T-cell line MOLT occurred in vitro (30). hCAP-18/LL-37 was also shown to cause DNA fragmentation in cultured smooth muscle cells comparable to hydrogen peroxide, which has been interpreted as a typical feature of programmed cell death (80). In addition, other host defense peptides like BMAP-27 and -28 were shown to be cytotoxic to proliferating lymphocytes (81), and our data showed high cytotoxicity of human β defensin 3 gene transfer to primary porcine keratinocytes in vitro (unpublished data, Tobias Hirsch, MD, 2007).
This finding induced the design of novel synthetic cationic peptides with less cytotoxic effects, but improved properties for the therapeutic use in sepsis. We showed that the ovispirin (porcine HDP)-derived designer peptide proline-novispirin G10 provides broad-spectrum antimicrobial activity against gram-positive and -negative bacteria with low hemolytic and cytotoxic activities in vitro. Antimicrobial activity was confirmed in a large animal wound healing model against clinically relevant strains of S. aureus (82) and in a P. aeruginosa infected burn model in rats (83). Ciornei et al. (84) tested two fragments of hCAP-18/LL-37 after removal of hydrophobic amino acids from the N-terminal end. During a radial diffusion assay, the researchers found antimicrobial activity of the two fragments to be unhampered or less hampered by sodium chloride at 150 mM or by the presence of serum compared with hCAP-18/LL-37 (84). Although LPS binding and neutralization was not affected or was hardly affected by truncation of hCAP-18/LL-37, the fragments caused significantly less hemolysis than hCAP-18/LL-37 (84). Finally, the similar concentration-dependent chemotactic activity for both fragments and hCAP-18/LL-37 was proved (84). Sigurdardottir et al. (85) identified an active domain of hCAP-18/LL-37, GKE, and showed that it is possible to use shorter and less toxic variants of hCAP-18/LL-37 with retained or improved antimicrobial and endotoxin-neutralizing activities.
Although modified peptides are emerging, the risk of toxic effects against eukaryotic host cells during application of therapeutic doses must be considered, and further studies are necessary to investigate the potential risk in therapeutic approaches.
In discovering new substances for the treatment of bacterial infections, we confront the problem of increasingly resistant germs against commonly used antimicrobial agents. However, research demonstrates that bacteria have also developed various ways to escape the direct killing by human host defense peptides (18, 86-88), which emphasizes the important and ancient role of host defense peptides in the innate immune system (87). One main extracellular resistance mechanism seems to be the reduction of negative surface charge by altering anionic components of the cell wall such as teichoic acids and LPS (18, 87). Changing the surface bacteria hinders antimicrobial peptides from penetrating the outer membrane and destabilizes and disrupts their cell wall (18, 87). In addition, intracellular modes of resistance exist (18). They are thought to work as efflux mechanisms comparable to ABC-type transporters; once the antimicrobial peptide has entered the microbial body, it is expelled before damaging intracellular targets (18). However, these mechanisms are inefficient compared with those that inhibit the effect of many therapeutic antibiotics (86). As far as we know, resistance mechanisms that are supposed to affect the effectiveness of host defense peptides in infections are generally related to their property to direct the killing of bacteria. Thinking of host defense peptides as potent immune modulators able to regulate proinflammatory and anti-inflammatory stimuli, the development of bacterial resistance against host defense peptides is not relevant. However, to prevent the development of resistance mechanisms and simultaneously gain the highest direct antimicrobial effectiveness to immune modulatory effects, the combination of common therapeutic antibiotics with host defense peptides must be investigated. Giacometti et al. investigated the therapeutic efficacy of the host defense peptide temporin L in the presence or absence of piperacillin or imipenem in a murine model of gram-negative sepsis (53). The authors found that combining temporin L with β lactams produced the lowest plasma endotoxin and TNF-α levels, the highest antimicrobial activities, and resulted in the highest survival rates (more than 90%) (53). In two different rat models used by Ghiselli et al. (48), β lactams even increased plasma endotoxin and TNF-α levels, whereas the combination with cecropin B was shown to be the most effective treatment, with the highest survival rates (more than 90%), the highest antimicrobial activities, and the strongest reduction in plasma endotoxin and TNF-α. Another study by Giacometti et al. (89) discussed that the combination of β lactams and the host defense peptide pexiganan, a maigainin analogue, represents one of the most potent therapies in rat models of sepsis. A recent study by Cirioni et al. (90) is of great importance and interest; they tested the efficacy of the combination between two α-helical antimicrobial peptides (cecropin A and maigainin II) and vancomycin against S.aureus with intermediate resistance to glycopeptides. As glycopeptides represent the only effective drug against methicillin-resistant staphylococci, strains with reduced sensitivity to these antibiotics is an alarming finding. In a murine sepsis model, they found that groups treated with a combination of the host defense peptide and vancomycin showed low lethality rates (5%-15%), low bacterial counts in blood, and the strongest reduction in TNF-α and IL-6 plasma concentrations.
Alternatively, Kristian et al. (91) reported impairments of host defense peptides by bacteriostatic antibiotics. They showed that when growth of species including E. coli and S.aureus is suppressed by the bacteriostatic agents chloramphenicol and erythromycin, the susceptibility of bacteria to cathelicidin antimicrobial peptides is markedly diminished. These results indicate the importance and relevance of host defense peptides in the future treatment of sepsis considering antibiotic resistances. Furthermore, it shows that we have to rethink currently used antibiotic treatments.
CLINICAL STUDIES
In general, there are only a few clinical studies of host defense peptide application to date (92). However, four cationic peptides have advanced into phase 3 clinical efficacy trials (93): one was tested in children with meningococcal sepsis (94). The authors investigated the effect of systemic delivery of recombinant 21-kd-modified fragment of human bactericidal/permeability-increasing protein (rBPI21) to 393 children (190 received rBPI21 and 203 received placebo, respectively) in the United Kingdom and United States with a clinical diagnosis of meningococcal sepsis. The BPI is a natural protein that binds to and neutralizes the effect of endotoxin, which is stored in the granules of neutrophils. The authors developed an rBPI21 containing the active antimicrobial and endotoxin-neutralizing moiety. The data from this randomized placebo-controlled trial indicate that rBPI21 reduces clinically significant morbidities and improves the functional outcome of children with severe meningococcemia. No statistically significant benefit in mortality was demonstrated; however, because of the rare incidence of the disease and the rapidity of death in this study, the trial was substantially underpowered to detect a statistically significant mortality advantage (95). In collaboration with investigators at the University of Texas Southwestern Medical Center, XOMA Ltd. is currently conducting a study of the therapeutic effects of rBPI21 treatment in patients with severe burn injury.
Other clinical trials investigating host defense peptides include the frog magainin derivative MSI-78 for studies dealing with diabetic foot ulcers and impetigo, the pig protegrin derivative IB-367 for oral mucositis, and the bovine indolicidin variant CP-226 for catheter-associated infections (92, 93, 96). To date, only two peptides showed beneficial results. MSI-78 was as effective as oral antibiotic treatment with ofloxacin for diabetic foot ulcers, leading to improvement in 90% of patients. However, the US Food and Drug Administration did not approve this drug for medical use. For the prevention of catheter-associated infections, a confirmatory study 3b is currently underway. Many other host defense peptides are proceeding through discovery, development, and clinical trials (92, 93) and are on their way from bench to bedside.
However, few data are available on host defense peptides in systemic or local inflammation in humans. Book et al. (98) reported a 3-fold increase in systemic plasma concentration of human β defensin 2 in patients with sepsis compared with healthy controls. Other studies reported significant systemic increases of plasma HNP-1-3, hBD-2, and -3 in patients with pneumonia (Table 2) (98, 99). More research is needed to gain further insights in human host defense peptide (HDP) kinetics during inflammatory response in the human host.
Table 2: Systemic concentrations of host defense peptides in patients with bacterial infections (i.e., pneumonia) and sepsis compared with healthy subjects
CONCLUSIONS
Sepsis is one of the most life-threatening complications in critical care units. There has been much effort to develop consensus lines for the treatment of sepsis that combine antimicrobial therapy with current findings in supportive care and pharmacology research. These therapies include lung-protective ventilation with low tidal volumes to prevent lung injury, and administration of pharmacologic substances like protein C and corticosteroids. Activated protein C has been shown to be effective in severe sepsis by lowering mortality (6), but the costs are comparable to those of organ transplantations and patients have to be chosen carefully (6). The mode of action of both corticosteroids and protein C is not well understood to date (2). The control of blood glucose seems to be another important aspect in the management of sepsis (6). There is no doubt that emergency management of sepsis requires a sufficient antimicrobial therapy (6). Because the site of infection and responsible germs is often unknown initially in patients with sepsis, broad-spectrum antibiotics should be applied. However, this treatment heightens the risk of increasing resistance against available antibiotics, one of the biggest problems in the future treatment of sepsis. Moreover, common antimicrobial substances have been shown to trigger the additional release of endotoxin (46-48), causing overwhelming immune response, sepsis, and shock. To overcome these problems, it is necessary to develop therapies that regard both the key determinants of sepsis, namely bacterial clearance and the control of inflammatory response (37). As this review shows, research reveals that host defense peptides could be used to control systemic inflammation and act as feedback regulators against immoderate immune responses (8). In recent years, focus has been on the innate immune response and its effector molecules. Host defense peptides have been recognized as potent signaling molecules for cellular effectors of both innate and adaptive immunity. Mammalian peptides in particular revealed different immunomodulatory functions, including chemotactic activities on dendritic and T cells, induction of cytokines and chemokines, regulation of host gene expression, and promotion of wound healing and angiogenesis. The multifunctional roles of these peptides and the possibility that indirect forms of antimicrobial activity may be as relevant, if not more so, than their direct antibiotic activity are now commonly accepted. Thus, host defense peptides present a family of natural substances that could be used in combination with antibiotics to complete a broad-spectrum antimicrobial regimen with endotoxin-neutralizing properties, preventing LPS-induced proinflammatory host response (9, 53). However, as discussed previously, there are side effects and limitations, such as toxicity, salt sensitivity, interference with other antimicrobial substances, and potential resistance, that must be considered in future clinical trials and in the development of "designer peptides." Host defense peptides will not be the magic bullet to solve all issues associated with increasing antimicrobial resistance and overwhelming immune response in sepsis. However, host defense peptides have the potential to be significant reinforcements to the currently available therapeutic options in the near future.
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