Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.
In mammals, secreted host defence peptides (HDPs) protect against a wide range of infectious pathogens. They also perform a range of immune modulatory functions which regulate the immune response to pathogens, ensuring that the protective inflammatory response is not exacerbated and that post-infection repair mechanisms are initiated. We identified a novel family of molecules secreted by medically-important helminth pathogens (termed helminth defence molecules; HDMs) that exhibit striking structural and biochemical similarities to the HDPs. To further investigate the extent of this similarity, we have performed a comparative functional study between several well characterized, anti-microbial, mammalian HDPs and a series of parasite-derived peptides. The parasite HDMs displayed immune modulatory properties that were similar to their HDP homologs in mammals, but possessed no antimicrobial or cytotoxic activity. We propose that HDMs of these helminth pathogens underwent specific adaptation, losing their anti-microbial activity but retaining their ability to regulate the immune responses of their mammalian hosts. This absence of cytotoxicity and retention of immune-modulatory activity offers an opportunity to design novel immunotherapeutics derived from the HDMs which could be used to combat destructive inflammatory responses associated with microbial infection and immune-related disorders.
Citation: Thivierge K, Cotton S, Schaefer DA, Riggs MW, To J, Lund ME, et al. (2013) Cathelicidin-like Helminth Defence Molecules (HDMs): Absence of Cytotoxic, Anti-microbial and Anti-protozoan Activities Imply a Specific Adaptation to Immune Modulation. PLoS Negl Trop Dis 7(7): e2307. doi:10.1371/journal.pntd.0002307
Editor: Edward Mitre, Uniformed Services University of the Health Sciences, United States of America
Received: January 24, 2013; Accepted: May 29, 2013; Published: July 11, 2013
Copyright: © 2013 Thivierge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: JPD is a Canadian Institute of Health Research (CIHR) Chair (Tier 1) in Infectious Diseases. JPD is recipient of a Discovery Grant for the National Science and Engineering Research Council (NSERC), Canada. MEL is supported by a University of Technology Sydney postgraduate scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Host defence peptides (HDPs) are found in all living organisms and play a pivotal role as effector components of the innate immune system , . They act as the first line of defence against pathogenic assaults from bacteria, fungi, eukaryotic parasites and viruses –. A range of HDPs with varied sequence lengths, structures and activities have been characterized  and since sequence identity between them is often very poor, their classification is based largely on homologous secondary structures. The two predominant HDP groups found in nature are the cathelicidins, characterized by α-helical secondary structure, and the defensins, which contain β-sheets stabilized by intra-molecular disulfide bridges –. Despite the diversity in their sequences and structures, HDPs are typically small amphipathic peptides (12–50 amino acids) with a net positive charge (+2 to +9) and consist of at least 50% hydrophobic amino acids . These biochemical properties are central to the HDPs antimicrobial function by allowing their interaction with, and disruption of, negatively charged bacterial membranes .
The contribution of mammalian HDPs to the innate immune response extends beyond direct bacterial killing. The elevated expression of HDPs in response to damage (injury or infection) has led to the suggestion that mammals utilize these peptides as ‘alarmins’ to activate the mobilization of a comprehensive immune response . Besides their antimicrobial activity, HDPs function as potent immune regulators, selectively altering host gene expression, inducing chemokine production, inhibiting bacterial- or hyaluronan-induced pro-inflammatory cytokine production, promoting wound healing and modulating T and B cell function [reviewed in –. The net result of these activities is a balance between pro- and anti-inflammatory immune responses which prevents an exacerbated inflammatory response while concurrently stimulating the resolution of infection and repair of damaged epithelia.
The immune response elicited by helminth (worm) parasites is akin to the innate immune response to tissue injury and wound healing , . Typically, this consists of a suppression of classical pro-inflammatory responses and the induction of anti-inflammatory regulatory Th2 type immune responses. While classical Th1-associated inflammatory mediators can provide protection from helminths , there is a substantial cost in collateral damage to host tissue , . In addition, due to their migration and feeding activities, helminth parasites cause considerable local tissue damage. Therefore, it has been proposed that on exposure to helminths, the most beneficial outcome is to shut down a destructive Th1-type response in favour of a Th2 response that rapidly and effectively heals tissue , , . Ultimately this means that the parasite is tolerated by the host, remaining in situ for many years and thus successfully completes its lifecycle.
Some advances have been made in identifying the signalling molecules that initiate helminth-associated Th2 responses. Many of these (such as IL-33 and Thymic stromal lymphopoietin (TSLP)) are thought to be released by epithelial cells damaged by migrating parasites , . However, a number of helminth-derived products have also been shown to modulate the function of innate immune cells and thus are potentially instrumental in the initiation of Th2 immune responses , . We have previously shown that a cysteine protease secreted by the trematode helminth Fasciola hepatica prevented the induction of pro-inflammatory macrophages and dendritic cells , . In addition, peroxiredoxin, also secreted by F. hepatica, promoted the development of Th2 host immune responses , . Importantly, homologues of these proteins are found in other medically-important trematode parasites which we have suggested reveals a common mechanism of immune-modulation employed by this class of pathogen.
As part of our on-going analysis of the secretome of F. hepatica we recently discovered a novel 8 kDa protein. On analysis, this protein shared structural and biochemical similarities to mammalian cathelicidins and was therefore termed F. hepatica Helminth Defence Molecule 1 (FhHDM-1) . Like the human cathelicidin LL-37 precursor CAP-18, FhHDM-1 is proteolytically processed (by a parasitic endopeptidase, cathepsin L1) to release a 34-residue C-terminal peptide previously named FhHDM-1 p2 . This peptide adopts an amphipathic helix structure and, like LL-37, can bind to Escherichia coli lipopolysaccharide (LPS) to prevent its interaction with the toll-like receptor (TLR) 4/MD2/CD14 complex on macrophages. Hence we proposed that F. hepatica utilized FhHDM-1 as a molecular mimic of mammalian cathelicidin-like HDPs as a means of controlling host innate immune responses .
Phylogenetic analysis showed that FhHDM-1 is a member of a family of HDMs conserved throughout several major animal and human trematodes such as Schistosoma, Fasciola, Opisthorchis, Clonorchis and Paragonimus species . Importantly, all HDM molecules in this family have preserved the C-terminal amphipathic helix. Here, we have performed a comparative functional study between several anti-microbial HDPs derived from well-characterized mammalian cathelicidins and parasite-derived peptides. For the helminth-derived peptides we selected FhHDM-1p2 and two homologs derived from Schistosoma mansoni which we term S. mansoni HDM-1 (SmHDM-1p146) and HDM-2 (SmHDM-2p58). In addition, we included a peptide derived from a previously characterized secretory molecule of S. mansoni, termed Sm16-p73 , which our phylogenetic studies suggest is a divergent member of the HDM superfamily . We show that, in contrast to the mammalian HDPs (LL-37, CRAMP, BMAP-28 and SMAP-29), the trematode-derived HDMs are not cytotoxic or bactericidal. However, like the mammalian HDPs, the trematode HDMs suppress the activation of macrophages by microbial stimuli and alter the isotype of immunoglobulin secreted by B cells. We propose that HDMs represent a novel family of HDPs that have undergone specific adaptation to retain potent immune modulatory properties in the absence of deleterious cytotoxic effects and are exploited by helminth pathogens to regulate the immune responses of their mammalian hosts.
Synthetic mammalian and trematode peptides and their biochemical properties
Four synthetic cathelicidin-derived peptides from diverse mammalian species were used: SMAP-29 from sheep , , CRAMP from mice , LL-37 from human , and BMAP-28 from cattle . The 34-residue C-terminal FhHDM-1 peptide, termed FhHDM-1p2, has been previously described . Sm16-p73 peptide is 35 residues in length, corresponding to residues 73–107 of the full-length protein S. mansoni Sm16 (GenBank accession number: AAD26122.1). SmHDM-1p146 is 35 residues in length and corresponds to residues 146–180 of the full length protein (GenBank accession number XP_002580563.1). Finally, SmHDM-2p58 is a 32 residue peptide that corresponds to residues 58–98 from the full length protein (GenBank accession number: XP_002576627.1). All mammalian and trematode peptides were synthesized by GenScript (NJ, USA) and supplied endotoxin-free. The single letter code sequence of each peptide is shown in Table 1.
The biochemical characteristics for each of the HDMs and HDPs were calculated using tools available from the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php)  and are presented in Table 1. Predicted properties were total net charge, Boman index, hydrophobic ratio and total hydrophobic residues on the same hydrophobic surface of the alpha helix.
HDM sequence analysis
We have previously shown, using circular dichroism (CD) spectroscopy, that FhHDM-1 has the propensity to adopt alpha-helical structure in solution . To assess whether HDMs from the related trematode parasite S. mansoni also form alpha helices, secondary structure prediction was performed using JPred 3 (; http://www.compbio.dundee.ac.uk/www-jpred/). Specific regions predicted to form alpha helices were then subjected to helical wheel analysis using Heliquest (; http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py) to identify those with distinct hydrophobic faces; i.e. are amphipathic. The atomic structures of the vertebrate HDPs LL-37 (PDB ID: 2K6O) and BMAP-28 (PDB ID: 2KET) were visualised for comparison using the PyMOL Molecular Graphics System, Version 126.96.36.199 Schrödinger, LLC. (http://pymol.org/).
Bacterial lipopolysaccharide binding assay
Lipopolysaccharide (LPS) binding was performed using a quantitative chromogenic Limulus amoebocyte assay (Chromo-LAL assay; Associates of Cape Cod Incorporated) following manufacturer's recommendations. Assays were performed in flat-bottom endotoxin- and glucan-free 96-well plates (Associates of Cape Cod Incorporated). Stock solutions of each peptide were prepared in endotoxin-free water (80 µg/ml) and diluted to a final concentration of 250 pmol/ml. In the first step, 25 µl of peptide solution was mixed with 25 µl of a solution containing 1 endotoxin U/ml of Escherichia coli O113:H10 LPS and incubated for 30 min at 37°C to allow peptide and LPS binding to occur. The second step involved the addition of 50 µl of the chromo-LAL reagent. The liberation of ρ-nitroaniline was monitored every 60 sec at 405 nm with a Synergy H1 hybrid reader (Biotek) while the temperature was maintained at 37°C. Each peptide concentration was also incubated with 25 µl of LPS-free water as a control to determine if the peptide itself could activate the Chromo-LAL assay. The experiment was conducted twice in triplicate. Standard deviation was calculated from these six replicates.
The minimal inhibitory concentration (MIC) of each peptide against various bacteria was determined using a standardized dilution method according to NCSLA guidelines . Overnight colonies of E. coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus epidermis and Staphylococcus aureus, were suspended to a turbidity of 0.5 OD units and further diluted in Mueller-Hinton broth (MHB). For determination of MIC, peptides were prepared in an acetic acid/BSA solution and used in graded concentrations (0, 1, 2, 4, 8, 16, 32, 64, and 128 µg/ml) from a stock solution. Ten microliters of each concentration was added to each corresponding well of a 96-well polypropylene microtiter plate and 1×105 bacteria in the volume of 90 µL. The plate was incubated at 37°C for 16 h and then read at 600 nm with a Synergy H1 hybrid reader (Biotek).
The peptide's haemolytic activities were determined using human red blood cells (RBCs; Research Blood Components, LLC) in 96-well polypropylene microtiter plates. One hundred µl of 0.5% RBC suspension was added to an equal volume of a peptide (8–256 µg/ml). After 1 h at 37°C, plates were centrifuged at 1420×g for 5 min and the optical density of the supernatant was measured at 414 nm with a Synergy H1 hybrid reader (Biotek). Values for 0% and 100% lysis were obtained by adding PBS or Triton X-100 (1%; final concentration) to RBCs, respectively. All assays were performed in triplicate and the values of percent lysis were within a 1% standard deviation range.
Measurement of cellular pore formation
RAW 264.7 murine macrophages (5×105 cells) were incubated in the presence of the fluorescent dye TO-PRO (Life Technologies) for 60 sec. After the peptides (50 µM) were added to the culture media the uptake of dye was measured for 360 sec by flow cytometry.
RAW 264.7 murine macrophages (1×106cells) were incubated with a range of concentrations (2.5–50 µM) of peptides for 1 h at 37°C. The culture supernatants were collected and assayed for LDH activity with the CytoTox LDH release kit (Promega) according to the manufacturer's instructions. The amount of LDH released is expressed as a percentage of the total amount of LDH released from cells treated with lysis buffer (regarded as 100% cytotoxicity).
Effect of peptides on Cryptosporidium spp. sporozoite viability
Oocysts of the Iowa C. parvum isolate  were propagated in experimentally infected newborn Cryptosporidium–free Holstein bull calves to obtain parasite material for study as previously described , . Oocysts were isolated by sucrose density gradient centrifugation, stored in 2.5% (W/V) potassium dichromate (4°C) and used within 6 weeks of isolation . Oocysts of the TU502 C. hominis isolate ,  were propagated in gnotobiotic piglets and isolated from feces at Tufts University  and used within 4 weeks of isolation. Prior to excystation, oocysts were treated with hypochlorite . In vitro excystation (37°C, 0.15% [W/V] taurocholate, 2 h) of oocysts used for all experiments was ≥90%. Sporozoites were isolated from excysted oocyst preparations by passage through a polycarbonate filter (2.0 µm pore size; Poretics, Livermore, California) and used immediately.
Sporozoite viability after incubation with peptides was assessed using fluorescein diacetate (FDA) and propidium iodide (PI) with modification . In brief, freshly excysted sporozoites were incubated (15 min, 37°C) in minimal essential medium (MEM) containing individual peptides (2.5, 0.25, 0.025 µM) or in MEM alone (n = 3). Peptide concentrations were selected based largely on studies by our group and others evaluating the effects of various antimicrobial peptides on C. parvum viability –. Heat-killed (20 sec, 100°C) sporozoites were used as a control. FDA (8 mg/ml final concentration) and PI (3 mg/ml final concentration) were then added to the sporozoite preparations and incubated further (5 min, 21°C). A minimum of 100 sporozoites were then examined by epifluorescence microscopy for each preparation, and the percent viability was determined. Percent reduction of viability was calculated as ([MEM-treated sporozoite viability−peptide-treated sporozoite viability]÷MEM treated sporozoite viability)×100. The mean values for test and control preparations were examined for significant differences using Student's t-test.
Purification and activation of bone marrow-derived murine macrophages
CD11b+F4/80+ macrophages were derived (99% purity) from the bone marrow of BALB/c mice by culturing with M-CSF (ebioscience) over 6 days and then seeded in RPMI (with 10% FBS v/v) at a concentration of 1×105 cells/ml. These cells were incubated with peptides (0.5 µM–5 µM) for 1 h at 37°C, in the absence of mCSF. Following two washes with ice-cold PBS, cells were incubated with a combination of E. coli LPS (10 ng/ml; Sigma) and IFNγ (10 ng/ml; BD Pharmingen) overnight. Supernatants were then collected and the amount of TNF measured by ELISA according to the manufacturer's instructions (BD Pharmingen).
Purification and activation of murine B cells
B cells were isolated from the spleens of BALB/c mice by negative selection using a B cell isolation kit containing biotin-conjugated mAbs to CD43, CD4, and Ter-119 (Miltenyi Biotec) and then seeded at a concentration of 1×106 cells/ml in RPMI (with 10% FBS v/v). The cells were treated with a range of concentrations of peptides (0.5 µM–5 µM) for 1 h at 37°C. After washing, the B cells were incubated with a combination of either E. coli LPS (10 µg/ml; Sigma) and IL-4 (10 ng/ml; BD Pharmingen) or E. coli LPS (10 µg/ml) and IFNγ (200 ng/ml; BD Pharmingen) for six days. Supernatants were then collected and the amount of IgG1 or IgG2a measured by ELISA (Sigma).
HDMs, like mammalian HDPs, are cationic, have a high percentage of hydrophobic amino acids and form amphipathic structures
The biochemical properties for each of the HDMs and HDPs are presented in Table 1. The cathelicidins are known to be highly cationic peptides. Except for the FhHDM-1p2 peptide, which has a net charge of 0, all the peptides in the present study have a net positive charge (+3 to +9) with a percentage of hydrophobic residues ranging from 34 to 44. The Boman index is an estimated potential of peptides to bind to other proteins. For this index, a low value (≤1) suggests that a peptide has more antibacterial activity, whereas values ranging from 2.5–3.0 indicate that a peptide is multifunctional with hormone-like activities . While BMAP-28 has a low Boman index (0.81) which correlates to its high antimicrobial activity, the Boman indices for the other peptides range from 1.34–3.11. Overall, there is no striking difference between the biochemical properties of HDMs and HDPs.
The atomic structures of the vertebrate HDPs LL-37, CRAMP, BMAP-28 and SMAP-29 have been previously solved and determined that each form an amphipathic helix , – (figure 1). The structures of LL-37 and BMAP-28 are shown in figure 1A as representative for this group of peptides. Secondary structure prediction of the parasite-derived peptides FhHDM-1p2, SmHDM-1p146, SmHDM-2p58 and Sm16-p73 revealed that all possessed regions likely to form alpha helices. Furthermore, helical wheel analysis showed that each molecule contained an alpha helix (32–35 amino acids) toward the C-terminal that was distinctly amphipathic. The number of residues forming the hydrophobic face of the parasite molecules ranged from 6–9 (figure 1B). Thus, like their vertebrate HDP counterparts, the secreted helminth parasite molecules also form distinct amphipathic helices.
(A) Most HDPs, including those used in the present study, form amphipathic helices. Here, the atomic structures of LL-37 (PDB ID: 2K6O) and BMAP-28 (PDB ID: 2KET) are shown as green ribbon with the residues comprising the hydrophobic face of the molecules coloured red. Structures were generated using Pymol (http://www.pymol.org). (B) Helical wheel analysis shows that peptides derived from FhHDM-1p2 (residues 14–34), Sm16-p73, SmHDM-1p146 and SmHDM-2p58 (residues 9–26 for each) also form distinct amphipathic structures.
Helminth defence molecules variably bind LPS
Mammalian HDPs have the ability to bind to and thus neutralize the bacterial endotoxin LPS –. The capacity of different mammalian and helminth peptides to bind LPS from E. coli O113:H10 was compared using the chromogenic Limulus amoebocyte assay (Chromo-LAL). The Limulus amoebocyte lysate contains enzymes that are activated in a cascade of reactions in the presence of LPS –. The final enzyme in the series splits the chromophore, ρ-nitroaniline (ρNA), from the chromogenic substrate, generating a yellow color. The amount of ρNA released is proportional to the amount of free LPS present in the system.
Consistent with that published in the literature, mammalian HDP LL-37, BMAP-28, SMAP-29 and CRAMP inhibited the activation of the Chromo-LAL assay at a concentration of 250 pmol/ml, indicating that they interact with LPS and thus prevent the activation of the enzymatic cascade , , ,  (figure 2). BMAP-28 was the most potent, preventing the activation of the enzyme cascade with a Vmax of 3.1 and SMAP-29 was less effective with a Vmax of 26.5. Of the helminth-derived HDPs, both Sm16-p73 and SmHDM-1p146 displayed no LPS-binding capacity with a Vmax of 40.4 and 31.1, respectively, which were above the control reaction with no peptide (Vmax of 28.5). By contrast, the helminth peptide SmHDM-2p58 was the second best inhibitor of the series with a Vmax of 12.5, just after BMAP-28.
25 µl of peptide (250 pmol/ml) was incubated with 25 µl of LPS from E. coli O113:H10 (1 EU/ml) for 30 min at 37°C to allow the interaction between both molecules. Chromo-LAL reagent was then added and the liberation of ρ-nitroaniline was monitored every 60 sec at 405 nm. The top panel is a representative of the curves obtained for each peptide. The data presented in the Table is the mean of three replicates from two separate experiments. The extent of binding of peptide to LPS prior to adding the mixture to the Chromo-LAL is reflected in the reduced rate of cleavage of the substrates and a corresponding inability of the reaction to reach maximum velocity (Vmax).
We found that FhHDM-1p2 was capable of directly activating the Chromo-LAL assay itself (data not shown) and, therefore, its binding capacity could not be evaluated using this test. However, using a plate-binding assay we have previously demonstrated that FhHDM-1p2 does indeed bind to LPS .
Helminth defence molecules do not possess antimicrobial or anti-protozoan activity
The bactericidal properties of mammalian HDPs are mediated by direct antimicrobial activities, and are therefore easily evaluated as the minimal concentration capable of inhibiting visible microbial growth (MIC) against a panel of bacterial species. Consistent with previous reports , , , , we found that SMAP-29 and BMAP-28 were effective against a broad group of gram-negative bacteria, including E. coli, P. aeruginosa, and S. typhimurium and two gram-positive bacteria, S. aureus and S. epidermis, with MIC values of <0.25–8 µg/ml (Table 2). LL-37 and its mouse counterpart, CRAMP, showed bactericidal activity against gram-negative bacteria with MIC ranging from 2 to 8 µg/ml, but were ineffective against the gram-positive bacteria tested (MIC<128 µg/ml). The inactivity of LL-37 against S. aureus and S. epidermis is consistent with other studies . However, the anti-microbial effect of LL-37 on Staphylococcus could be strain dependent as several studies have reported an effect of this peptide on both S epidermis  and S. aureus , . Despite structural similarities with the mammalian peptides tested, none of the HDMs demonstrated bactericidal activity against any species of bacteria at the concentrations tested (<0.25 to 128 µg/ml).
We have recently shown that cationic peptides, including the cathelicidin LL-37, are highly parasiticidal against the apicomplexan parasite C. parvum in vitro . Using the same methodology, we compared the anti-parasite activity of the mammalian HDPs to that of the four helminth-derived peptides. In keeping with our previous data , LL-37 exhibited parasiticidal activity at a concentration of 2.5 µM against C. parvum and the related species C. hominis (figure 3). The other mammalian cathelicidins showed greater parasiticidal activity, reducing the viability of protozoans at lower concentrations of 0.025 and 0.25 µM. Of particular note, BMAP-28 demonstrated the highest potency against both species of protozoan at 2.5 µM (P<0.01). In stark contrast to these results, was the relative absence of parasiticidal activity of the helminth-derived peptides, even at the highest concentration of 2.5 µM.
The viability of C. parvum (A) and C. hominis (B) sporozoites was assessed after incubation with peptides or in medium alone (untreated). Following the addition of FDA and PI, the percent viability for each preparation was determined by epifluorescence. The data shown are means ± SD.
Helminth defence molecules are not cytotoxic to mammalian cells
The predominant mechanism of HDP bactericidal activity is the formation of pores in the membrane lipid bilayer, destroying its integrity and causing cell death . However, this effect is not specific to bacterial cells and HDPs have also been shown to be cytolytic to eukaryotic cells, particularly at high concentrations . TO-PRO is a membrane impermeant dye and therefore its detection within cells is indicative of pore formation. Using this dye, we demonstrated that, as expected, all mammalian peptides at a concentration of 50 µM (equivalent to the highest concentration tested in the bactericidal assays) induced the formation of pores in a murine macrophage cell line (figure 4). However, at the same concentration none of the helminth peptides exhibited this effect.
RAW macrophages were incubated in the presence of TO-PRO. After 60 sec, 50 µM of mammalian (left panel) or helminth (right panel) peptides was added to the medium. The uptake of fluorescent dye was measured over a period of 360 sec.
To more completely assess the cytotoxicity of the peptides, we first examined their haemolytic activity against human RBCs at various concentrations (8–256 µg/ml). After one hour of co-incubation, all of the mammalian peptides induced concentration dependent hemolysis (Table 3). BMAP-28 was the most potent of all the peptides, with 50% of RBCs lysed at the lowest concentration of peptide tested (8 µg/ml) and 70% at the highest concentration of 256 µg/ml. In comparison, at this highest concentration, the other mammalian peptides were much less cytotoxic, lysing only 14.5–29.3% of RBCs. Notably, under the same experimental conditions, the S. mansoni-derived peptides did not lyse the cells at any concentration tested. The F. hepatica-derived FhHDM-1p2 showed some low-level cytolytic activity, with 11.4% of cells lysed at the highest concentration of 256 µg/ml.
Lactate Dehydrogenase (LDH) is a soluble cytosolic enzyme that is released into culture media following loss of membrane integrity resulting from either apoptosis or necrosis. Therefore LDH release is an indicator of cell membrane integrity and acts as a measure to assess cytotoxicity. Consistent with the demonstration of hemolysis, higher concentrations (>10 µM) of mammalian peptides also resulted in the death of murine macrophages, with the highest concentration tested (50 µM) resulting in 100% cytotoxicity, compared to the effect of a lysis buffer (figure 5). By contrast, none of the helminth peptides induced cell death at any concentration tested. This lack of LDH detection was not due to enzyme inhibition by the helminth peptides; when the peptides were added directly to culture supernatant from lysed cells the LDH activity was unchanged. Consistent with these data, the cells treated with helminth peptides (10–50 µM) looked morphologically normal by light microscopy (data not shown).
RAW macrophages were incubated with peptides for 1 h at 37°C. The release of LDH by cells was measured and expressed as the percentage of LDH released after treatment of cells with lysis buffer (regarded as 100%). Data shown are the means ± SEM of triplicate samples and representative of three independent experiments.
Both mammalian- and helminth-derived peptides suppress secretion of the inflammatory cytokine TNF from macrophages
Activation of macrophages by microbial stimuli is central to the induction of innate immune responses. IFNγ is one of the key cytokines in the innate immune response to intracellular pathogens, and augments cellular responses to TLR ligands such as bacterial LPS , . To prevent excessive inflammation potentially leading to sepsis, HDPs have been shown to inhibit the response of macrophages to these inflammatory mediators using mechanisms that are independent of direct binding to LPS . Significantly, macrophages isolated from animals and humans infected with helminth parasites are also hyporesponsive to stimulation with LPS and IFNγ , . Therefore, here we investigated whether helminth-derived peptides, like mammalian HDPs, could suppress the activation of inflammatory macrophages. At concentrations below cytotoxic levels (<5 µM), all mammalian peptides significantly inhibited TNF production in response to the combined stimulation with LPS and IFNγ (figure 6). Titration of the peptide concentration showed that even at concentrations as low as 0.5 µM, the inhibitory activity was preserved. In contrast, at the lowest concentration tested, the helminth-derived peptides had no effect on the activation of macrophages. However, as the concentration was increased, the helminth peptides significantly suppressed the inflammatory response of macrophages and in most cases more effectively than the mammalian peptides (figure 6). It is worth noting that the helminth-derived peptides could be tested at concentrations up to 50 µM as they are non-toxic to cells, whereas due to their cytotoxicity the HDPs were not tested at concentrations above 10 µM (figures 4,5). At these higher concentrations (10, 25 and 50 µM) the helminth-derived peptides significantly reduced the production of TNF from activated macrophages in a concentration dependent manner (data not shown).
Primary murine bone marrow derived macrophages were treated with peptides for 1 h, washed then subsequently stimulated with a combination of E. coli LPS (10 ng/ml) and IFNγ (10 ng/ml) for 16 h. The amount of TNF (pg/ml) secreted into the culture media was measured by ELISA. The data shown are means ± SEM of triplicate samples and are representative of three independent experiments.
Both mammalian- and helminth-derived peptides alter the secretion of immunoglobulin from activated B cells
In addition to directly inhibiting inflammatory innate immune responses, there is evidence that mammalian HDPs have an additional role in regulating the magnitude of the adaptive antibody responses. For example, it has been shown that CRAMP functions to positively regulate the level of IgG1 produced by B cells , and LL-37 reportedly decreased the production of IgG2a from mouse splenic B cells activated with LPS and IFNγ . Consistent with these reports, our analyses showed that with the exception of BMAP-28, all the mammalian HDPs significantly increased the production of IgG1 in response to a Th2 biased environment (LPS and IL-4) (figure 7A). The apparent reduction in IgG1 production recorded for the higher concentration of BMAP-28, likely reflects some level of cell death rather than a reduction in antibody production. Due to this cytotoxicity the HDPs were not tested at concentrations above 5 µM. While there was greater variability between the HDMs, peptides from F. hepatica and S. mansoni significantly increased the production of IgG1 in response to LPS and IL-4 even at low concentrations.
Murine splenic B cells were treated with a range of concentrations of peptides for 1 h at 37°C, washed and then incubated with a combination of either (A) E. coli LPS (10 µg/ml) and IL-4 (10 ng/ml) or (B) E. coli LPS (10 µg/ml) and IFNγ (200 ng/ml) for six days. Levels of IgG1 (A) and IgG2a (B) in cell supernatants were measured by ELISA. The data shown are from triplicate samples, corresponds to the means ± SEM, and are representative of two independent experiments.
Conversely, mammalian peptides reduced the production of IgG2a in a Th1 (LPS and IFNγ) biased environment (figure 7B). For the helminth peptides, only concentrations above 5 µM had the same effect on B cells, significantly (p<0.001) inhibiting IgG2a secretion (data not shown), suggesting a lower potency than mammalian-derived peptides.
Parasitic helminths secrete molecules that modulate host immune responses to establish an environment that facilitates their survival and a prolonged reproductive phase , , . Co-evolution of helminths with their hosts means that these parasites are well adapted to the host's immune system, making use of endogenous regulation mechanisms to manipulate the immune response to their benefit. In this study, we compared the biological activities of a series of helminth-derived cathelicidin-like peptides to that of their mammalian homologues and suggest how their production by helminths can facilitate a successful parasitic life cycle.
In vitro, most mammalian HDPs are effective antimicrobial agents against a range of organisms including gram-negative and gram-positive bacteria, protozoa, viruses and fungi , , . In general, the expression of HDPs is increased at the onset of an infection and therefore the anti-pathogenic activity was thought to be one of the most important immediate responses that the mammalian host evolved to deal with invading pathogens. It has been proposed that the specificity of HDPs for particular microbes is subjected to significant variation and is particularly influenced by the types of microbial biotas to which each HDP species is exposed . Consistent with this theory, we showed some variation in the anti-microbial capabilities of the mammalian HDPs examined. While sheep and bovine derived peptides were effective against a broad range of both gram-positive and gram-negative bacteria, the mouse and human HDPs were largely ineffective against the gram-positive species tested. However, we found that none of the helminth-derived peptides displayed gram positive or negative bactericidal activity, even at the highest concentration tested, implying that their specialised function is not anti-microbial. However, we cannot exclude the possibility of the peptides having anti-microbial activity on other, untested pathogenic bacteria.
Despite lacking bactericidal activity, we showed previously that FhHDM1-p2, like the mammalian peptides, interacts with LPS, thus effectively neutralising the ability of infecting bacteria to induce an inflammatory response . We suggested that this may be a mechanism used by the parasite to prevent excessive activation of innate cells in response to the translocation of microbes into circulation occurring as a result of damage to the skin and/or gut epithelium during migration of the parasite . As the two major factors mediating interaction between LPS and HDPs are hydrophobicity and cationicity , inspection of the sequence of the other HDMs would predict a universal ability to bind to LPS. However, while SmHDM-1p146 appeared to bind LPS as efficiently as the mammalian peptides, neither SmHDM-2p58 or Sm16-p73 were particularly potent, indicating that the neutralization of LPS may not be a common function of the helminth-derived peptides.
A number of studies have demonstrated the ability of amphibian and mammalian HDPs to kill protozoan parasites in vitro , . For example, BMAP-28 possesses potent activity against the agent of human leishmaniasis, Leishmania major , and we have shown that LL-37 can reduce the viability and infectivity of sporozoites of C. parvum, an intestinal infection of humans and agricultural animals . In the present study we confirm the activity of LL-37 against C. parvum and the related parasite C. hominis and show that the other mammalian cathelicidins tested also have anti-protozoan activity; BMAP-28 exhibited the most potent in vitro activity. By contrast, and consistent with their lack of antibacterial activity, the helminth-derived HDMs did not kill either parasite in vitro. Clearly, the particular physico-chemical properties of HDMs do not confer an ability to penetrate and disrupt the surface membrane of these parasitic organisms.
Although widely defined as antimicrobial, in fact, at the concentrations normally found at human mucosal surfaces and in physiological salt conditions, the mammalian cathelicidin-like peptides do not display bactericidal activity. However, at these same concentrations and under the same conditions, the peptides exhibit a variety of immune modulatory functions , . This has led to the suggestion that the cathelicidins are principally immune modulators rather than antimicrobials, and like the mammalian defensins, have traded their bactericidal capacities to acquire the ability to broadly regulate the immune response , . The mammalian cathelicidins have a diverse effect on the cellular immune response, but in particular, the peptides have a crucial role in regulating TLR-dependent innate inflammatory responses. This means that they function to maintain homeostasis in response to natural shedding of microflora-TLR agonists as well as controlling the systemic inflammatory response to infection or tissue damage , , . We have shown here that the helminth-derived cathelicidin-like peptides also regulated the innate immune response to TLR stimulation by inhibiting TNF release from macrophages stimulated with bacterial LPS. While the helminth HDMs were not as effective as their mammalian homologues at the lowest concentration tested, they were correspondingly more potent as their concentration was increased towards quantities that are likely secreted during infection. It is probable that the function of these secreted HDMs is similar to the predicted role for the mammalian HDPs, i.e. prevention of an excessive inflammatory response, which acts to prevent the expulsion of the worm and to protect the host from exacerbated tissue damage.
The role of HDPs in regulating the adaptive immune response has been less extensively studied. Recent studies have shown that LL-37 decreased the production of IgG2a from murine B cells stimulated with LPS and IFNγ , and that CRAMP increased the amount of IgG1 in response to IL-4 . These results are consistent with the suggestion that HDPs are engaged in the process of infection resolution and wound healing, as autoreactive IgG1 antibody production is central to tissue repair processes , while IgG2a are associated with IFNγ/Th1-mediated inflammatory responses. Consistent with these reports, the helminth-derived peptides performed in a similar manner to their mammalian counterparts, successfully enhancing the production of IgG1. At high concentrations, the helminth-derived peptides were far more effective than mammalian HDMs at suppressing the release of IgG2a in response to IFNγ, supporting their role as potent anti-inflammatory agents.
Due to their immune modulatory activities, there is considerable interest in developing HDPs as therapeutics such as anti-inflammatory agents, adjuvants and wound healing agents. The therapeutic potential of HDPs has been demonstrated and a number of peptides are being developed as anti-inflammatory agents . However, the clinical use of these peptides as injectable therapeutics has been hampered by indications of toxic side-effects on mammalian cells and their ability to lyse eukaryotic cells , . This had led to intense research into understanding how HDPs function in terms of their physico-chemical properties. Among the factors that appear to influence specificity between their activity against prokaryotic and eukaryotic cells are the ability to form an amphipathic α-helical structure, hydrophobicity, overall charge distribution, and minimal peptide length . At first sight, the biochemical characteristics of the helminth-derived HDMs would predict an inherent cytotoxic activity: they form amphipathic helices (figure 1), they have a comparable proportion of hydrophobic amino acids to mammalian HDPs and most of them are cationic (Table 1). However, based on the assays employed in this study, we found no correlation between the level of hydrophobicity and cytotoxic activity.
The majority of mammalian HDPs have an overall net charge ranging from +4 to +6.37 , implying an optimal range for biological activity. HDPs with a net positive charge of <+4 are found to be inactive, whereas increasing the net charge from +4 to +8 confers antimicrobial activity and some haemolytic activity . Three of the HDMs used in this study, FhHDM-1p2, SmHDM-1p146 and SmHDM-2p58, all had a net charge <+4 which is consistent with a non-bactericidal, non-haemolytic peptide. However, despite possessing a net charge of +5, Sm16-p73 displayed neither antimicrobial nor cytotoxic activities. Recent studies have proposed that rather than a simple correlation between net charge and haemolysis it is the localisation of the positively charged amino acids within the peptide that also dictates membrane interaction and selectivity. By increasing the charge of an amphipathic HDP analog from +8 to +9, by the addition of one positive charge on the polar face, the haemolytic activity of the peptide was enhanced 32-fold . Using this parameter, we calculated that Sm16 has four positively charged amino acids on its polar face compared to six for LL-37, CRAMP and BMAP-28 and seven for SMAP-29, which may provide some clue as to the difference in cytotoxicity between these peptides. Likewise, all the helminth HDMs used in this study have less positive charges on their polar face compared to LL-37, which was the least cytotoxic of all the mammalian peptides examined in this study.
It is generally accepted that the cytoplasmic membrane is the main target of many mammalian HDPs, whereby peptide accumulation in the membrane causes increased permeability and a loss of barrier function, resulting in the leakage of cytoplasmic components and cell death . However, the cytotoxic concentrations of HDPs are higher than the concentrations required for the destruction of microbes, which, some authors suggest, reveals a cell-selective killing mechanism , . The physiological concentration of HDPs at mucosal sites is typically less than 2 µg/ml , , well below the concentration that is cytotoxic to mammalian cells in vitro. We have found that helminth HDMs are abundant molecules within the secretions of helminth parasites, which during a multi-parasite infection would likely be at relatively high concentrations in circulation. Therefore, it is essential for the success of the parasite that these peptides do not possess cytotoxic activity, whilst at the same time retain the beneficial immune modulatory properties. The complete absence of antimicrobial activity by helminth-derived peptides is likely linked to this need to prevent host cell death during infection.
The extraordinary capacity of helminths to regulate the immune response is central to their longevity in the mammalian host and thus underpins their success as parasitic organisms , . Therefore, it is perhaps unsurprising that helminth secretory products contain homologues of components of the host immune system that target the same mammalian pathways. In addition to the HDMs identified here, helminth parasites express highly conserved cytokine gene families that, like their mammalian counterparts, ligate specific receptors on immune cells. Brugia malayi and Ancylostoma ceylanicum express homologues of the mammalian cytokine macrophage migration inhibitory factor (MIF) , and in a Th2 environment, such as that activated by helminth infection, Brugia MIF synergises with IL-4 to induce the development of regulatory M2 macrophages . Helminths also express members of the Tumour Growth Factor-(TGF)β and TGF-β receptor superfamilies –, and similar to the mammalian cytokine, Heligmosomoides polygyrus TGF-β homologue has been shown to directly induce the differentiation of regulatory T cells, demonstrating a key role in parasite immune regulation .
The cysteine protease inhibitors, cystatins, are an ancient and conserved family of peptides in the animal and plant kingdoms . The cystatins of parasitic worms differ substantially from those produced by free-living nematodes with regard to their immune modulatory properties . In particular, the acquisition of an asparaginyl endopeptidase site, similar to that of vertebrate cystatin C, confers an ability to reduce the activation of host T cell responses by directly inhibiting the presentation of antigen by dendritic cells , , suggesting a specific adaption to regulate host immune responses , . Similar to the cystatins, HDPs are conserved in all organisms, including plants, animals and humans . However, we show here that while the helminth-derived HDMs are effective immune modulators, they display no bactericidal activity. These observations would suggest that like the cystatins, HDMs have become specifically adapted to support a parasitic lifestyle, losing the more ancient property of direct antimicrobial killing but acquiring the ability to regulate immune responses in order to promote their survival within the mammalian host. It is possible that HDM immune modulation arose in trematodes following their divergence from the chordate lineage (as acoelomates) and their subsequent specialisation to an endoparasitic lifestyle, distinct from the free-living acoelomate turbellarian flatworms from which HDM homologues have yet to be identified (unpublished observation).
The immune-modulatory properties of mammalian HDPs, and in particular their ability to prevent excessive inflammatory induced pathology associated with bacterial sepsis, has attracted interest in exploiting these as anti-infectives. However, their cytotoxicity, as also shown in the present study, has presented a major drawback for their in vivo use. Accordingly, the absence of cytotoxicity and retention of immune-modulatory activity observed for the helminth-derived HDMs offer an opportunity to design novel immunotherapeutics to combat microbial pathogens and immune-related disorders.
Conceived and designed the experiments: K. Thivierge, S. Cotton, M. Riggs, M. Robinson, J. Dalton, S. Donnelly. Performed the experiments: K. Thivierge, S. Cotton, D. Schaefer, M. Riggs, J. To, M. Lund, M. Robinson, S. Donnelly. Analyzed the data: K. Thivierge, S. Cotton, D. Schaefer, M. Riggs, J. To, M. Lund, M. Robinson, J. Dalton, S. Donnelly. Contributed reagents/materials/analysis tools: M. Riggs, J. Dalton. Wrote the paper: K. Thivierge, S. Cotton, D. Schaefer, M. Riggs, J. To, M. Lund, M. Robinson, J. Dalton, S. Donnelly.
- 1. Hancock RE, Diamond G (2000) The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8: 402–410.
- 2. Bowdish DM, Davidson DJ, Speert DP, Hancock RE (2004) The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 172: 3758–3765.
- 3. Mookherjee N, Hancock RE (2007) Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections. Cell Mol Life Sci 64: 922–933.
- 4. Andes D, Craig W, Nielsen LA, Kristensen HH (2009) In vivo pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine infection model. Antimicrob Agents Chemother 53: 3003–3009.
- 5. Hsu KH, Pei C, Yeh JY, Shih CH, Chung YC, et al. (2009) Production of bioactive human alpha-defensin 5 in Pichia pastoris. J Gen Appl Microbiol 55: 395–401.
- 6. Fjell CD, Hancock RE, Cherkasov A (2007) AMPer: a database and an automated discovery tool for antimicrobial peptides. Bioinformatics 23: 1148–1155.
- 7. Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75: 39–48.
- 8. Lehrer RI (2004) Primate defensins. Nat Rev Microbiol 2: 727–738.
- 9. Ganz T (2003) Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 3: 710–720.
- 10. Hancock RE, Chapple DS (1999) Peptide antibiotics. Antimicrob Agents Chemother 43: 1317–1323.
- 11. Oppenheim JJ, Yang D (2005) Alarmins: chemotactic activators of immune responses. Curr Opin Immunol 17: 359–365.
- 12. Lai Y, Gallo RL (2009) AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 30: 131–141.
- 13. Steinstraesser L, Kraneburg UM, Hirsch T, Kesting M, Steinau HU, et al. (2009) Host defense peptides as effector molecules of the innate immune response: a sledgehammer for drug resistance? Int J Mol Sci 10: 3951–3970.
- 14. Choi KY, Mookherjee N (2012) Multiple immune-modulatory functions of cathelicidin host defense peptides. Front Immunol 3: 149.
- 15. Allen JE, Maizels RM (2011) Diversity and dialogue in immunity to helminths. Nat Rev Immunol 11: 375–388.
- 16. Jackson JA, Friberg IM, Little S, Bradley JE (2009) Review series on helminths, immune modulation and the hygiene hypothesis: immunity against helminths and immunological phenomena in modern human populations: coevolutionary legacies? Immunology 126: 18–27.
- 17. James SL, Glaven J (1989) Macrophage cytotoxicity against schistosomula of Schistosoma mansoni involves arginine-dependent production of reactive nitrogen intermediates. J Immunol 143: 4208–4212.
- 18. Allen JE, Wynn TA (2011) Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathog 7: e1002003.
- 19. Perrigoue JG, Marshall FA, Artis D (2008) On the hunt for helminths: innate immune cells in the recognition and response to helminth parasites. Cell Microbiol 10: 1757–1764.
- 20. Harnett W, Harnett MM (2010) Helminth-derived immunomodulators: can understanding the worm produce the pill? Nat Rev Immunol 10: 278–284.
- 21. Donnelly S, O'Neill SM, Stack CM, Robinson MW, Turnbull L, et al. (2010) Helminth cysteine proteases inhibit TRIF-dependent activation of macrophages via degradation of TLR3. J Biol Chem 285: 3383–3392.
- 22. Dowling DJ, Hamilton CM, Donnelly S, La Course J, Brophy PM, et al. (2010) Major secretory antigens of the helminth Fasciola hepatica activate a suppressive dendritic cell phenotype that attenuates Th17 cells but fails to activate Th2 immune responses. Infect Immun 78: 793–801.
- 23. Donnelly S, Stack CM, O'Neill SM, Sayed AA, Williams DL, et al. (2008) Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages. FASEB J 22: 4022–4032.
- 24. Donnelly S, O'Neill SM, Sekiya M, Mulcahy G, Dalton JP (2005) Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages. Infect Immun 73: 166–173.
- 25. Robinson MW, Donnelly S, Hutchinson AT, To J, Taylor NL, et al. (2011) A family of helminth molecules that modulate innate cell responses via molecular mimicry of host antimicrobial peptides. PLoS Pathog 7: e1002042.
- 26. Rao KV, Ramaswamy K (2000) Cloning and expression of a gene encoding Sm16, an anti-inflammatory protein from Schistosoma mansoni. Mol Biochem Parasitol 108: 101–108.
- 27. Bagella L, Scocchi M, Zanetti M (1995) cDNA sequences of three sheep myeloid cathelicidins. FEBS Lett 376: 225–228.
- 28. Skerlavaj B, Benincasa M, Risso A, Zanetti M, Gennaro R (1999) SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Lett 463: 58–62.
- 29. Gallo RL, Kim KJ, Bernfield M, Kozak CA, Zanetti M, et al. (1997) Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse. J Biol Chem 272: 13088–13093.
- 30. Agerberth B, Gunne H, Odeberg J, Kogner P, Boman HG, et al. (1995) FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci USA 92: 195–199.
- 31. Skerlavaj B, Gennaro R, Bagella L, Merluzzi L, Risso A, et al. (1996) Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. J Biol Chem 271: 28375–28381.
- 32. Wang G, Li X, Wang Z (2009) APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 37: D933–937.
- 33. Cole C, Barber JD, Barton GJ (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36: W197–201.
- 34. Gautier R, Douguet D, Antonny B, Drin G (2008) HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24: 2101–2102.
- 35. Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3: 163–175.
- 36. Heine J, Pohlenz JF, Moon HW, Woode GN (1984) Enteric lesions and diarrhea in gnotobiotic calves monoinfected with Cryptosporidium species. J Infect Dis 150: 768–775.
- 37. Riggs MW, McGuire TC, Mason PH, Perryman LE (1989) Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites. J Immunol 143: 1340–1345.
- 38. Riggs MW, Perryman LE (1987) Infectivity and neutralization of Cryptosporidium parvum sporozoites. Infect Immun 55: 2081–2087.
- 39. Arrowood MJ, Sterling CR (1987) Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. J Parasitol 73: 314–319.
- 40. Akiyoshi DE, Feng X, Buckholt MA, Widmer G, Tzipori S (2002) Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species. Infect Immun 70: 5670–5675.
- 41. Chappell CL, Okhuysen PC, Langer-Curry R, Widmer G, Akiyoshi DE, et al. (2006) Cryptosporidium hominis: experimental challenge of healthy adults. Am J Trop Med Hyg 75: 851–857.
- 42. Akiyoshi DE, Mor S, Tzipori S (2003) Rapid displacement of Cryptosporidium parvum type 1 by type 2 in mixed infections in piglets. Infect Immun 71: 5765–5771.
- 43. Arrowood MJ, Jaynes JM, Healey MC (1991) In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum. Antimicrob Agents Chemother 35: 224–227.
- 44. Giacometti A, Cirioni O, Del Prete MS, Barchiesi F, Scalise G (2000) Short-term exposure to membrane-active antibiotics inhibits Cryptosporidium parvum infection in cell culture. Antimicrob Agents Chemother 44: 3473–3475.
- 45. Giacometti A, Cirioni O, Del Prete MS, Skerlavaj B, Circo R, et al. (2003) In vitro effect on Cryptosporidium parvum of short-term exposure to cathelicidin peptides. J Antimicrob Chemother 51: 843–847.
- 46. Zaalouk TK, Bajaj-Elliott M, George JT, McDonald V (2004) Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection. Infect Immun 72: 2772–2779.
- 47. Carryn S, Schaefer DA, Imboden M, Homan EJ, Bremel RD, et al. (2012) Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms. J Parasitol 98: 199–204.
- 48. Boman HG (2003) Antibacterial peptides: basic facts and emerging concepts. J Intern Med 254: 197–215.
- 49. Wang G (2008) Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles. J Biol Chem 283: 32637–32643.
- 50. Yu K, Park K, Kang SW, Shin SY, Hahm KS, et al. (2002) Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy. J Pept Res 60: 1–9.
- 51. Tack BF, Sawai MV, Kearney WR, Robertson AD, Sherman MA, et al. (2002) SMAP-29 has two LPS-binding sites and a central hinge. Eur J Biochem 269: 1181–1189.
- 52. Rosenfeld Y, Shai Y (2006) Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: role in bacterial resistance and prevention of sepsis. Biochim Biophys Acta 1758: 1513–1522.
- 53. Hirsch T, Metzig M, Niederbichler A, Steinau HU, Eriksson E, et al. (2008) Role of host defense peptides of the innate immune response in sepsis. Shock 30: 117–126.
- 54. Larrick JW, Hirata M, Balint RF, Lee J, Zhong J, et al. (1995) Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein. Infect Immun 63: 1291–1297.
- 55. Scott A, Weldon S, Buchanan PJ, Schock B, Ernst RK, et al. (2011) Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo. PLoS One 6: e26525.
- 56. Dzik JM (2006) Molecules released by helminth parasites involved in host colonization. Acta Biochim Pol 53: 33–64.
- 57. D'Este F, Tomasinsig L, Skerlavaj B, Zanetti M (2012) Modulation of cytokine gene expression by cathelicidin BMAP-28 in LPS-stimulated and -unstimulated macrophages. Immunobiology 217: 962–971.
- 58. Nakamura S, Morita T, Iwanaga S, Niwa M, Takahashi K (1977) A sensitive substrate for the clotting enzyme in horseshoe crab hemocytes. J Biochem 81: 1567–1569.
- 59. Iwanaga S, Morita T, Harada T, Nakamura S, Niwa M, et al. (1978) Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis 7: 183–188.
- 60. Hochstein HD (1987) The LAL Test versus the Rabbit Pyrogen Test for endotoxin detection: update '87. Pharma Technol 11: 124–129.
- 61. Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI (1998) Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42: 2206–2214.
- 62. Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD, et al. (2000) Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immun 68: 2748–2755.
- 63. Tomasinsig L, De Conti G, Skerlavaj B, Piccinini R, Mazzilli M, et al. (2010) Broad-spectrum activity against bacterial mastitis pathogens and activation of mammary epithelial cells support a protective role of neutrophil cathelicidins in bovine mastitis. Infect Immun 78: 1781–1788.
- 64. Wang G, Epand RF, Mishra B, Lushnikova T, Thomas VC, et al. (2012) Decoding the functional roles of cationic side chains of the major antimicrobial region of human cathelicidin LL-37. Antimicrob Agents Chemother 56: 845–856.
- 65. Nelson A, Hultenby K, Hell E, Riedel HM, Brismar H, et al. (2009) Staphylococcus epidermidis isolated from newborn infants express pilus-like structures and are inhibited by the cathelicidin-derived antimicrobial peptide LL37. Pediatr Res 66: 174–178.
- 66. Kim SJ, Quan R, Lee SJ, Lee HK, Choi JK (2009) Antibacterial activity of recombinant hCAP18/LL37 protein secreted from Pichia pastoris. J Microbiol 47: 358–362.
- 67. Werth BJ, Sakoulas G, Rose WE, Pogliano J, Tewhey R, et al. (2013) Ceftaroline increases membrane binding and enhances the activity of daptomycin against daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus in a pharmacokinetic/pharmacodynamic model. Antimicrob Agents Chemother 57: 66–73.
- 68. Oren Z, Shai Y (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47: 451–463.
- 69. Ciornei CD, Sigurdardottir T, Schmidtchen A, Bodelsson M (2005) Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37. Antimicrob Agents Chemother 49: 2845–2850.
- 70. Schroder K, Sweet MJ, Hume DA (2006) Signal integration between IFNgamma and TLR signalling pathways in macrophages. Immunobiology 211: 511–524.
- 71. Held TK, Weihua X, Yuan L, Kalvakolanu DV, Cross AS (1999) Gamma interferon augments macrophage activation by lipopolysaccharide by two distinct mechanisms, at the signal transduction level and via an autocrine mechanism involving tumor necrosis factor alpha and interleukin-1. Infect Immun 67: 206–212.
- 72. Brown KL, Poon GF, Birkenhead D, Pena OM, Falsafi R, et al. (2011) Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses. J Immunol 186: 5497–5505.
- 73. Sasisekhar B, Aparna M, Augustin DJ, Kaliraj P, Kar SK, et al. (2005) Diminished monocyte function in microfilaremic patients with lymphatic filariasis and its relationship to altered lymphoproliferative responses. Infect Immun 73: 3385–3393.
- 74. Flynn RJ, Irwin JA, Olivier M, Sekiya M, Dalton JP, et al. (2007) Alternative activation of ruminant macrophages by Fasciola hepatica. Vet Immunol Immunopathol 120: 31–40.
- 75. Kin NW, Chen Y, Stefanov EK, Gallo RL, Kearney JF (2011) Cathelin-related antimicrobial peptide differentially regulates T- and B-cell function. Eur J Immunol 41: 3006–3016.
- 76. Nijnik A, Pistolic J, Wyatt A, Tam S, Hancock RE (2009) Human cathelicidin peptide LL-37 modulates the effects of IFN-gamma on APCs. J Immunol 183: 5788–5798.
- 77. Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, et al. (2004) Helminth parasites–masters of regulation. Immunol Rev 201: 89–116.
- 78. van Riet E, Hartgers FC, Yazdanbakhsh M (2007) Chronic helminth infections induce immunomodulation: consequences and mechanisms. Immunobiology 212: 475–490.
- 79. Jiang Z, Vasil AI, Hale JD, Hancock RE, Vasil ML, et al. (2008) Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides. Biopolymers 90: 369–383.
- 80. Chen Y, Mant CT, Farmer SW, Hancock RE, Vasil ML, et al. (2005) Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index. J Biol Chem 280: 12316–12329.
- 81. Zelezetsky I, Pontillo A, Puzzi L, Antcheva N, Segat L, et al. (2006) Evolution of the primate cathelicidin. Correlation between structural variations and antimicrobial activity. J Biol Chem 281: 19861–19871.
- 82. Porro M (1994) Structural basis of endotoxin recognition by natural polypeptides. Trends Microbiol 2: 65–66 discussion 66–67.
- 83. Lynn MA, Kindrachuk J, Marr AK, Jenssen H, Pante N, et al. (2011) Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival. PLoS Negl Trop Dis 5: e1141.
- 84. Haines LR, Thomas JM, Jackson AM, Eyford BA, Razavi M, et al. (2009) Killing of trypanosomatid parasites by a modified bovine host defense peptide, BMAP-18. PLoS Negl Trop Dis 3: e373.
- 85. Bowdish DM, Davidson DJ, Lau YE, Lee K, Scott MG, et al. (2005) Impact of LL-37 on anti-infective immunity. J Leukoc Biol 77: 451–459.
- 86. Selsted ME, Tang YQ, Morris WL, McGuire PA, Novotny MJ, et al. (1993) Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem 268: 6641–6648.
- 87. Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, et al. (2006) Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol 176: 2455–2464.
- 88. Nishio N, Ito S, Suzuki H, Isobe K (2009) Antibodies to wounded tissue enhance cutaneous wound healing. Immunology 128: 369–380.
- 89. Easton DM, Nijnik A, Mayer ML, Hancock RE (2009) Potential of immunomodulatory host defense peptides as novel anti-infectives. Trends Biotechnol 27: 582–590.
- 90. Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 341 (Pt 3) 501–513.
- 91. Risso A, Zanetti M, Gennaro R (1998) Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cell Immunol 189: 107–115.
- 92. Dathe M, Wieprecht T (1999) Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta 1462: 71–87.
- 93. Tossi A, Sandri L, Giangaspero A (2000) Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55: 4–30.
- 94. Kai-Larsen Y, Agerberth B (2008) The role of the multifunctional peptide LL-37 in host defense. Front Biosci 13: 3760–3767.
- 95. Vermeire JJ, Cho Y, Lolis E, Bucala R, Cappello M (2008) Orthologs of macrophage migration inhibitory factor from parasitic nematodes. Trends Parasitol 24: 355–363.
- 96. Prieto-Lafuente L, Gregory WF, Allen JE, Maizels RM (2009) MIF homologues from a filarial nematode parasite synergize with IL-4 to induce alternative activation of host macrophages. J Leukoc Biol 85: 844–854.
- 97. Gomez-Escobar N, Gregory WF, Maizels RM (2000) Identification of tgh-2, a filarial nematode homolog of Caenorhabditis elegans daf-7 and human transforming growth factor beta, expressed in microfilarial and adult stages of Brugia malayi. Infect Immun 68: 6402–6410.
- 98. Osman A, Niles EG, LoVerde PT (2001) Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. J Biol Chem 276: 10072–10082.
- 99. McSorley HJ, Grainger JR, Harcus Y, Murray J, Nisbet AJ, et al. (2010) daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns. Parasitology 137: 159–171.
- 100. Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y, et al. (2010) Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway. J Exp Med 207: 2331–2341.
- 101. Margis R, Reis EM, Villeret V (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch Biochem Biophys 359: 24–30.
- 102. Gregory WF, Maizels RM (2008) Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship. Int J Biochem Cell Biol 40: 1389–1398.
- 103. Dainichi T, Maekawa Y, Ishii K, Zhang T, Nashed BF, et al. (2001) Nippocystatin, a cysteine protease inhibitor from Nippostrongylus brasiliensis, inhibits antigen processing and modulates antigen-specific immune response. Infect Immun 69: 7380–7386.
- 104. Manoury B, Gregory WF, Maizels RM, Watts C (2001) Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing. Curr Biol 11: 447–451.
- 105. Schierack P, Lucius R, Sonnenburg B, Schilling K, Hartmann S (2003) Parasite-specific immunomodulatory functions of filarial cystatin. Infect Immun 71: 2422–2429.
- 106. Zhu S (2008) Did cathelicidins, a family of multifunctional host-defense peptides, arise from a cysteine protease inhibitor? Trends Microbiol 16: 353–360.