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Divergent evolution for more than 310 million years has resulted in an avian immune system that is complex and more compact than that of primates, sharing much of its structure and functions. Not surprisingly, well conserved ancient host defense molecules, such as defensins and cathelicidins, have diversified over time. In this review, we describe how evolution influenced the host defense peptides repertoire, its distribution, and the relationship between structure and biological functions. Marked features of primate and avian HDPs are linked to species-specific characteristics, biological requirements, and environmental challenge.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), a coronavirus of domestic fowl. IB is a major concern in the poultry industry, causing worldwide economic losses through decreased egg production and quality and by increasing the chicken's susceptibility for secondary bacterial infections, particularly Escherichia coli. In this study, the anti-IBV activity of cathelicidins, small antimicrobial peptides of the innate immune system was investigated. The cell culture adapted (nonvirulent) IBV strain Beaudette was effectively inhibited by the human cathelicidin LL-37 in bovine hamster kidney-21 cells at nontoxic concentrations. The peptide needed to be present during virus inoculation to effectively inhibit the infection of IBV-Beaudette, indicating that LL-37 likely bound viral particles. However, no clear morphological changes in the IBV virion upon binding were observed by electron microscopy. In this cell culture model, chicken cathelicidins (CATH1-3) were inactive against IBV-Beaudette. In contrast, in multicellular infection models using the virulent IBV-M41 strain the activities of human and chicken cathelicidins were different. In particular, upon inoculation of 10-day-old embryonic eggs with IBV-M41, CATH-2 reduced the viral load to a higher extend than LL-37. Similarly, viral infection of chicken tracheal organ cultures with IBV-M41 was significantly reduced in the presence of CATH-2 but not LL-37. These results indicate a potential antiviral role for CATH-2 upon IBV infection in vivo.

Graphical abstract, Highlights • D-CATH-2 has strong antimicrobial activities towards multiple S.suis strains. • D-CATH-2 ameliorates macrophage function. • DCATH-2 binds LTA. • DCATH-2 has prophylactic effect against S. suis infection in vivo., Introduction Due to the increase of antibiotic resistant bacterial strains, there is an urgent need for development of alternatives to antibiotics. Cathelicidins can be such an alternative to antibiotics having both a direct antimicrobial capacity as well as an immunomodulatory function. Previously, the full d-enantiomer of chicken cathelicidin-2 (d-CATH-2) has shown to prophylactically protect chickens against infection 7 days post hatch when administered in ovo three days before hatch. Objectives To further evaluate d-CATH-2 in mammals as a candidate for an alternative to antibiotics. In this study, the prophylactic capacity of d-CATH-2 and two truncated derivatives, d-C(1–21) and d-C(4–21), was determined in mammalian cells. Methods Antibacterial assays; immune cell differentiation and modulation; cytotoxicity, isothermal titration calorimetry; in vivo prophylactic capacity of peptides in an S. suis infection model. Results d-CATH-2 and its derivatives were shown to have a strong direct antibacterial capacity against four different S. suis serotype 2 strains (P1/7, S735, D282, and OV625) in bacterial medium and even stronger in cell culture medium. In addition, d-CATH-2 and its derivatives ameliorated the efficiency of mouse bone marrow-derived macrophages (BMDM) and skewed mouse bone marrow-derived dendritic cells (BMDC) towards cells with a more macrophage-like phenotype. The peptides directly bind lipoteichoic acid (LTA) and inhibit LTA-induced activation of macrophages. In addition, S. suis killed by the peptide was unable to further activate mouse macrophages, which indicates that S. suis was eliminated by the previously reported silent killing mechanism. Administration of d-C(1–21) at 24 h or 7 days before infection resulted in a small prophylactic protection with reduced disease severity and reduced mortality of the treated mice. Conclusion d-enantiomers of CATH-2 show promise as anti-infectives against pathogenic S. suis for application in mammals.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica, i.e. pagP and lpxL1, could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

Background Antimicrobial peptides are considered potential alternatives for antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. Methods Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of Pepbiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. Results A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using isothermal titration calorimetry and 31P solid state NMR. Conclusions PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. General significance These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

Establishing the rapid and accurate diagnosis of sepsis is a key component to the improvement of clinical outcomes. The ability of analytical platforms to rapidly detect pathogen-associated molecular patterns (PAMP) in blood could provide a powerful host-independent biomarker of sepsis. A novel concept was investigated based on the idea that a pre-bound and fluorescent ligand could be released from lectins in contact with high-affinity ligands (such as PAMPs). To create fluorescent ligands with precise avidity, the kinetically followed TEMPO oxidation of yeast mannan and carbodiimide coupling were used. The chemical modifications led to decreases in avidity between mannan and human collectins, such as the mannan-binding lectin (MBL) and human surfactant protein D (SP-D), but not in porcine SP-D. Despite this effect, these fluorescent derivatives were captured by human lectins using highly concentrated solutions. The resulting fluorescent beads were exposed to different solutions, and the results showed that displacements occur in contact with higher affinity ligands, proving that two-stage competition processes can occur in collectin carbohydrate recognition mechanisms. Moreover, the fluorescence loss depends on the discrepancy between the respective avidities of the recognized ligand and the fluorescent mannan. Chemically modulated fluorescent ligands associated with a diversity of collectins may lead to the creation of diagnostic tools suitable for multiplex array assays and the identification of high-avidity ligands.

Trained innate immunity can be induced in human macrophages by microbial ligands, but it is unknown if exposure to endogenous alarmins such as cathelicidins can have similar effects. Previously, we demonstrated sustained protection against infection by the chicken cathelicidin-2 analog DCATH-2. Thus, we assessed the capacity of cathelicidins to induce trained immunity. PMA-differentiated THP-1 (dTHP1) cells were trained with cathelicidin analogs for 24 hours and restimulated after a 3-day rest period. DCATH-2 training of dTHP-1 cells amplified their proinflammatory cytokine response when restimulated with TLR2/4 agonists. Trained cells displayed a biased cellular metabolism towards mTOR-dependent aerobic glycolysis and long-chain fatty acid accumulation and augmented microbicidal activity. DCATH-2-induced trained immunity was inhibited by histone acetylase inhibitors, suggesting epigenetic regulation, and depended on caveolae/lipid raft-mediated uptake, MAPK p38 and purinergic signaling. To our knowledge, this is the first report of trained immunity by host defense peptides.

Cathelicidins are short cationic peptides that are part of the innate immune system. At first, these peptides were studied mostly for their direct antimicrobial killing capacity, but nowadays they are more and more appreciated for their immunomodulatory functions. In this review, we will provide a comprehensive overview of the various effects cathelicidins have on the detection of damage- and microbe-associated molecular patterns, with a special focus on their effects on Toll-like receptor (TLR) activation. We review the available literature based on TLR ligand types, which can roughly be divided into lipidic ligands, such as LPS and lipoproteins, and nucleic-acid ligands, such as RNA and DNA. For both ligand types, we describe how direct cathelicidin-ligand interactions influence TLR activation, by for instance altering ligand stability, cellular uptake and receptor interaction. In addition, we will review the more indirect mechanisms by which cathelicidins affect downstream TLR-signaling. To place all this information in a broader context, we discuss how these cathelicidin-mediated effects can have an impact on how the host responds to infectious organisms as well as how these effects play a role in the exacerbation of inflammation in auto-immune diseases. Finally, we discuss how these immunomodulatory activities can be exploited in vaccine development and cancer therapies.

Cathelicidins have antimicrobial and immunomodulatory activities. Previous studies have shown that chicken cathelicidin-2 (CATH-2) exerts strong anti-inflammatory activity through LPS neutralization. However, it is still unclear whether other intracellular signaling pathways are involved in CATH-2 immunomodulation. Therefore, the CATH-2-meadiated immune response was investigated in LPS-primed neutrophils. Firstly, inflammatory cytokines release was determined in LPS-primed neutrophils. The results showed that CATH-2 significantly promoted secretion of IL-1β and IL-1α while IL-6 and TNF-α were not affected. IL-1β is the key indicator of inflammasome activation. Next, NLRP3 inflammasome signaling pathway was explored using neutrophils of Nlrp3 -/-, Asc -/- and Casp1 -/- mice and the results showed that the CATH-2-enhanced IL-1β release was completely abrogated, indicating it is NLRP3-dependent. Moreover, CATH-2 significantly induced activation of caspase-1 and gasdermin D (GSDMD) but did not affect LPS-induced mRNA expression of IL-1β and NLRP3, demonstrating that CATH-2 serves as the second signal activating the NLRP3 inflammasome. Furthermore, CATH-2-mediated IL-1β secretion and caspase-1 activation is dependent on potassium efflux but independent of P2X7R. In addition, other signaling pathways including JNK, ERK and SyK were investigated using different inhibitors and the results showed that these signaling pathway inhibitors partially attenuated CATH-2-enhanced IL-1β secretion, especially the JNK inhibitor. Finally, the role of serine protease in CATH-2-mediated NLRP3 inflammasome activation was investigated in neutrophils and the results showed that serine protease activity is involved in CATH-2-enhanced IL-1β secretion and caspase-1 activation. In conclusion, after LPS priming in neutrophils, CATH-2 can be an agonist of the NLRP3 inflammasome. Our study increases the understanding on immunomodulatory effects of chicken cathelicidins and provides new insight on chicken cathelicidins-mediated immune response.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes., The natural antibiotic teixobactin kills bacteria by direct binding to their cognate cell wall precursors (Lipid II and III). Here authors use solid-state NMR to reveal the native binding mode of teixobactins and show that teixobactins only weakly bind to Lipid II in anionic cellular membranes.

Encrypted peptides have been recently found in the human proteome and represent a potential class of antibiotics. Here we report three peptides derived from the human apolipoprotein B (residues 887-922) that exhibited potent antimicrobial activity against drug-resistant Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococci both in vitro and in an animal model. The peptides had excellent cytotoxicity profiles, targeted bacteria by depolarizing and permeabilizing their cytoplasmic membrane, inhibited biofilms, and displayed anti-inflammatory properties. Importantly, the peptides, when used in combination, potentiated the activity of conventional antibiotics against bacteria and did not select for bacterial resistance. To ensure translatability of these molecules, a protease resistant retro-inverso variant of the lead encrypted peptide was synthesized and demonstrated anti-infective activity in a preclinical mouse model. Our results provide a link between human plasma and innate immunity and point to the blood as a source of much-needed antimicrobials.

Highlights • Sub-lethal PMAP-36 treatment of bacteria increases outer membrane vesicle release. • Lipidomic analysis revealed the OMV lipidome upon PMAP-36 or heat treatment. • Supplementation with PMAP-36 attenuated undesirable OMV-induced immune responses., Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines., Graphical abstract Image, graphical abstract

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs.

Objectives: Our group recently developed a new group of antimicrobial peptides termed PepBiotics, of which peptides CR-163 and CR-172 showed optimized antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus without inducing antimicrobial resistance. In this study, the antibacterial mechanism of action and the immunomodulatory activity of these two PepBiotics was explored. Methods: RAW264.7 cells were used to determine the ability of PepBiotics to neutralize Lipopolysaccharide (LPS)-and Lipoteichoic acid (LTA)-induced activation of macrophages. Isothermal titration calorimetry and competition assays with dansyl-labeled polymyxin B determined binding characteristics to LPS and LTA. Combined bacterial killing with subsequent macrophage activation assays was performed to determine so-called ‘silent killing’. Finally, flow cytometry of peptide-treated genetically engineered Escherichia coli expressing Green Fluorescent Protein (GFP) and mCherry in the cytoplasm and periplasm, respectively, further established the antimicrobial mechanism of PepBiotics. Results: Both CR-163 and CR-172 were shown to have broad-spectrum activity against ESKAPE pathogens and E. coli using a membranolytic mechanism of action. PepBiotics could exothermically bind LPS/LTA and were able to replace polymyxin B. Finally, it was demonstrated that bacteria killed by PepBiotics were less prone to stimulate immune cells, contrary to gentamicin and heat-killed bacteria that still elicited a strong immune response. Conclusions: These studies highlight the multifunctional nature of the two peptide antibiotics as both broad-spectrum antimicrobial and immunomodulator. Their ability to kill bacteria and reduce unwanted subsequent immune activation is a major advantage and highlights their potential for future therapeutic use.

Gram-negative bacteria release vesicular structures from their outer membrane, so called outer membrane vesicles (OMVs). OMVs have a variety of functions such as waste disposal, communication, and antigen or toxin delivery. These vesicles are the promising structures for vaccine development since OMVs carry many surface antigens that are identical to the bacterial surface. However, isolation is often difficult and results in low yields. Several methods to enhance OMV yield exist, but these do affect the resulting OMVs. In this review, our current knowledge about OMVs will be presented. Different methods to induce OMVs will be reviewed and their advantages and disadvantages will be discussed. The effects of the induction and isolation methods used in several immunological studies on OMVs will be compared. Finally, the challenges for OMV-based vaccine development will be examined and one example of a successful OMV-based vaccine will be presented.

Fungal infections in humans are increasing worldwide and are currently mostly treated with a relative limited set of antifungals. Resistance to antifungals is increasing, for example, in Aspergillus fumigatus and Candida auris, and expected to increase for many medically relevant fungal species in the near future. We have developed and patented a set of cathelicidin-inspired antimicrobial peptides termed ‘PepBiotics’. These peptides were initially selected for their bactericidal activity against clinically relevant Pseudomonas aeruginosa and Staphylococcus aureus isolates derived from patients with cystic fibrosis and are active against a wide range of bacteria (ESKAPE pathogens). We now report results from studies that were designed to investigate the antifungal activity of PepBiotics against a set of medically relevant species encompassing species of Aspergillus, Candida, Cryptococcus, Fusarium, Malassezia, and Talaromyces. We characterized a subset of PepBiotics and show that these peptides strongly affected metabolic activity and/or growth of a set of medically relevant fungal species, including azole-resistant A. fumigatus isolates. PepBiotics showed a strong inhibitory activity against a large variety of filamentous fungi and yeasts species at low concentrations (≤1 μM) and were fungicidal for at least a subset of these fungal species. Interestingly, the concentration of PepBiotics required to interfere with growth or metabolic activity varied between different fungal species or even between isolates of the same fungal species. This study shows that PepBiotics display strong potential for use as novel antifungal compounds to fight a large variety of clinically relevant fungal species.

Abstract Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents. Key points Human cryptides identified in ApoB are effective antifungal agents. ApoB-derived cryptides exert fungicidal effects towards C. albicans cells. ApoB-derived cryptides affect different stages of growth of A. niger. Graphical abstract

Current vaccines against Bordetella pertussis do not prevent colonization and transmission of the bacteria, and vaccine-induced immunity wanes rapidly. Besides, efficacy of vaccines for Bordetella bronchiseptica remains unclear. Novel vaccines could be based on outer-membrane vesicles (OMVs), but vesiculation of bordetellae needs to be increased for cost-effective vaccine production. Here, we focused on increasing OMV production by reducing the anchoring of the outer membrane to the peptidoglycan layer. Inactivation of rmpM, tolR, and pal failed, presumably because their products are essential in bordetellae. Conditional pal mutants were constructed, which were hypervesiculating under Pal-depletion conditions. SDS-PAGE and Western blot analyses showed that the protein composition of OMVs produced under Pal-depletion conditions resembled that of the outer membrane but differed from that of OMVs released by the wild type. Pal depletion affected the cell morphology and appeared to increase the amounts of cell-surface-exposed phospholipids, possibly reflecting a role for the Tol-Pal system in retrograde phospholipid transport. We also identified additional lipoproteins in bordetellae with a putative peptidoglycan-anchoring domain. However, their inactivation did not influence OMV production. We conclude that the conditional pal mutants could be valuable for the development of OMV-based vaccines.

Antibiotic resistance is increasing and one strategy to prevent resistance development is the use of bacterial vaccines. For Gram-negative bacteria, natural outer membrane vesicles (OMVs) could be used for vaccine development. These vesicular structures are naturally produced by all Gram-negative bacteria and contain several antigens in their native environment. However, despite that the presence of lipopolysaccharide (LPS) may aid as intrinsic adjuvant, there is a risk that it may also cause undesired immune responses. Therefore, molecules to dampen LPS-induced toll-like receptor (TLR) 4 activation may be needed. Here host defense peptides (HDPs), like cathelicidins, can play an important role. They have been shown to interact with LPS and thereby neutralize LPS-induced TLR4 activation. However, there is currently no knowledge about neutralization in an OMV-based setting. Therefore, in this paper the immune modulating capacity of HDPs was investigated after macrophage stimulation with either spontaneous or heat-induced B. bronchiseptica OMVs. This revealed that the cathelicidins LL-37, CATH-2, PMAP-36 and K9CATH were able to modulate immune responses. Interestingly, immune modulation by these cathelicidins was different for spontaneous compared to heat-induced OMVs. Interaction studies revealed that the mode of binding of cathelicidins to OMVs slightly differed between OMV classes. Furthermore, TLR screening revealed that TLR2, 4, 5 and 9 were involved in stimulation of macrophages by OMVs, with TLR4-mediated activation being the most important pathway. Uptake of OMVs did not play a major role in macrophage activation. Taken together, this study shows how OMVs can activate macrophages and how cathelicidins may modulate these immune responses.

Background Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Methods Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM3CSK4 stimulation. Human monocyte-derived macrophages were used as control. Results Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Conclusion Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research.

Infectious diseases in pigs cause monetary loss to farmers and pose a zoonotic risk. Therefore, it is important to obtain more porcine specific immunological knowledge as a measure to protect against infectious diseases, for example by exploring immunomodulators that are usable as vaccine adjuvants. Cathelicidins are a class of host defence peptides (HDPs) able to directly kill microbes as well as exert a diverse range of effects on the immune system. The peptides have shown promise as immunomodulatory peptides in many applications, including vaccines. However, it is currently unknown what the precise effect of these peptides is on porcine immune cells and whether peptides of other species might also have a strong immunomodulatory effect on porcine macrophages. Mononuclear bone marrow cells of pigs, aged 5-6 months, were cultured into M1 or M2 macrophages and stimulated with LPS or whole bacteria in the presence of host defence peptides (HDPs). CATH-2 and LL-37 strongly inhibited LPS-induced activation of M1 macrophages, the inhibition of LPS-induced activation of M2 macrophages by HDPs was milder, showing that the peptides have selective effects on different cell types. Upon stimulation with whole bacteria, only CATH-2 could effectively inhibit macrophage activation, showing the potent anti-inflammatory potential of this peptide. These results show that porcine peptides are not necessarily the most active in a porcine system, and that CATH-2 is effective in a porcine system as an anti-inflammatory immune modulator, which can be used, for example, in inactivated pathogen vaccines.

Pertussis, also known as whooping cough, is caused by the Gram-negative bacterium Bordetella pertussis, an obligate human pathogen. Despite high vaccination rates in high-income countries, resurgence of pertussis cases is an occurring problem that urges the necessity of developing an improved vaccine. Likewise, the efficacy of vaccines for Bordetella bronchiseptica, which causes similar disease in pigs and companion animals, is debatable. A promising approach for novel vaccines is the use of outer membrane vesicles (OMVs). However, spontaneous OMV (sOMV) release by Bordetella spp. is too low for cost-effective vaccine production. Therefore, we investigated the influence of growth in various media commonly used for culturing Bordetella in the Bvg+, i.e. virulent, phase and of a heat shock applied to inactivate the cells on OMV production. Inactivation of the bacterial cells at 56 °C before OMV isolation greatly enhanced OMV release in both Bordetella spp. without causing significant cell lysis. The growth medium used barely affected the efficiency of OMV release but did affect the protein pattern of the OMVs. Differences were found to be related, at least in part, to different availability of the nutrient metals iron and zinc in the media and include expression of potentially relevant vaccine antigens, such as the receptors FauA and ZnuD. The protein content of OMVs released by heat shock was comparable to that of sOMVs as determined by SDS-PAGE and Western blot analysis, and their heat-modifiable electrophoretic mobility suggests that also protein conformation is unaffected. However, significant differences were noticed between the protein content of OMVs and that of a purified outer membrane fraction, with two major outer membrane proteins, porin OmpP and the peptidoglycan-associated RmpM, being underrepresented in the OMVs. Altogether, these results indicate that the application of a heat shock is potentially an important step in the development of cost-effective, OMV-based vaccines for both Bordetella spp.

The rising incidence of antibiotic-resistant lung infections has instigated a much-needed search for new therapeutic strategies. One proposed strategy is the use of exogenous surfactants to deliver antimicrobial peptides (AMPs), like CATH-2, to infected regions of the lung. CATH-2 can kill bacteria through a diverse range of antibacterial pathways and exogenous surfactant can improve pulmonary drug distribution. Unfortunately, mixing AMPs with commercially available exogenous surfactants has been shown to negatively impact their antimicrobial function. It was hypothesized that the phosphatidylglycerol component of surfactant was inhibiting AMP function and that an exogenous surfactant, with a reduced phosphatidylglycerol composition would increase peptide mediated killing at a distal site. To better understand how surfactant lipids interacted with CATH-2 and affected its function, isothermal titration calorimetry and solid-state nuclear magnetic resonance spectroscopy as well as bacterial killing curves against Pseudomonas aeruginosa were utilized. Additionally, the wet bridge transfer system was used to evaluate surfactant spreading and peptide transport. Phosphatidylglycerol was the only surfactant lipid to significantly inhibit CATH-2 function, showing a stronger electrostatic interaction with the peptide than other lipids. Although diluting the phosphatidylglycerol content in an existing surfactant, through the addition of other lipids, significantly improved peptide function and distal killing, it also reduced surfactant spreading. A synthetic phosphatidylglycerol-free surfactant however, was shown to further improve CATH-2 delivery and function at a remote site. Based on these in vitro experiments synthetic phosphatidylglycerol-free surfactants seem optimal for delivering AMPs to the lung.

© 2020, Springer Nature Limited. Cationic host defence peptides (CHDP), also known as antimicrobial peptides, are naturally occurring peptides that can combat infections through their direct microbicidal properties and/or by influencing the host’s immune responses. The unique ability of CHDP to control infections as well as resolve harmful inflammation has generated interest in harnessing the properties of these peptides to develop new therapies for infectious diseases, chronic inflammatory disorders and wound healing. Various strategies have been used to design synthetic optimized peptides, with negligible toxicity. Here, we focus on the progress made in understanding the scope of functions of CHDP and the emerging potential clinical applications of CHDP-based therapies.

Cathelicidins (CATHs) are host defense peptides (HDPs) that play an important role in the innate immune response against infections. Although multiple functions of cathelicidins have been described, including direct antimicrobial activity and several immunomodulatory effects on the host, relatively little is known about their antiviral activity. Therefore, in vitro antiviral activity of chicken cathelicidins and the underlying mechanism was investigated in this study against different influenza A virus (IAV) strains. Our results show that chicken CATH-B1 has broad anti-IAV activity compared to other cathelicidins (CATH-1, -2, -3, LL-37, PMAP-23, and K9CATH) with an inhibition of viral infection up to 80% against three tested IAV strains (H1N1, H3N1, and H5N1). In agreement herewith, CATH-B1 affected virus-induced inflammatory cytokines expression (IFN-β, IL-1β, IL-6, and IL-8). Incubation of cells with CATH-B1 prior to or after their inoculation with virus did not reduce viral infection indicating that direct interaction of virus with the peptide was required for CATH-B1's antiviral activity. Experiments using combined size exclusion and affinity-based separation of virus and peptide also indicated that CATH-B1 bound to viral particles. In addition, using electron microscopy, no morphological change of the virus itself was seen upon incubation with CATH-B1 but large aggregates of CATH-B1 and viral particles were observed, indicating that aggregation might be the mechanism of action reducing IAV infectivity. Neuraminidase (NA) activity assays using monovalent or multivalent substrates, indicated that CATH-B1 did not affect NA activity per se, but negatively affected the ability of virus particles to interact with multivalent receptors, presumably by interfering with hemagglutinin activity. In conclusion, our results show CATH-B1 has good antiviral activity against IAV by binding to the viral particle and thereby blocking viral entry.

Background: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. Objective: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. Method: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. Results: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, markers for antigen presentation, on primary chicken monocytes, whereas LL-37 did not. D-CATH- 2 also increased the MRC1 and MHC-II expression if a chicken macrophage cell line (HD11 cells) was used. In addition, LPS-induced NO production by HD11 cells was inhibited by CATH-2 and D-CATH-2. Conclusion: These results are a clear indication that CATH-2 (and D-CATH-2) affect the activation state of monocytes and macrophages, which leads to optimization of the innate immune response and enhancement of the adaptive immune response.

Macrophages are part of the first line of defense against invading pathogens. In mammals, the in vitro culture of macrophages from blood monocytes or bone marrow cells is well established, including culturing conditions to differentiate them towards M1 or M2-like macrophages. In chicken, monocyte-derived macrophages have been used in several studies, but there is no uniform protocol or actual characterization of these cells. Therefore, to generate proinflammatory M1-like macrophages, in this study blood monocytes were differentiated using GM-CSF for 4 days and characterized based on cell morphology, surface marker expression and cytokine expression response to TLRs stimulation at each (daily) time point. Cell morphology showed that one-day-cultured cells contained a mixture of cell populations, while the homogenous population of cells on day 3 and day 4 were flat and had a 'fried-egg' like shape, similar to human M1 macrophages. In addition, cell surface marker staining showed that 3 and 4- days-cultured cells expressed a high level of MRC1L-B (KUL01) and MHC-II. Furthermore, LPS stimulation of the cultured cells induced gene expression of the proinflammatory cytokines IL-1β, IL-6 and IL-8 after 3 days of culture. Finally, it was shown that day 3 macrophages were able to phagocytose avian pathogenic E. coli (APEC) and respond by nitric oxide production. Overall, our systematic characterization of the monocyte derived cells from blood showed that a 3-days culture was optimal to obtain pro-inflammatory M1 like macrophages, increasing our knowledge about chicken macrophage polarization and providing useful information for studies on chicken macrophage phenotypes.

Cathelicidins (CATHs) play an important role in the innate immune response against microbial infections. Among the four chicken cathelicidins, CATH-B1 is studied the least. In this study, the effect of CATH-B1 on the macrophage response towards avian pathogenic E. coli (APEC) and bacterial ligands was investigated. Our results show that APEC induced CATH-B1 gene expression in both a chicken macrophage cell line (HD11 cells) and primary macrophages, while expression of the other three CATHs was virtually unaffected. While the antimicrobial activity of CATH-B1 is very low under cell culture conditions, it enhanced bacterial phagocytosis by macrophages. Interestingly, CATH-B1 downregulated APEC-induced gene expression of pro-inflammatory cytokines (IFN-β, IL-1β, IL-6 and IL-8) in primary macrophages. In addition, CATH-B1 pre-incubated macrophages showed a significantly higher gene expression of IL-10 after APEC challenge, indicating an overall anti-inflammatory profile for CATH-B1. Using isothermal titration calorimetry (ITC), CATH-B1 was shown to bind LPS. This suggests that CATH-B1 reduces toll like receptor (TLR) 4 dependent activation by APEC which may partly explain the decreased production of pro-inflammatory cytokines by macrophages. On the contrary, direct binding of CATH-B1 to ODN-2006 enhanced the TLR21 dependent activation of macrophages as measured by nitric oxide production. In conclusion, our results show for the first time that CATH-B1 has several immunomodulatory activities and thereby could be an important factor in the chicken immune response.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Cationic Host Defense Peptides (HDPs) are endowed with a broad variety of activities, including direct antimicrobial properties and modulatory roles in the innate immune response. Even if it has been widely demonstrated that bacterial membrane represents the main target of peptide antimicrobial activity, the molecular mechanisms underlying membrane perturbation by HDPs have not been fully clarified yet. Recently, two cryptic HDPs have been identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, and with anti-biofilm, wound healing and immunomodulatory properties. Moreover, ApoB derived HDPs are able to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, by using a multidisciplinary approach, including time killing curves, Zeta potential measurements, membrane permeabilization assays, electron microscopy analyses, and isothermal titration calorimetry studies, the antimicrobial effects of ApoB cryptides have been analysed on bacterial strains either susceptible or resistant to peptide toxicity. Intriguingly, it emerged that even if electrostatic interactions between negatively charged bacterial membranes and positively charged HDPs play a key role in mediating peptide toxicity, they are strongly influenced by the composition of negatively charged bacterial surfaces and by defined extracellular microenvironments.

Salmonella Infantis is a poultry-adapted Salmonella enterica serovar that is increasingly reported in broilers and is also regularly identified among human salmonellosis cases. An emerging S. Infantis mega-plasmid (pESI), carrying fitness, virulence and antimicrobial resistance genes, is also increasingly found. We investigated the prevalence, genetic characteristics and risk factors for (pESI-carrying) S. Infantis in broilers. Faecal samples from 379 broiler flocks (in 198 farms with ≥3000 birds) in the Netherlands were tested. A questionnaire about farm characteristics was also administered. Sampling was performed in July 2018-May 2019, three weeks before slaughter. Fourteen flocks (in 10 farms) were S. Infantis-positive, resulting in a 3.7 % flock-level and 5.1 % farm-level prevalence. Based on multi-locus sequence typing (MLST), all isolates belonged to sequence type 32. All but one isolate carried a pESI-like mega-plasmid. Core-genome MLST showed considerable heterogeneity among the isolates, even within the same farm, with a few small clusters detected. The typical pESI-borne multi-resistance pattern to aminoglycosides, sulphonamide and tetracycline (93 %), as well as trimethoprim (71 %), was found. Additionally, resistance to (fluoro)quinolones based on gyrA gene mutations was detected. S. Infantis was found more often in flocks using salinomycin as coccidiostat, where flock thinning was applied or litter quality was poor, whereas employing external cleaning companies, wheat in feed, and vaccination against infectious bronchitis, were protective. Suggestive evidence for vertical transmission from hatcheries was found. A heterogeneous (pESI-carrying) S. Infantis population has established itself in Dutch broiler flocks, calling for further monitoring of its spread and a comprehensive appraisal of control options.