Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Effects of Escherichia coli LPS Structure on Antibacterial and Anti-Endotoxin Activities of Host Defense Peptides.

The binding of Host Defense Peptides (HDPs) to the endotoxin of Gram-negative bacteria has important unsolved aspects. For most HDPs, it is unclear if binding is part of the antibacterial mechanism or whether LPS actually provides a protective layer against HDP killing. In addition, HDP binding to LPS can block the subsequent TLR4-mediated activation of the immune system. This dual activity is important, considering that HDPs are thought of as an alternative to conventional antibiotics, which do not provide this dual activity. In this study, we systematically determine, for the first time, the influence of the O-antigen and Lipid A composition on both the antibacterial and anti-endotoxin activity of four HDPs (CATH-2, PR-39, PMAP-23, and PMAP36). The presence of the O-antigen did not affect the antibacterial activity of any of the tested HDPs. Similarly, modification of the lipid A phosphate (MCR-1 phenotype) also did not affect the activity of the HDPs. Furthermore, assessment of inner and outer membrane damage revealed that CATH-2 and PMAP-36 are profoundly membrane-active and disrupt the inner and outer membrane of Escherichia coli simultaneously, suggesting that crossing the outer membrane is the rate-limiting step in the bactericidal activity of these HDPs but is independent of the presence of an O-antigen. In contrast to killing, larger differences were observed for the anti-endotoxin properties of HDPs. CATH-2 and PMAP-36 were much stronger at suppressing LPS-induced activation of macrophages compared to PR-39 and PMAP-23. In addition, the presence of only one phosphate group in the lipid A moiety reduced the immunomodulating activity of these HDPs. Overall, the data strongly suggest that LPS composition has little effect on bacterial killing but that Lipid A modification can affect the immunomodulatory role of HDPs. This dual activity should be considered when HDPs are considered for application purposes in the treatment of infectious diseases.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens.

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

Chimeric Peptidomimetic Antibiotic Efficiently Neutralizes Lipopolysaccharides (LPS) and Bacteria-Induced Activation of RAW Macrophages.

Peptide antibiotics have gathered attention given the urgent need to discover antimicrobials with new mechanisms of action. Their extended role as immunomodulators makes them interesting candidates for the development of compounds with dual mode of action. The objective of this study was to test the anti-inflammatory capacity of a recently reported chimeric peptidomimetic antibiotic (CPA) composed of polymyxin B nonapeptide (PMBN) and a macrocyclic ß-hairpin motif (MHM). We investigated the potential of CPA to inhibit lipopolysaccharide (LPS)-induced activation of RAW264.7 macrophages. In addition, we elucidated which structural motif was responsible for this activity by testing CPA, its building blocks, and their parent compounds separately. CPA showed excellent LPS neutralizing activity for both smooth and rough LPSs. At nanomolar concentrations, CPA completely inhibited LPS-induced nitric oxide, TNF-α, and IL-10 secretion. Murepavadin, MHM, and PMBN were incapable of neutralizing LPS in this assay, while PMB was less active compared to CPA. Isothermal titration calorimetry showed strong binding between the CPA and LPS with similar binding characteristics also found for the other compounds, indicating that binding does not necessarily correlate with neutralization of LPS. Finally, we showed that CPA-killed bacteria caused significantly less macrophage activation than bacteria killed with gentamicin, heat, or any of the other compounds. This indicates that the combined killing activity and LPS neutralization of CPA can prevent unwanted inflammation, which could be a major advantage over conventional antibiotics. Our data suggests that immunomodulatory activity can further strengthen the therapeutic potential of peptide antibiotics and should be included in the characterization of novel compounds.

Avian surfactant protein (SP)-A2 first arose in an early tetrapod before the divergence of amphibians and gradually lost the collagen domain.

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP -D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SP-A-like gene. Here we investigated the evolution of the SP-A and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SP-A analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SP-A. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SP-A. In fact, the CRD of SP-A is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

Probing Cell-Surface Interactions in Fungal Cell Walls by High-Resolution 1 H-Detected Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.

Chicken cathelicidin-2 promotes NLRP3 inflammasome activation in macrophages.

Chicken cathelicidin-2 (CATH-2) as a host defense peptide has been identified to have potent antimicrobial and immunomodulatory activities. Here, we reported the mechanism by which CATH-2 modulates NLRP3 inflammasome activation. Our results show that CATH-2 and ATP as a positive control induced secretion of IL-1ß and IL-1α in LPS-primed macrophages but did not affect secretion of IL-6, IL-12 and TNF-α. Furthermore, CATH-2 induced caspase-1 activation and oligomerization of apoptosis-associated speck-like protein containing a carboxy- terminal caspase recruitment domain (ASC), which is essential for NLRP3 inflammasome activation. However, CATH-2 failed to induce IL-1ß secretion in Nlrp3-/-, Asc-/- and Casp1-/- macrophages. Notably, IL-1ß and NLRP3 mRNA expression were not affected by CATH-2. In addition, CATH-2-induced NLRP3 inflammasome activation was mediated by K+ efflux but independent of the P2X7 receptor that is required for ATP-mediated K+ efflux. Gene interference of NEK7 kinase which has been identified to directly interact with NLRP3, significantly reduced IL-1ß secretion and caspase-1 activation induced by CATH-2. Furthermore, confocal microscopy shows that CATH-2 significantly induced lysosomal leakage with the diffusion of dextran fluorescent signal. Cathepsin B inhibitors completely abrogated IL-1ß secretion and caspase-1 activation as well as attenuating the formation of ASC specks induced by CATH-2. These results all indicate that CATH-2-induced activation of NLRP3 inflammasome is mediated by K+ efflux, and involves the NEK7 protein and cathepsin B. In conclusion, our study shows that CATH-2 acts as a second signal to activate NLRP3 inflammasome. Our study provides new insight into CATH-2 modulating immune response.

Innate Immune Training of Human Macrophages by Cathelicidin Analogs.

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.

Modulation of outer membrane vesicle-based immune responses by cathelicidins.

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.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Chicken cathelicidin-2 promotes IL-1ß secretion via the NLRP3 inflammasome pathway and serine proteases activity in LPS-primed murine neutrophils.

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-1ß and IL-1α while IL-6 and TNF-α were not affected. IL-1ß 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-1ß 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-1ß and NLRP3, demonstrating that CATH-2 serves as the second signal activating the NLRP3 inflammasome. Furthermore, CATH-2-mediated IL-1ß 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-1ß 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-1ß 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.

Synthetic Antibiotic Derived from Sequences Encrypted in a Protein from Human Plasma.

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.

d-enantiomers of CATH-2 enhance the response of macrophages against Streptococcus suis serotype 2.

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.

The cathelicidin CATH-2 efficiently neutralizes LPS- and E. coli-induced activation of porcine bone marrow derived macrophages.

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.

The Interplay between Salmonella and Intestinal Innate Immune Cells in Chickens.

Salmonellosis is a common infection in poultry, which results in huge economic losses in the poultry industry. At the same time, Salmonella infections are a threat to public health, since contaminated poultry products can lead to zoonotic infections. Antibiotics as feed additives have proven to be an effective prophylactic option to control Salmonella infections, but due to resistance issues in humans and animals, the use of antimicrobials in food animals has been banned in Europe. Hence, there is an urgent need to look for alternative strategies that can protect poultry against Salmonella infections. One such alternative could be to strengthen the innate immune system in young chickens in order to prevent early life infections. This can be achieved by administration of immune modulating molecules that target innate immune cells, for example via feed, or by in-ovo applications. We aimed to review the innate immune system in the chicken intestine; the main site of Salmonella entrance, and its responsiveness to Salmonella infection. Identifying the most important players in the innate immune response in the intestine is a first step in designing targeted approaches for immune modulation.

PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles.

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.

Outer Membrane Vesicles Protect Gram-Negative Bacteria against Host Defense Peptides.

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.

PepBiotics, novel cathelicidin-inspired antimicrobials to fight pulmonary bacterial infections.

BACKGROUND: Antimicrobial peptides are considered potential alternatives to 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 ITC 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.

Activity of Mannose-Binding Lectin on Bacterial-Infected Chickens-A Review.

In recent years, diseases caused by pathogenic bacteria have profoundly impacted chicken production by causing economic loss in chicken products and by-product revenues. MBL (mannose-binding lectin) is part of the innate immune system (IIS), which is the host's first line defense against pathogens. The IIS functions centrally by identifying pathogen-specific microorganism-associated molecular patterns (MAMPs) with the help of pattern recognition receptors (PRRs). Studies have classified mannose-binding lectin (MBL) as one of the PRR molecules which belong to the C-type lectin family. The protective role of MBL lies in its ability to activate the complement system via the lectin pathway and there seems to be a direct link between the chicken's health status and the MBL concentration in the serum. Several methods have been used to detect the presence, the level and the structure of MBL in chickens such as Enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR) among others. The concentration of MBL in the chicken ranges from 0.4 to 35 µg/mL and can be at peak levels at three to nine days at entry of pathogens. The variations observed are known to depend on the bacterial strains, breed and age of the chicken and possibly the feed manipulation strategies. However, when chicken MBL (cMBL) becomes deficient, it can result in malfunctioning of the innate immune system, which can predispose chickens to diseases. This article aimed to discuss the importance and components of mannose-binding lectin (MBL) in chickens, its mode of actions, and the different methods used to detect MBL. Therefore, more studies are recommended to explore the causes for low and high cMBL production in chicken breeds and the possible effect of feed manipulation strategies in enhancing cMBL production.

Host defence peptides identified in human apolipoprotein B as promising antifungal agents.

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.