Antimicrobial peptide LL‐37 promotes antigen‐specific immune responses in mice by enhancing Th17‐skewed mucosal and systemic immunities

The human antimicrobial peptide LL‐37 is known to have chemotactic and modulatory activities on various cells including monocytes, T cells, and epithelial cells. Given that LL‐37 enhances chemotactic attraction and modulates the activity of DCs, it is conceivable that it might play a role as an immune adjuvant by skewing the immune environment toward immunostimulatory conditions. In this study, we characterized the mucosal adjuvant activity of LL‐37 using model and pathogenic Ags. When LL‐37‐conjugated Ag was administered orally to mice, a tolerogenic Peyer's patch environment was altered to cell populations containing IL‐6‐secreting CD11c⁺, CD11c⁺CD70⁺, and Th17 cells capable of evoking a subsequent LL‐37‐conjugated Ag‐specific immune response in both systemic and mucosal immune compartments. In addition, we showed presentation of formyl peptide receptor, an LL‐37 receptor, on M cells, which may aid the initiation of an LL‐37‐mediated enhanced immune response through targeting and transcytosis of the conjugated Ag. Based on our findings, we conclude that LL‐37 has potential as an oral mucosal adjuvant, not only by enhancing the delivery of LL‐37‐conjugated Ag to M cells, but also by triggering T‐cell‐mediated Ag‐specific immune responses through modulation of the mucosal immune environment.     

Pulmonary defense and the human cathelicidin hCAP-18/LL-37

Antimicrobial peptides form an important component of the innate immune system. The cathelicidin family, a key member of the antimicrobial peptide defenses, has been highly conserved throughout evolution. Though widespread in mammals, there is currently only one identified human example, hCAP-18/LL-37. The cathelicidins have been found to have multiple functions, in addition to their known antimicrobial and lipopolysaccharide-neutralizing effects. As a result, they profoundly affect both innate and adaptive immunity. Currently, antimicrobial peptides are being evaluated as therapeutic drugs in disease states as diverse as oral mucositis, cystic fibrosis, and septic shock. One such peptide, the cathelicidin hCAP-18/LL-37, is reviewed in detail in the context of its role in lung physiology and defense.     

Expression of human cathelicidin peptide LL‐37 in inflammatory bowel disease

Cathelicidin peptide LL‐37 plays an important role in the early host response against invading pathogens via its broad‐spectrum anti‐microbial activity. In this study, we investigated LL‐37 expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, the regulatory mechanism of LL‐37 induction was investigated in human colonic subepithelial myofibroblasts (SEMFs). LL‐37 mRNA expression and protein secretion were analysed using real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Intracellular signalling pathways were analysed using immunoblotting and specific small interference RNA (siRNA). The expression of LL‐37 mRNA was increased significantly in the inflamed mucosa of ulcerative colitis and Crohn's disease. The Toll‐like receptor (TLR)‐3 ligand, polyinosinic‐polycytidylic acid (poly(I:C), induced LL‐37 mRNA expression and stimulated LL‐37 secretion in colonic SEMFs. The transfection of siRNAs specific for intracellular signalling proteins [Toll/IL‐1R domain‐containing adaptor‐inducing interferon (IFN) (TRIF), tumour necrosis factor receptor‐associated factor (TRAF)6, transforming growth factor β‐activated kinase (TAK)1] suppressed the poly(I:C)‐induced LL‐37 mRNA expression significantly. Poly(I:C)‐induced phosphorylation of mitogen‐activated protein kinases (MAPKs) and activated nuclear factor kappa B (NF‐κB) and activating factor protein (AP)‐1. siRNAs specific for NF‐κB and c‐Jun inhibited poly(I:C)‐induced LL‐37 mRNA expression. LL‐37 suppressed lipopolysaccharide (LPS)‐induced interleukin (IL)‐6 and IL‐8 expression significantly in colonic SEMFs. The expression of LL‐37 was up‐regulated in the inflamed mucosa of IBD patients. LL‐37 was induced by TLR‐3 stimulation and exhibited an anti‐microbial effect via interaction with lipopolysaccharide (LPS).     

Host defense peptides and their antimicrobial-immunomodulatory duality

Host defence peptides (HDPs) are short cationic molecules produced by the immune systems of most multicellular organisms and play a central role as effector molecules of innate immunity. Host defence peptides have a wide range of biological activities from direct killing of invading pathogens to modulation of immunity and other biological responses of the host. HDPs have important functions in multiple, clinically relevant disease processes and their imbalanced expression is associated with pathology in different organ systems and cell types. Furthermore, HDPs are now evaluated as model molecules for the development of novel natural antibiotics and immunoregulatory compounds. This review provides an overview of HDPs focused on their antimicrobial-immunomodulatory duality.     

Too much of a good thing: How modulating LTB4 actions restore host defense in homeostasis or disease

The ability to regulate inflammatory pathways and host defense mechanisms is critical for maintaining homeostasis and responding to infections and tissue injury. While unbalanced inflammation is detrimental to the host; inadequate inflammation might not provide effective signals required to eliminate pathogens. On the other hand, aberrant inflammation could result in organ damage and impair host defense. The lipid mediator leukotriene B₄ (LTB₄) is a potent neutrophil chemoattractant and recently, its role as a dominant molecule that amplifies many arms of phagocyte antimicrobial effector function has been unveiled. However, excessive LTB₄ production contributes to disease severity in chronic inflammatory diseases such as diabetes and arthritis, which could potentially be involved in poor host defense in these groups of patients. In this review we discuss the cellular and molecular programs elicited during LTB₄ production and actions on innate immunity host defense mechanisms as well as potential therapeutic strategies to improve host defense.     

Multifunctional antimicrobial peptides: therapeutic targets in several human diseases

Antimicrobial peptides have emerged as promising agents against antibiotic-resistant pathogens. They represent essential components of the innate immunity and permit humans to resist infection by microbes. These gene-encoded peptides are found mainly in phagocytes and epithelial cells, showing a direct activity against a wide range of microorganisms. Their role has now broadened from that of simply endogenous antibiotics to multifunctional mediators, and their antimicrobial activity is probably not the only primary function. Although antimicrobial peptide deficiency, dysregulation, or overproduction is not known to be a direct cause of any single human disease, numerous studies have now provided compelling evidence for their involvement in the complex network of immune responses and inflammatory diseases, thereby influencing diverse processes including cytokine release, chemotaxis, angiogenesis, wound repair, and adaptive immune induction. The purpose of this review is to highlight recent literature, showing that antimicrobial peptides are associated with several human conditions including infectious and inflammatory diseases, and to discuss current clinical development of peptide-based therapeutics for future use.     

Understanding regulation of the host-mediated gut symbiont population and the symbiont-mediated host immunity in the Riptortus-Burkholderia symbiosis system

Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris, is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia. Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut.     

Anti-endotoxic and antibacterial effects of a dermal substitute coated with host defense peptides

Biomaterials used during surgery and wound treatment are of increasing importance in modern medical care. In the present study we set out to evaluate the addition of thrombin-derived host defense peptides to human acellular dermis (hAD, i.e. epiflex^®). Antimicrobial activity of the functionalized hAD was demonstrated using radial diffusion and viable count assays against Gram-negative Escherichia coli, Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Electron microscopy analyses showed that peptide-mediated bacterial killing led to reduced hAD degradation. Furthermore, peptide-functionalized hAD displayed endotoxin-binding activity in vitro, as evidenced by inhibition of NF-κB activation in human monocytic cells (THP-1 cells) and a reduction of pro-inflammatory cytokine production in whole blood in response to lipopolysaccharide stimulation. The dermal substitute retained its anti-endotoxic activity after washing, compatible with results showing that the hAD bound a significant amount of peptide. Furthermore, bacteria-induced contact activation was inhibited by peptide addition to the hAD. E. coli infected hAD, alone, or after treatment with the antiseptic substance polyhexamethylenebiguanide (PHMB), yielded NF-κB activation in THP-1 cells. The activation was abrogated by peptide addition. Thus, thrombin-derived HDPs should be of interest in the further development of new biomaterials with combined antimicrobial and anti-endotoxic functions for use in surgery and wound treatment.     

Purification and characterization of tenecin 4, a new anti-Gram-negative bacterial peptide, from the beetle Tenebrio molitor

The biochemical characterization of novel antimicrobial peptides (AMPs) and the determination of ligand molecules that induce AMP production are essential for understanding the host innate immune response in insects. Here, we purified a new 14-kDa AMP, named tenecin 4, from the larval hemolymph of the beetle Tenebrio molitor. Tenecin 4 contains 14% glycine residues and has moderate similarities both to the C-terminal region of Drosophila attacin and to silk-moth gloverin proteins. Purified tenecin 4 showed bactericidal activity against Gram-negative Escherichia coli but not against Gram-positive Bacillus subtilis or the fungus Candida albicans. Tenecin 4 production was induced by Toll cascade-activating ligands, such as β-1,3-glucan, lysine-type peptidoglycan and active Spätzle, and by the probable Imd pathway-activating ligand monomeric meso-diaminopimelic acid-type peptidoglycan. Taken together, these data show that tenecin 4 is a defense protein against Gram-negative pathogens and is induced by multiple ligands in Tenebrio larvae.     

Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps

The formation of extracellular traps (ETs) by phagocytic cells has been recognized as a novel and important mechanism of the host innate immune response against infections. ETs are formed by different host immune cells such as neutrophils, mast cells, and eosinophils after stimulation with mitogens, cytokines, or pathogens themselves, in a process dependent upon induction of a reactive-oxygen-species-mediated signaling cascade. ETs consist of nuclear or mitochondrial DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases and thereby provide a matrix to entrap and kill microbes and to induce the contact system. This review summarizes the latest research on ETs and their role in innate immunity and host innate defense. Attention is also given to mechanisms by which certain leading bacterial pathogens have evolved to avoid entrapment and killing in these specialized structures.     

Evidence that cathelicidin peptide LL‐37 may act as a functional ligand for CXCR2 on human neutrophils

LL‐37, derived from human cathelicidin, stimulates immune responses in neutrophils. Although FPR2 and P2X7 were proposed as LL‐37 receptors, we have shown that among 21 neutrophil receptors only CXCR2 was down‐regulated by LL‐37. LL‐37 functions similarly to CXCR2‐specific chemokines CXCL1 and CXCL7 in terms of receptor down‐regulation and intracellular calcium mobilization on freshly isolated neutrophils. Neutrophils pretreated with CXCL8, a chemokine that binds both CXCR1/2, completely blocked the calcium mobilization in response to LL‐37, while LL‐37 also partially inhibited ¹²⁵I‐CXCL8 binding to neutrophils. SB225002, a selective CXCR2 antagonist, blocked LL‐37‐induced calcium mobilization and migration of neutrophils. LL‐37 stimulates calcium mobilization in CXCR2‐transfected HEK293 cells, CXCR2⁺ THP‐1 cells and monocytes, but not in CXCR1‐transfected HEK293 cells. WKYMVm peptide (ligand for FPR2) does not block LL‐37‐stimulated calcium flux in either THP‐1 (FPR2^?) or monocytes (FPR2^high), further confirming the specificity of LL‐37 for CXCR2 and not FPR2. Among all ligands tested (ATP, BzATP, WKYMVm, CXCL1, and LL‐37), only LL‐37 stimulated migration of monocytes (CXCR2⁺ and FPR2⁺) and migration was inhibited by the CXCR2 inhibitor SB225002. Moreover, CXCR2 but not CXCR1 was internalized in LL‐37‐treated neutrophils. Thus, our data provide evidence that LL‐37 may act as a functional ligand for CXCR2 on human neutrophils.     

Eosinophils, ribonucleases and host defense: Solving the puzzle

The eosinophil ribonucleases eosinophil-derived neurotoxin (EDN/ RNase 2) and eosinophil cationic protein (ECP/RNase 3) are among the major secretory effector proteins of human eosinophilic leukocytes, cells whose role in host defense remains controversial and poorly understood. We have recently described the unusual manner in which this ribonuclease lineage has evolved, with extraordinary diversification observed in primate as well as in rodent EDNs and ECPs. The results of our evolutionary studies suggest that the EDN/ ECP ribonucleases are in the process of being tailored for a specific, ribonuclease-related goal. With this in mind, we have begun to look carefully at some of the intriguing associations that link eosinophils and their ribonucleases to disease caused by the single-stranded RNA viral pathogen, respiratory syncytial virus (RSV). Recent work in our laboratory has demonstrated that eosinophils can mediate a direct, ribonuclease-dependent reduction in infectivity of RSV in vitro, and that EDN can function alone as an independent antiviral agent. The results of this work have led us to consider the possibility that the EDN/ECP ribonucleases represent a heretofore unrecognized element of innate and specific antiviral host defense.     

Antimicrobial mechanisms of fish phagocytes and their role in host defense

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.     

Reciprocal regulation of IL‐23 and IL‐12 following co‐activation of Dectin‐1 and TLR signaling pathways

Recognition of microbial products by germ‐line‐encoded PRR initiates immune responses, but how PRR mediate specific host responses to infectious agents is poorly understood. We and others have proposed that specificity is achieved by collaborative responses mediated between different PRR. One such example comprises the fungal β‐glucan receptor Dectin‐1, which collaborates with TLR to induce TNF production. We show here that collaborative responses mediated by Dectin‐1 and TLR2 are more extensive than first appreciated, and result in enhanced IL‐23, IL‐6 and IL‐10 production in DC, while down‐regulating IL‐12 relative to the levels produced by TLR ligation alone. Such down‐regulation occurred with multiple MyD88‐coupled TLR, was dependent on signaling through Dectin‐1 and also occurred in macrophages. These findings explain how fungi can induce IL‐23 and IL‐6, while suppressing IL‐12, a combination which has previously been shown to contribute to the development of Th17 responses found during fungal infections. Furthermore, these data reveal how the collaboration of different PRR can tailor specific responses to infectious agents.     

Downregulation of the Musca domestica peptidoglycan recognition protein SC (PGRP-SC) leads to overexpression of antimicrobial peptides and tardy pupation

PGRP (peptidoglycan recognition protein) is a conserved protein family that recognizes the peptidoglycan in bacterial cell wall and causes the activation of various innate immune responses. Previous studies have reported that PGRP-SCs in Drosophila dampen the activation of Immune Deficiency (Imd) pathway to microbial infection, and participate in the lifespan extension of the insects. To facilitate understanding the function of PGRP-SCs from an evolutionary angle, we identified and functionally characterized the PGRP-SC gene in the housefly Musca domestica, a species that has adapted to a septic environment much harsher than the natural habitat of Drosophila. The gene designated as MdPGRP-SC was found most abundantly expressed in the 3rd instar larvae, and is expressed at this developmental stage predominantly in the gut. MdPGRP-SC was virtually unchanged in whole larvae after a septic injury at the second larval instar, while two antimicrobial peptides (AMPs), diptericin and attacin, were upregulated in the first 24 h but not later. Through dsRNA microinjection, MdPGRP-SC was knocked down by RNA interference (RNAi), and caused the significant increased expression of diptericin and attacin. The pupation of MdPGRP-SC-depleted larvae was severely suppressed compared to controls. Opposite to the expression trend of MdPGRP-SC, a spontaneous active expression of diptericin and attacin was found in pre-pupae but not in third instar larvae. Taken together, our study reveals that downregulation of MdPGRP-SC leads to the overexpression of the AMPs, and is involved in the larvae-to-pupa transition of housefly.     

Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain

Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.     

Activation of an innate immune response in large numbers of permeabilized Drosophila embryos

Innate immunity in Drosophila involves the inducible expression and synthesis of antimicrobial peptides. We have previously shown that not only Drosophila larvae and adults, but also embryos, are capable of mounting an immune response after injection of bacterial substances. To simplify genetic dissection of the signaling pathways involved in immune-gene regulation we developed a procedure for permeabilization of large number of embryos and subsequent infiltration with bacterial fragments. This approach, which promoted expression of CecropinA1- and Diptericin-driven β-gal expression in the epidermis of more than 90% of the treated embryos, will enable analysis of mutants that are embryonic lethal. Thus, genes that are involved in essential pleiotrophic functions, in addition to being candidates in immune-regulation will be amenable for analysis of their involvement in the fly's immune defense.