The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors.

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents.

Antiviral Effect of hBD-3 and LL-37 during Human Primary Keratinocyte Infection with West Nile Virus.

West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.

Emerging Roles of Vitamin D-Induced Antimicrobial Peptides in Antiviral Innate Immunity.

Vitamin D deficiency, characterized by low circulating levels of calcifediol (25-hydroxyvitamin D, 25D) has been linked to increased risk of infections of bacterial and viral origin. Innate immune cells produce hormonal calcitriol (1,25-dihydroxyvitamin D, 1,25D) locally from circulating calcifediol in response to pathogen threat and an immune-specific cytokine network. Calcitriol regulates gene expression through its binding to the vitamin D receptor (VDR), a ligand-regulated transcription factor. The hormone-bound VDR induces the transcription of genes integral to innate immunity including pattern recognition receptors, cytokines, and most importantly antimicrobial peptides (AMPs). Transcription of the human AMP genes ß-defensin 2/defensin-ß4 (HBD2/DEFB4) and cathelicidin antimicrobial peptide (CAMP) is stimulated by the VDR bound to promoter-proximal vitamin D response elements. HDB2/DEFB4 and the active form of CAMP, the peptide LL-37, which form amphipathic secondary structures, were initially characterized for their antibacterial actively. Notably, calcitriol signaling induces secretion of antibacterial activity in vitro and in vivo, and low circulating levels of calcifediol are associated with diverse indications characterized by impaired antibacterial immunity such as dental caries and urinary tract infections. However, recent work has also provided evidence that the same AMPs are components of 1,25D-induced antiviral responses, including those against the etiological agent of the COVID-19 pandemic, the SARS-CoV2 coronavirus. This review surveys the evidence for 1,25D-induced antimicrobial activity in vitro and in vivo in humans and presents our current understanding of the potential mechanisms by which CAMP and HBD2/DEFB4 contribute to antiviral immunity.

Update on the aetiology and mechanisms of generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a chronic disease characterized by non-bacterial pustules. Variants in several genes, such as IL36RN, AP1S3, and CARD14, are involved in the pathogenesis of GPP. The prevalence of different gene variants varies among ethnicities, and some variants are related to concurrent psoriasis vulgaris or age at onset. Flares can be triggered by medications (most commonly corticosteroids), infections (possibly due to Toll-like receptor [TLR] and antimicrobial peptides), pregnancy (the onset of GPP has been attributed to endocrine abnormalities such as hypoparathyroidism and hypocalcaemia), hypocalcaemia (presumably due to low levels of calcium and vitamin D regulating the proliferation and differentiation of keratinocytes), and other factors including stress and sun exposure. The mechanisms of pustule formation involve: 1) the LL37/TLR pathway, in which LL37 acts as an alarmin, interacting with TLR and activating the NF-κB and MAPK pathways; 2) the balance between calcium and 1,25(OH)2D levels, and 3) neutrophils and the complement system.

Anti-Carbamylated LL37 Antibodies Promote Pathogenic Bone Resorption in Rheumatoid Arthritis.

Objective: Antibodies against carbamylated proteins (anti-CarP) are associated with poor prognosis and the development of bone erosions in rheumatoid arthritis (RA). RA neutrophils externalize modified autoantigens through the formation of neutrophil extracellular traps (NETs). Increased levels of the cathelicidin LL37 have been documented in the synovium of RA patients, but the cellular source remains unclear. We sought to determine if post-translational modifications of LL37, specifically carbamylation, occur during NET formation, enhance this protein's autoantigenicity, and contribute to drive bone erosion in the synovial joint. Methods: ELISA and Western blot analyses were used to identify carbamylated LL37 (carLL37) in biological samples. Anti-carLL37 antibodies were measured in the serum of HLA-DRB1*04:01 transgenic mice and in human RA synovial fluid. Results: Elevated levels of carLL37 were found in plasma and synovial fluid from RA patients, compared to healthy controls. RA NETs release carLL37 and fibroblast-like synoviocytes (FLS) internalized NET-bound carLL37 and loaded it into their MHCII compartment. HLA-DRB1*04:01 transgenic mice immunized with FLS containing NETs developed autoantibodies against carLL37. Anti-carLL37 antibodies were present in RA sera and synovial fluid and they correlated with radiologic bone erosion scores of the hands and feet in RA patients. CarLL37-IgG immune complexes enhanced the ability of monocytes to differentiate into osteoclasts and potentiated osteoclast-mediated extracellular matrix resorption. Conclusions: NETs are a source of carLL37 leading to induction of anti-carbamylated autoantibody responses. Furthermore, carLL37-IgG immune complexes may be implicated in the bone damage characteristic of RA. These results support that dysregulated NET formation has pathogenic roles in RA.

Mast cell phenotypic plasticity and their activity under the influence of cathelicidin-related antimicrobial peptide (CRAMP) and IL-33 alarmins.

Invading pathogens are contained/eliminated by orchestrated actions of different humoral components of the innate immune response. One of them is endogenous molecules called alarmins, which contribute to diverse processes from danger sense until the infection extinction. Considering the participation of mast cells (MCs) in many aspects of the body's defense and, on the other hand, the importance of alarmins as molecules that signal damage/danger, in this study, we evaluated the effect of alarmins on MC phenotype and activity. We found that cathelicidin CRAMP and cytokine IL-33 significantly affect the appearance of Dectin-1, Dectin-2, RIG-I, and NOD1 receptors in mature MCs and modulate their inflammatory response. We established that chosen alarmins might stimulate MCs to release pro-inflammatory and immunoregulatory mediators and induce a migratory response. In conclusion, our data highlight that alarmins CRAMP and IL-33 might strongly influence MC features and activity, mainly by strengthening their role in the inflammatory mechanisms and controlling the activity of cells participating in antimicrobial processes.

Molecular characterization of cathelicidin in tiger frog (Hoplobatrachus rugulosus): Antimicrobial activity and immunomodulatory activity.

Cathelicidins are an important antimicrobial peptide family and are expressed in many different vertebrates. They play an important role in the innate immune system of the host. However, amphibian cathelicidins are poorly understood. In this study, the cDNA of the cathelicidin gene was obtained from the skin transcriptome of tiger frog (Hoplobatrachus rugulosus). The predicted amino acid sequence of tiger frog cathelicidin (HR-CATH) comprises a signal peptide, a cathelin domain, and a mature peptide. The HR-CATH amino acid sequence alignment with other frog cathelicidins showed that the functional mature peptide is highly variable in amphibians, whereas the cathelin domain is conserved. A phylogenetic tree analysis showed that HR-CATH is most closely related to cathelicidin-NV from Nanorana ventripunctata. HR-CATH was chemically synthesized and its in vitro activity was determined. It had high antibacterial activity against Vibrio parahaemolyticus, Staphylococcus aureus, and the pathogenic bacterium Aeromonas hydrophila. HR-CATH damaged the cell membrane integrity of A. hydrophila according to a lactate dehydrogenase release assay and was able to hydrolyze the genomic DNA from A. hydrophila in a dose-dependent manner. Furthermore, in RAW264.7 cells (mouse leukemic monocyte/macrophage cell line), HR-CATH induced chemotaxis and enhanced respiratory burst. Our study shows that amphibian cathelicidin has antimicrobial activity and an immunomodulatory effect on immune cells.

Peritoneal macrophages are impaired in cathelicidin-deficient mice systemically challenged with Escherichia coli.

Cathelicidins are small, cationic peptides produced by macrophages with protective effects against infection although their involvement in phagocytosis is not fully understood. This study demonstrates that fewer macrophages were recruited in mice genetically deficient in cathelicidin (Camp-/-) during acute Escherichia coli-induced peritonitis and those macrophages had impaired phagocytosis. These defects seem due to endogenous functions of murine cathelicidin (CRAMP) as phagocytosis was not improved by synthetic human cathelicidin (LL-37) in a murine phagocytic cell line. This knowledge contributes to understanding the function of cathelicidins in the recruitment and function of phagocytic cells and differential roles between endogenous and exogenous cathelicidins.

The immunomodulatory effect of cathelicidin-B1 on chicken macrophages.

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-ß, IL-1ß, 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.

Cathelicidins Modulate TLR-Activation and Inflammation.

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.

Citrullination Alters the Antiviral and Immunomodulatory Activities of the Human Cathelicidin LL-37 During Rhinovirus Infection.

Human rhinoviruses (HRV) are the most common cause of viral respiratory tract infections. While normally mild and self-limiting in healthy adults, HRV infections are associated with bronchiolitis in infants, pneumonia in immunocompromised patients, and exacerbations of asthma and COPD. The human cathelicidin LL-37 is a host defense peptide (HDP) with broad immunomodulatory and antimicrobial activities that has direct antiviral effects against HRV. However, LL-37 is known to be susceptible to the enzymatic activity of peptidyl arginine deiminases (PAD), and exposure of the peptide to these enzymes results in the conversion of positively charged arginines to neutral citrullines (citrullination). Here, we demonstrate that citrullination of LL-37 reduced its direct antiviral activity against HRV. Furthermore, while the anti-rhinovirus activity of LL-37 results in dampened epithelial cell inflammatory responses, citrullination of the peptide, and a loss in antiviral activity, ameliorates this effect. This study also demonstrates that HRV infection upregulates PAD2 protein expression, and increases levels of protein citrullination, including histone H3, in human bronchial epithelial cells. Increased PADI gene expression and HDP citrullination during infection may represent a novel viral evasion mechanism, likely applicable to a wide range of pathogens, and should therefore be considered in the design of therapeutic peptide derivatives.

Expression and Function of Host Defense Peptides at Inflammation Sites.

There is a growing interest in the complex role of host defense peptides (HDPs) in the pathophysiology of several immune-mediated inflammatory diseases. The physicochemical properties and selective interaction of HDPs with various receptors define their immunomodulatory effects. However, it is quite challenging to understand their function because some HDPs play opposing pro-inflammatory and anti-inflammatory roles, depending on their expression level within the site of inflammation. While it is known that HDPs maintain constitutive host protection against invading microorganisms, the inducible nature of HDPs in various cells and tissues is an important aspect of the molecular events of inflammation. This review outlines the biological functions and emerging roles of HDPs in different inflammatory conditions. We further discuss the current data on the clinical relevance of impaired HDPs expression in inflammation and selected diseases.

Diversity, immunoregulatory action and structure-activity relationship of green sea turtle cathelicidins.

Cathelicidins are a family of gene-encoded immune effectors in vertebrate innate immunity. Here, we reported the diversity and biological activity of cathelicidins in green sea turtle, a marine reptile species known for long lifespan and disease resistance. Four novel cathelicidins (Cm-CATH1-4) were identified from green sea turtle. All of them, especially Cm-CATH2, exhibited potent, broad-spectrum and rapid bactericidal and anti-biofilm activities by inducing the disruption of cell membrane integrity. Additionally, Cm-CATH2 effectively induced the macrophages/monocytes and neutrophils trafficking to the infection site, and inhibited the LPS-induced production of inflammatory cytokines, by blocking TLR4/MD2 complex and the downstream signaling pathway activation. In mouse peritonitis and pneumonia models, Cm-CATH2 exhibited evident protection against drug-resistant bacterial infections. Taken together, the diverse structures and functions of Cm-CATHs indicated their pleiotropic role in innate immunity of green sea turtle, and the potent antimicrobial, anti-biofilm and immunomodulatory properties make them ideal candidates for the development of novel anti-infective drugs.

Modulation of cathelicidins, IFNß and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

In vitro immunomodulatory activities of peptides derived from Salmo salar NK-lysin and cathelicidin in fish cells.

Antimicrobial peptides (AMPs) are amphipathic peptides, which play an important role in innate defence. These peptides are gene-encoded and either constitutively expressed and/or upregulated during an infection. NK-lysins are AMPs with a three-dimensional globular structure. They are larger molecules, which comprise 74-78 amino acid residues and six conserved cysteine residues forming three disulphide bonds. Cathelicidins are a family of antimicrobial peptides that act as important components of the innate immune system with a broad spectrum of antimicrobial activity and immunomodulatory properties. Although they are widely studied in mammals, little is known about their immunomodulatory function. In the present study, we identified and characterized for the first time four NK-lysin-like transcripts from Atlantic salmon (Salmo salar) based on EST reported sequences. In vitro, NK-lysin derived peptides were able to induce the expression of IL-1ß and IL-8 in Salmo salar head kidney leukocytes. We also tested Salmo salar cathelicidin 1 derived peptide in a similar assay, showing its ability to induce the expression of IFN-γ. These results indicate that NK-lysin and cathelicidin 1 derived peptides are able to modulated immune response, suggesting their potential use to enhance immune response in fish.

G.I. pros: Antimicrobial defense in the gastrointestinal tract.

The gastrointestinal tract is a complex environment in which the host immune system interacts with a diverse array of microorganisms, both symbiotic and pathogenic. As such, mobilizing a rapid and appropriate antimicrobial response depending on the nature of each stimulus is crucial for maintaining the balance between homeostasis and inflammation in the gut. Here we focus on the mechanisms by which intestinal antimicrobial peptides regulate microbial communities during dysbiosis and infection. We also discuss classes of bacterial peptides that contribute to reducing enteric pathogen outgrowth. This review aims to provide a comprehensive overview on the interplay of diverse antimicrobial responses with enteric pathogens and the gut microbiota.

Modulation of toll-like receptor signaling by antimicrobial peptides.

Antimicrobial peptides (AMPs) are typically thought of as molecular hole punchers that directly kill pathogens by membrane permeation. However, recent work has shown that AMPs are pleiotropic, multifunctional molecules that can strongly modulate immune responses. In this review, we provide a historical overview of the immunomodulatory properties of natural and synthetic antimicrobial peptides, with a special focus on human cathelicidin and defensins. We also summarize the various mechanisms of AMP immune modulation and outline key structural rules underlying the recently-discovered phenomenon of AMP-mediated Toll-like receptor (TLR) signaling. In particular, we describe several complementary studies demonstrating how AMPs self-assemble with nucleic acids to form nanocrystalline complexes that amplify TLR-mediated inflammation. In a broader scope, we discuss how this new conceptual framework allows for the prediction of immunomodulatory behavior in AMPs, how the discovery of hidden antimicrobial activity in known immune signaling proteins can inform these predictions, and how these findings reshape our understanding of AMPs in normal host defense and autoimmune disease.

Human antimicrobial peptides and cancer.

Antimicrobial peptides (AMPs) have long been a topic of interest for entomologists, biologists, immunologists and clinicians because of these agents' intriguing origins in insects, their ubiquitous expression in many life forms, their capacity to kill a wide range of bacteria, fungi and viruses, their role in innate immunity as microbicidal and immunoregulatory agents that orchestrate cross-talk with the adaptive immune system, and, most recently, their association with cancer. We and others have theorized that surveillance through epithelial cell-derived AMPs functions to keep the natural flora of microorganisms in a steady state in different niches such as the skin, the intestines, and the mouth. More recently, findings related to specific activation pathways of some of these AMPs have led investigators to associate them with pro-tumoral activity; i.e., contributing to a tumorigenic microenvironment. This area is still in its infancy as there are intriguing yet contradictory findings demonstrating that while some AMPs have anti-tumoral activity and are under-expressed in solid tumors, others are overexpressed and pro-tumorigenic. This review will introduce a new paradigm in cancer biology as it relates to AMP activity in neoplasia to address the following questions: Is there evidence that AMPs contribute to tumor promoting microenvironments? Can an anti-AMP strategy be of use in cancer therapy? Do AMPs, expressed in and released from tumors, contribute to compositional shifting of bacteria in cancerous lesions? Can specific AMP expression characteristics be used one day as early warning signs for solid tumors?

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.