Cathelicidin Research Articles

Emergency care for life-threatening arrhythmias

Background. Sepsis is often accompanied by arrhythmias and conduction disorders. It can be assumed that pacemaker cells of the sinoatrial node, strongly sensitized by massive stimulation with β1-adrenergic catecholamines, tend to trigger arrhythmias. The importance of the inflammatory component in the development of new atrial fibrillation (AF) events is also confirmed by the existence of a strong correlation between increased levels of C-reactive protein, interleukin-6 and tumor necrosis factor and the onset of fibrillation. Under the conditions of the new-onset AF, the hospital mortality of patients of general profile in the intensive care unit (ICU) significantly exceeds that for people without AF.
 Objective. To describe the features of treatment of life-threatening arrhythmias.
 Materials and methods. Analysis of literature data on this issue.
 Results and discussion. Amiodarone, diltiazem and lidocaine are the most commonly used treatments for life-threatening arrhythmias. According to a UK-wide study, amiodarone is used to treat new-onset AF in ICU in 80.94 % of cases, β-blockers (BB) – in 11.60 %, other antiarrhythmic drugs (AAD) – in 3.87 %, and digoxin – in 3.31 %. However, this tactic is not in line with the existing guidelines. According to the recommendations for the heart rate (HR) control in emergency care for AF (Bokeria L.A. et al., 2017), in an acute situation in the absence of ventricular pre-excitation syndrome intravenous administration of BB or non-dihydropyridine calcium channel blockers (CCB) is recommended to slow ventricular rhythm in patients with AF. Caution should be taken in patients with hypotension or heart failure. For the last group of patients intravenous administration of cardiac glycosides or amiodarone is recommended. In patients with ventricular pre-excitation syndrome, class I AAD or amiodarone are the drugs of choice. In presence of the pre-excitation syndrome and AF BB, non-dihydropyridine CCB, digoxin and adenosine are contraindicated. The guidelines for the management of AF patients, developed in 2017 by the European Society of Cardiology in collaboration with the European Association of Cardiothoracic Surgery, recommend to use different management tactics depending on the left ventricular ejection fraction (LV EF). In case of LV EF <40 % or signs of heart failure, the lowest effective dose of BB should be prescribed to achieve rhythm control. Amiodarone is prescribed to hemodynamically unstable patients or to individuals with severely reduced LV EF. The primary goal of treatment is to achieve a HR <110 beats/min. In the absence of this result, digoxin should be added. In case of LV EF ≥40 %, BB, or diltiazem, or verapamil should be administered. In the absence of clinical result, digoxin should be added. Practical models of AF treatment in sepsis have demonstrated the superiority of BB over CCB, digoxin and amiodarone (Walkey A.J. et al., 2016). BB weaken the stimulating effect of the sympathetic part of the autonomic nervous system on the myocardium, have a negative chronotropic effect, improve the contractility of ischemized cardiomyocytes, slow atrioventricular conduction, reduce myocardial oxygen demand, and apoptosis. Esmolol (Biblok, “Yuria-Pharm”) is indicated for supraventricular tachycardia (except for ventricular pre-excitation syndrome) and for the rapid control of ventricular rhythm in patients with AF or atrial flutter in the pre- and postoperative periods or in other circumstances when it is necessary to normalize ventricular rhythm with a short-acting drug. Studies show that esmolol inhibits inflammation in sepsis by increasing the expression of the antimicrobial peptide cathelicidin. Kaplan – Mayer analysis shows better survival for experimental animals with sepsis receiving esmolol compared to animals in the 0.9 % NaCl group (Ibrahim-Zada I. et al., 2014).
 Conclusions. 1. Sepsis is often accompanied by arrhythmias and conduction disorders. 2. Under the conditions of new-onset AF, the hospital mortality of patients of general somatic profile in ICU significantly exceeds the number for people without AF. 3. In case of AF and LV EF <40 % or signs of heart failure, the lowest effective dose of BB should be prescribed to achieve rhythm control. 4. In case of LV EF ≥40 %, BB, or diltiazem, or verapamil should be administered. 5. Esmolol is indicated for supraventricular tachycardia and for the rapid control of ventricular rhythm in patients with AF or atrial flutter. 6. Esmolol inhibits inflammation in sepsis by increasing the expression of the antimicrobial peptide cathelicidin.

DNase-mediated eDNA removal enhances D-LL-31 activity against biofilms of bacteria isolated from chronic rhinosinusitis patients

Chronic rhinosinusitis (CRS) is a chronic infection of the nasal cavity and paranasal sinuses associated with the presence of a microbial biofilm. Extracellular DNA (eDNA) is an important component of the biofilm matrix. Antimicrobial peptides (AMPs) are natural peptides with the ability to kill microorganisms. D-LL-31 is a synthetic variant of the AMP cathelicidin with increased resistance to proteolytic breakdown. In this study it is shown for 3 clinical CRS isolates that treatment of 24 h biofilms with DNase I enhanced the antimicrobial activity of D-LL-31. Conversely, co-incubation of D-LL-31 at the IC50 value with exogenous DNA resulted in reduced antimicrobial activity. DNase I alone did not show antimicrobial activity against the isolates tested but caused dispersal of an established biofilm. Hence, the presence of eDNA in the biofilm matrix reduced AMP-mediated killing. These results suggest that combination therapy with proteolysis resistant AMP D-LL-31 and DNase could be considered for effective treatment of CRS.

Role of 4-hydroxybutyrate in increased resistance to surgical site infections associated with surgical meshes

An increased resistance to surgical site infections has been associated with surgical meshes composed of naturally occurring materials, including poly-4-hydroxybutrate (4HB). 4HB is a naturally occurring short-chain fatty acid that has been shown to promote endogenous expression of the Cramp gene coding for the antimicrobial peptide (AMP) cathelicidin LL-37 in murine bone marrow-derived macrophages. The molecular pathways involved in the 4HB-induced cathelicidin LL-37 expression have not yet been identified. The present study showed that transcriptional activation of the Cramp gene by 4HB is independent of inhibition of histone deacetylase (HDAC) activity, and that upregulation of Cramp is modulated by the G-protein coupled receptor GPR109A. Furthermore, an intracellular signaling cascade that promotes the activation of the MAP kinases, p38 and JNK, and a subsequent NF-κB phosphorylation downstream from p38 is essential for the AMP transcriptional response in 4HB-stimulated macrophages. The findings provide a solid scientific basis and rationale for the decreased incidence of surgical site infections with the use of this type of surgical meshes. Further clinical significance is found in the fact that the 4HB activated molecular pathway includes common targets of frequently used nonsteroidal anti-inflammatory drugs (NSAIDs) and other FDA approved drugs recognizing G-protein coupled receptors.

Antimicrobial peptides cathelicidin, PMAP23, and PMAP37: Expression in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy and regulation by steroid hormones and calcitriol in pigs

The endometrium, which regulates the reproductive cyclicity and the establishment and maintenance of pregnancy, is a type of mucosal tissue and is involved in the regulation of immunity. Antimicrobial peptides, called cathelicidins, play critical roles in innate immunity by killing various microbes and modulating host immune properties. The expression and regulation of cathelicidin in female reproductive tissues have been studied in some species, but not determined in pigs, which form a true epitheliochorial placenta. Thus, we determined the expression of Cathelicidin (the conserved common cathelin domain for all porcine cathelicidin family members), PMAP23, and PMAP37 in the endometrium throughout the estrous cycle and pregnancy. The expression of Cathelicidin, PMAP23, and PMAP37 in the endometrium changed during the estrous cycle and pregnancy in a stage-specific manner. The mRNA expression of Cathelicidin, PMAP23, and PMAP37 was also detected in conceptus and chorioallantoic tissues during pregnancy. Cathelicidin protein was mainly localized to luminal epithelial cells and some immune cells in blood vessels and to chorionic epithelial cells of chorioallantoic membranes during mid-to late pregnancy. The expression of Cathelicidin was induced by calcitriol but increasing doses of estradiol-17β and progesterone did not affect the expression of Cathelicidin, PMAP23, or PMAP37 in endometrial tissues. These results indicate that members of the cathelicidin family were dynamically expressed at the maternal-conceptus interface in pigs, suggesting that the cathelicidin family may play important roles in the regulation of innate immunity by mediating antimicrobial activity to protect the maternal and/or conceptus tissues to conserve pregnancy capacity in pigs.

Reducing Risks from COVID-19: Cost-Effective Ways of Strengthening Individual's and the Population Immunity with Vitamin D

In addition to being involved in the regulation of calcium and phosphate metabolism and the musculoskeletal functions, vitamin D has immune modulatory effects through several independent pathways. Its active hormone, calcitriol [1,25(OH)2D] effect both innate and adaptive immune systems essential for optimal immune functions. Vitamin D deficiency exacerbates immune-related disorders, including type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriasis, respiratory infections, including COVID-19. In those with COVID-19, complications and the number of deaths is higher in those who are older than 70 years, persons with a darker skin colour and/or ethnic minorities living in colder climatic regions, institutionalized persons, and with pre-existing chronic diseases. These groups of people have exceedingly high prevalence of severe vitamin D deficiency and thereof weaker immune systems. Collectively, these increases the vulnerability to microbial infections, particularly respiratory viruses, and for developing severe complications and deaths. Vitamin D related immune protective effects includes, the generation of anti-microbial peptides cathelicidin and defensins and antibodies against invading pathogens; the initiation of immune defences via natural killer cells, macrophages, and epithelial cells; the enhanced expression of angiotensin-converting enzyme 2 (ACE-2) and diminish expression of inflammatory cytokines; and reduce replication and enhance elimination of viruses from the body. The severity of complications and deaths associated with COVID-19 markedly increases in the presence of severe hypovitaminosis D: serum 25(OH)D concentration of less than 10 ng/mL. Excess complications and deaths from COVID-19 can be cost-effectively prevented with rapidly boosting the immunity using upfront loading, high doses of vitamin D; this will create an equivalent of internal “body armour”-defence system, that protects against COVID-19.

Vitamin D3 Inhibits Helicobacter pylori Infection by Activating the VitD3/VDR-CAMP Pathway in Mice.

Helicobacter pylori (H. pylori) infection is closely associated with the occurrence and development of gastric diseases. Therefore, eliminating H. pylori infection should help to prevent gastric diseases. Vitamin D3 (VitD3, 1,25(OH)2D3) was previously observed to exhibit anti-H. pylori infection activity in clinic, but these results were reported in heterogeneous in vivo studies without elucidation of the underlying mechanisms. In the present study, we established H. pylori infection models in both wild-type and VDR knockdown (VDR-KD) mice, which were used to demonstrate that VitD3 inhibits H. pylori infection by enhancing the expression of VitD receptor (VDR) and cathelicidin antimicrobial peptide (CAMP). Furthermore, VDR-KD mice that exhibited lower VDR expression were more susceptible to H. pylori infection. In cultured mouse primary gastric epithelial cells, we further demonstrated that the VitD3/VDR complex binds to the CAMP promoter region to increase its expression. These data provide a mechanistic explanation of the anti-H. pylori infection activity of VitD3 at the molecular level in mice and suggest a new avenue for the clinical management of H. pylori eradication therapy.

Human cathelicidin antimicrobial peptide suppresses proliferation, migration and invasion of oral carcinoma HSC-3 cells via a novel mechanism involving caspase-3 mediated apoptosis.

Human cathelicidin antimicrobial peptide and its active product, LL-37 (CAMP/LL-37), exhibit a broad spectrum of antimicrobial effects. An increasing number of studies have shown that human CAMP/LL-37 also serves significant roles in various types of cancer. The primary aims of the present study were to investigate the roles and mechanisms of human CAMP/LL-37 in oral squamous cell carcinoma (OSCC) cells. The results indicated that either LL-37 C-terminal deletion mutants (CDEL) or CAMP stable expression in HSC-3 cells reduced colony formation, proliferation, migration and invasion ability of the cells. Expression analysis demonstrated that either CDEL or CAMP stable expression in HSC-3 cells induced caspase-3 mediated apoptosis via the P53-Bcl-2/BAX signalling pathway, whereas the levels of cell cycle-related proteins, cyclin B1 and PKR-like ER kinase, were significantly upregulated in the CAMP, but not in the CDEL overexpressing cells. Transcriptional profile comparisons revealed that CDEL or CAMP stable expression in HSC-3 cells upregulated expression of genes involved in the IL-17-dependent pathway compared with the control. Taken together, these results suggest that CAMP may act as a tumour suppressor in OSCC cells, and the underlying mechanism involves the induction of caspase-3 mediated apoptosis via the P53-Bcl-2/BAX signalling pathway.

LL-37-Induced Autophagy Contributed to the Elimination of Live Porphyromonas gingivalis Internalized in Keratinocytes.

Porphyromonas gingivalis (P. gingivalis), one of the most important pathogens of periodontitis, is closely associated with the aggravation and recurrence of periodontitis and systemic diseases. Antibacterial peptide LL-37, transcribed from the cathelicidin antimicrobial peptide (CAMP) gene, exhibits a broad spectrum of antibacterial activity and regulates the immune system. In this study, we demonstrated that LL-37 reduced the number of live P. gingivalis (ATCC 33277) in HaCaT cells in a dose-dependent manner via an antibiotic-protection assay. LL-37 promoted autophagy of HaCaT cells internalized with P. gingivalis. Inhibition of autophagy with 3-methyladenine (3-MA) weakened the inhibitory effect of LL-37 on the number of intracellular P. gingivalis. A cluster of orthologous groups (COGs) and a gene ontology (GO) functional analysis were used to individually assign 65 (10%) differentially expressed genes (DEGs) to an “Intracellular trafficking, secretion, and vesicular transport” cluster and 306 (47.08%) DEGs to metabolic processes including autophagy. Autophagy-related genes, a tripartite motif-containing 22 (TRIM22), and lysosomal-associated membrane protein 3 (LAMP3) were identified as potentially involved in LL-37-induced autophagy. Finally, bioinformatics software was utilized to construct and predict the protein–protein interaction (PPI) network of CAMP-TRIM22/LAMP3-Autophagy. The findings indicated that LL-37 can reduce the quantity of live P. gingivalis internalized in HaCaT cells by promoting autophagy in these cells. The transcriptome sequencing and analysis also revealed the potential molecular pathway of LL-37-induced autophagy.

The Role of T Cells Reactive to the Cathelicidin Antimicrobial Peptide LL-37 in Acute Coronary Syndrome and Plaque Calcification

The human cationic anti-microbial peptide LL-37 is a T cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. However, the role of LL-37 as a T cell self-antigen in the context of atherosclerosis remains unclear. The objective of this study was to test for the presence of T cells reactive to LL-37 in patients with acute coronary syndrome (ACS). Furthermore, the role of T cells reactive to LL-37 in atherosclerosis was assessed using apoE−/− mice immunized with the LL-37 mouse ortholog, mCRAMP. Peripheral blood mononuclear cells (PBMCs) from patients with ACS were stimulated with LL-37. PBMCs from stable coronary artery disease (CAD) patients or self-reported subjects served as controls. T cell memory responses were analyzed with flow cytometry. Stimulation of PBMCs with LL-37 reduced CD8+ effector T cell responses in controls and patients with stable CAD but not in ACS and was associated with reduced programmed cell death protein 1 (PDCD1) mRNA expression. For the mouse studies, donor apoE−/− mice were immunized with mCRAMP or adjuvant as controls, then T cells were isolated and adoptively transferred into recipient apoE−/− mice fed a Western diet. Recipient mice were euthanized after 5 weeks. Whole aortas and hearts were collected for analysis of atherosclerotic plaques. Spleens were collected for flow cytometric and mRNA expression analysis. Adoptive transfer experiments in apoE−/− mice showed a 28% reduction in aortic plaque area in mCRAMP T cell recipient mice (P < 0.05). Fifty six percent of adjuvant T cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mCRAMP T cell recipient mice (Fisher’s exact test P = 0.003). Recipients of T cells from mice immunized with mCRAMP had increased IL-10 and IFN-γ expression in CD8+ T cells compared to controls. In conclusion, the persistence of CD8+ effector T cell response in PBMCs from patients with ACS stimulated with LL-37 suggests that LL-37-reactive T cells may be involved in the acute event. Furthermore, studies in apoE−/− mice suggest that T cells reactive to mCRAMP are functionally active in atherosclerosis and may be involved in modulating plaque calcification.

DNA microarray analysis of hypothermia-exposed murine lungs for identification of forensic biomarkers.

We used DNA microarray technology to analyze the pulmonary transcriptome of mice killed by hypothermia. This analysis identified significant differential regulation of 4094 genes; specifically, 1699 genes were upregulated, and 2395 were downregulated in response to hypothermia. The gene encoding cathelicidin antimicrobial peptide was the most upregulated gene, and that encoding BAI1-associated protein 2-like 1 was the most downregulated. Gene-set analysis identified significant hypothermia-induced variations in 101 pathways, and we discovered that pathways related to immunity are involved in the pulmonary pathogenesis of hypothermia. The present findings demonstrate some of the acute pulmonary responses to hypothermia and indicate several pulmonary genes as candidate forensic biomarkers of hypothermia. Furthermore, the present findings suggest that host defense is induced in hypothermic lungs. The present microarray data may facilitate the development of protein analyses for human forensics by immunohistochemistry, western blotting and enzyme-linked immunosorbent assay and may be beneficial in clinical research of hypothermia.

Dispersed liquid crystals as pH-adjustable antimicrobial peptide nanocarriers

HypothesispH-responsive nanocarriers have the potential to provide targeted delivery of antimicrobial peptides (AMPs) to sites of bacterial infection with typically abnormal pH levels in the body. However, the local pH of the infected sites varies substantially among different infection-related diseases, calling for the development of delivery systems capable of targeting local pathological conditions in an adjustable pH range. ExperimentsIn this study, a highly versatile pH-responsive nanocarrier platform, based on dispersions of oleic acid (OA) and glycerol monooleate (GMO) self-assemblies with the human cathelicidin AMP LL-37, was designed and characterized. FindingsA detailed pH-composition phase diagram was constructed from small angle X-ray scattering and cryogenic transmission electron microscopy data. In addition, the protonation state and apparent pKa of OA embedded in these nano-self-assemblies were investigated by electrophoretic mobility measurements at different pHs and found to be strongly dependent on nanocarrier composition. By varying composition of these nanocarriers, the apparent pKa of embedded OA molecules could be tuned from 7.8 to 6.3, shifting the range of nanocarriers' pH-response. The study advances our fundamental understanding of self-assembly and pH-responsiveness in lipid-peptide systems containing monounsaturated long-chain fatty acids. The results may guide the future design of highly adaptable nanocarriers for patient-optimized pH-targeted AMP delivery.

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-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.

Enhancement of Immune Response and Anti-Infection of Mice by Porcine Antimicrobial Peptides and Interleukin-4/6 Fusion Gene Encapsulated in Chitosan Nanoparticles.

In order to develop a novel and effective immunoregulator to enhance both the immune response and antimicrobial function, a recombinant eukaryotic expression plasmid-pVAX1 co-expressing fusion cathelicidin antimicrobial peptides (CAMPs) and fusion porcine interleukin-4/6 gene (IL-4/6) was constructed and encapsulated in chitosan nanoparticles (CS-VAP4/6), prepared by the ionotropic gelation method. Four-week-old female Kunming mice were divided into three groups and intramuscularly injected, respectively, with CS-VAP, CS-VAP4/6, and CS-pVAX1. On 28 days post-inoculation, the mice were challenged by intraperitoneal injection with Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922); IgG, IgG1 and IgG2a, CD4+, and CD8+ T cells increased significantly in the VAP- and VAP4/6- treated mice, detected by ELISA and flow cytometry, correspondingly (p < 0.05). As analyzed by qPCR, expression levels of Toll-like receptor (TLR) 1, TLR4, TLR6, TLR9, IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-23, Tumor Necrosis Factor (TNF)-α, and Interferon-gamma (IFN-γ) genes were also significantly up-regulated in comparison with those of the control mice (p < 0.05). Their immunological markers were elevated significantly to different degrees in CS-VAP4/6-treated mice compared with CS-VAP in different days post-inoculation (p < 0.05). After challenge with E. coli and Staphylococcus aureus, most of the VAP- and VAP4/6- treated mice survived, and no symptoms of bacterial infection were observed. In contrast, 80% of control mice died of infection. Among the treated groups, VAP4/6 had a stronger resistance against challenge with E. coli infection. These results demonstrated that the fusion gene of antimicrobial peptide and interleukin-4/6 has the promising potential as a safe and effective immunomodulator for the control of bacterial infections.

Diminishing reactive adipogenesis leads to disease progression of oral submucous fibrosis

Oral fibroblasts, similar to dermal fibroblasts, have the potential to resist the local insults like trauma to the oral mucosa by differentiating into adipocytes and secreting antimicrobial peptide cathelicidin (Camp) and this physiologic process in known as reactive adipogenesis. We hypothesize that in oral submucous fibrosis (OSF), due to constant secretion and up-streaming of transforming growth factor-beta (TGF- β), oral fibroblast lose their adipogenic differentiation potential and Camp production, which leads to progressive fibrosis in OSF. The implication of this hypothesis could open some promising vistas on still unexplored innate immune systems harboured by oral mucosa. Restoring and maintaining the adipogenic and protective potential of oral fibroblasts by inhibiting TGF- β receptors could hinder the disease progression of OSF.

Does peri-implant bone loss affect the LL-37 and proteinase 3 levels in peri-implant sulcus fluid?

BackgroundInactive human cathelicidin antimicrobial peptide is present in neutrophils, and proteinase 3 activates this peptide by producing active LL-37 peptide. LL-37 acts as a defensive peptide in the oral tissues. In the present study, the aim was to evaluate LL-37 and proteinase 3 levels in peri-implant sulcus fluid (PISF) in implants with and without peri-implantitis.MethodsPatients who simultaneously had dental implants with peri-implantitis and without peri-implantitis were included in the study. Forty-four samples with peri-implantitis and 34 samples without peri-implantitis from 16 patients were obtained. Intraoral evaluations such as pocket depth, modified sulcus bleeding index, and modified plaque index were noted. Enzyme-linked immunosorbent assay was used for the evaluation of PISF LL-37 and proteinase 3 levels.ResultsPISF volume was significantly increased in the implants with peri-implantitis than those without peri-implantitis (p < 0.05). No difference was present between PISF LL-37 and proteinase 3 total amounts between the implants with and without peri-implantitis (p > 0.05). Pocket depths and PISF LL-37 and proteinase 3 levels were not correlated in the groups (p > 0.05).ConclusionsPISF volume might be increased in response to peri-implant bone destruction. However, peri-implant tissue destruction caused by peri-implantitis does not seem to affect PISF LL-37 and proteinase 3 levels.

Cathelicidin-mediated lipopolysaccharide signaling via intracellular TLR4 in colonic epithelial cells evokes CXCL8 production

ABSTRACT We hypothesized that the antimicrobial peptide cathelicidin has a physiological role in regulating gut inflammatory homeostasis. We determined that cathelicidin synergizes with LPS to facilitate its internalization and signaling via endosomic TLR4 in colonic epithelium, evoking synthesis of the human neutrophil chemoattractant, CXCL8 (or murine homolog, CXCL1). Interaction of cathelicidin with LPS in the control of CXCL8/CXCL1 synthesis was assessed in human colon epithelial cells, murine colonoids and cathelicidin-null mice (Camp−/- ). Mechanistically, human cathelicidin (LL-37), as an extracellular complex with LPS, interacted with lipid raft-associated GM1 gangliosides to internalize and activate intracellular TLR4. Two signaling pathways converged on CXCL8/CXCL1 production: (1) a p38MAPK-dependent pathway regulated by Src-EGFR kinases; and, (2) a p38MAPK-independent, NF-κB-dependent pathway, regulated by MEK1/2-MAPK. Increased cathelicidin-dependent CXCL8 secretion in the colonic mucosa activated human blood-derived neutrophils. These cathelicidin effects occurred in vitro at concentrations well below those needed for microbicidal function. The important immunomodulatory role of cathelicidins was evident in cathelicidin-null/Camp−/- mice, which had diminished colonic CXCL1 secretion, decreased neutrophil recruitment-activation and reduced bacterial clearance when challenged with the colitis-inducing murine pathogen, Citrobacter rodentium. We conclude that in addition to its known microbicidal action, cathelicidin has a unique pathogen-sensing role, facilitating LPS-mediated intestinal responses, including the production of CXCL8/CXCL1 that would contribute to an integrated tissue response to recruit neutrophils during colitis.

Scavenger Receptor Class B Type I is Required for 25-Hydroxycholecalciferol Cellular Uptake and Signaling in Myeloid Cells.

Vitamin D3 is a critical molecule for the properly controlled activity of the immune system. In myeloid-derived cells, vitamin D3 induces the production of the antimicrobial and antitumor peptide cathelicidin. In this study, the mechanism of the entry of 25-hydroxycholecalciferol (25(OH)D) in myeloid-derived cells is explored. Here, a novel regulatory pathway of vitamin D3 biology is described. Using a polyclonal antibody, two different chemical inhibitors, and a high-density lipoprotein as a competing ligand, it is demonstrated here that the 25(OH)D signaling pathway in myeloid cells depends on scavenger receptor class B type I (SR-B1). This effect is observed in the THP-1 monocytic cell line and in human primary monocytes. SR-B1 blockade abrogates the cellular uptake of 25(OH)D leading to a general shut down of the gene transcription program modulated by 25(OH)D. The results obtained at the transcriptional level are confirmed at the protein and functional level for CD14 in the THP-1 cell line. In conclusion, SR-B1 plays a critical role in vitamin D3 biology, paving the way for novel therapeutic interventions.

Hypovitaminosis D and reduced cathelicidin are strongly correlated during the multidrug therapy against leprosy

Mycobacterium leprae infection depends on the competence of the host immune defense to induce effective protection against this intracellular pathogen. The present study investigated the serum levels of vitamin D and the antimicrobial peptide cathelicidin, to determine the statistical correlation between them in leprosy patients before and post-six months of multidrug therapy (MDT), household contacts, and healthy individuals. Previous studies associated these molecules with high risks to develop mycobacterial diseases, such as tuberculosis and leprosy. A total of 34 leprosy patients [paucibacillary (n = 14), multibacillary (n = 20)], and 25 household contacts were recruited. Eighteen healthy adults were selected as a control group. Serum concentrations of vitamin D (25(OH)VD3) and cathelicidin were measured using high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit, respectively. There were no significant differences in serum levels of 25(OH)VD3 between all groups, and the overall prevalence rate of vitamin D deficiency was 67.1%. Cathelicidin levels were significantly lower in both untreated and treated patients when compared to controls and household contacts (p < 0.05). Strong correlations between hypovitaminosis D and reduced cathelicidin in untreated (r = 0.86) and post-six months of MDT (r = 0.79) leprosy patients were observed. These results suggest that vitamin D status and cathelicidin levels are strongly correlated during multidrug therapy for leprosy and nutritional supplementation from the beginning of treatment could strengthen the immune response against leprosy.

Murine cathelicidin: as a host defensive response against Leishmania major infection.

Leishmaniasis is a serious global challenge with neither efficacious prophylactic vaccine nor effective and safe therapeutic measures. Cathelicidins, members of antimicrobial peptides family, are small proteins of innate immunity system, which represent a protective barrier against a number of potential pathogens in living organisms. The murine cathelicidin or cathelin-related antimicrobial peptide (CRAMP) is expressed by a variety of cells or tissues, and highly resembles to human cathelicidin (LL-37). It is naturally expressed at a low concentration in adolescent age, but extensively increases during cutaneous infections. Despite its important role, it has less been investigated in parasitic infections. Among all cells, macrophages and skin cells are the two important cells that directly have a relationship with Leishmania major parasites. The present study aimed to show whether cathelicidins protect their hosts following cutaneous leishmaniasis due to L. major parasites. Both in vitro and in vivo models of L. major infection were established by exposing of J744 cell line (murine macrophages) and BALB/c mice with the stationary phase of L. major promastigotes for 24h and 7days. The findings revealed that both macrophages and skin cells significantly (p < 0.05) expressed a high level of CRAMP gene and peptide after challenging with L. major parasites. Thus, our data suggest a protective role for cathelicidins against infections caused by L. major parasites. This experimental model could be considered as a novel potential vaccine candidate for planning future control strategy against human leishmaniasis.

Antimicrobial peptide LL-37 ameliorates a murine sepsis model via the induction of microvesicle release from neutrophils.

Sepsis is a life-threatening disease caused by systemic dys-regulated inflammatory response to infection. We previously revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of cecal ligation and puncture septic mice. Ectosomes, microvesicles released from neutrophils, are reported to be elevated in sepsis survivors; however, the functions of ectosomes in sepsis remain largely unknown. Therefore, we herein elucidated the protective action of LL-37 on sepsis, by focusing on LL-37-induced ectosome release in a cecal ligation and puncture model. The results demonstrated the enhancement of ectosome levels by LL-37 administration, accompanied by a reduction of bacterial load. Importantly, ectosomes isolated from LL-37-injected cecal ligation and puncture mice contained higher amounts of antimicrobial proteins/peptides and exhibited higher antibacterial activity, compared with those from PBS-injected cecal ligation and puncture mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. Actually, LL-37 stimulated mouse bone-marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed antibacterial potential. Furthermore, administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of cecal ligation and puncture mice. Together these observations suggest LL-37 induces the release of antimicrobial ectosomes in cecal ligation and puncture mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.