Cathelicidin Antimicrobial Peptide LL-37 Research Articles

Abstract 14475: Intrinsic T Cell Tolerance to the Autoantigen LL-37 is Mediated in Part by Platelets

Introduction: An autoimmune response to human cathelicidin antimicrobial peptide LL-37, a component of neutrophil extracellular traps (NETs), may play a role in athero-thrombosis. We have reported that acute coronary syndrome (ACS) patients have increased LL-37 auto-reactive CD8+CD69+CD137+ T cells compared to healthy controls suggesting breach of tolerance to LL-37. Platelets present antigens to CD8+ T cells via HLA Class-I but it is unknown if this pathway is involved in maintaining intrinsic tolerance to the autoantigen LL-37. In this study, we investigated the role of platelets in maintaining immune tolerance to LL-37 in healthy subjects. Methods and Results: Platelets and autologous peripheral blood mononuclear cells (PBMCs) were isolated from remnant blood of platelet donor Leukocyte Reduction System cones from the institutional blood bank. Platelet enrichment was confirmed by CD41+ flow cytometry and over 95% were HLA Class-I(+). PBMCs were co-cultured with autologous platelets that were either non-primed or primed with LL-37 peptide at a ratio of 25:1 platelet-to-PBMC. Cells were collected after 16 hours and subjected to fluorescent staining for activation markers by flow cytometry. Compared to non-primed platelets, co-culture of LL-37-primed platelets significantly reduced T cell activation in autologous PBMCs including CD8+CD69+CD137+ T cells, CD4+CD134+ T cells and CD4+CD134+CD137+ T cells (Table), supporting platelet-mediated T cell tolerance to LL-37. We then evaluate if the breach in T cell tolerance to LL-37 in ACS is related to platelet counts. CD8+CD69+CD137+ T cell response to LL-37 in PBMCs of ACS patients was negatively correlated with platelet count (N=21; Pearson r = -0.438, P=0.047). Conclusions: The results demonstrate that intrinsic immune tolerance to the autoantigen LL-37 in healthy subjects is mediated in part by platelets. Reduced platelet counts are implicated in the breach of immune tolerance to LL-37 in ACS.

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

Nafcillin Augmentation of Daptomycin and Cathelicidin LL-37 Killing of Methicillin-resistant Staphylococcus epidermidis: Foundations of Successful Therapy of Endocarditis

Methicillin-resistant Staphylococcus epidermidis (MRSE) endocarditis failing conventional therapy has been successfully treated with nafcillin plus daptomycin in the clinic. In vitro studies showed that nafcillin enhanced daptomycin killing of MRSE in both planktonic cells and biofilm. Nafcillin exposure also sensitized MRSE to killing by human neutrophils and cathelicidin antimicrobial peptide LL-37. Fluorescent microscopy showed increased daptomycin and LL-37 binding to the MRSE bacterial surface upon nafcillin treatment. Ceftaroline also increased MRSE killing by daptomycin in planktonic cultures and biofilms, as well as daptomycin and LL-37 binding on the bacterial surface. Nafcillin, ceftaroline, and possibly other β-lactams, may serve an important role in the therapy of MRSE endocarditis through augmentation of cationic peptide, the innate immune system, and daptomycin killing. Clinical studies will be needed to determine how early these regimens should be deployed to optimize clinical outcome.

Bicarbonate Effects on Antibacterial Immunity and Mucus Glycobiology in the Cystic Fibrosis Lung: A Review With Selected Experimental Observations.

The primary defect in cystic fibrosis (CF) is abnormal chloride and bicarbonate transport in the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial ion channel. The apical surface of the respiratory tract is lined by an airway surface liquid layer (ASL) composed of mucin comprising mainly MUC5A and MUC5B glycoproteins. ASL homeostasis depends on sodium bicarbonate secretion into the airways and secretion deficits alter mucus properties leading to airway obstruction, inflammation, and infections. Downstream effects of abnormal ion transport in the lungs include altered intrinsic immune defenses. We observed that neutrophils killed Pseudomonas aeruginosa more efficiently when it had been exposed to sodium bicarbonate, and formation of neutrophil extracellular traps (NETs) by neutrophils was augmented in the presence of increasing bicarbonate concentrations. Physiological levels of bicarbonate sensitized P. aeruginosa to the antimicrobial peptide cathelicidin LL-37, which is present in both lung ASL and in NETs. Sodium bicarbonate has various uses in clinical medicine and in the care of CF patients, and could be further explored as a therapeutic adjunct against Pseudomonas infections.

Role of LL-37 in thrombotic complications in patients with COVID-19

Blood clot formation induced by dysfunctional coagulation is a frequent complication of coronavirus disease 2019 (COVID-19) and a high-risk factor for severe illness and death. Neutrophil extracellular traps (NETs) are implicated in COVID-19-induced immunothrombosis. Furthermore, human cathelicidin, a NET component, can perturb the interaction between the SARS-CoV-2 spike protein and its ACE2 receptor, which mediates viral entry into cells. At present, however, the levels of cathelicidin antimicrobial peptides after SARS-CoV-2 infection and their role in COVID-19 thrombosis formation remain unclear. In the current study, we analyzed coagulation function and found a decrease in thrombin time but an increase in fibrinogen level, prothrombin time, and activated partial thromboplastin time in COVID-19 patients. In addition, the cathelicidin antimicrobial peptide LL-37 was upregulated by the spike protein and significantly elevated in the plasma of patients. Furthermore, LL-37 levels were negatively correlated with thrombin time but positively correlated with fibrinogen level. In addition to platelet activation, cathelicidin peptides enhanced the activity of coagulation factors, such as factor Xa (FXa) and thrombin, which may induce hypercoagulation in diseases with high cathelicidin peptide levels. Injection of cathelicidin peptides promoted the formation of thrombosis, whereas deletion of cathelicidin inhibited thrombosis in vivo. These results suggest that cathelicidin antimicrobial peptide LL-37 is elevated during SARS-CoV-2 infection, which may induce hypercoagulation in COVID-19 patients by activating coagulation factors.

Upregulating Human Cathelicidin Antimicrobial Peptide LL-37 Expression May Prevent Severe COVID-19 Inflammatory Responses and Reduce Microthrombosis.

COVID-19 is characterized by hyperactivation by inflammatory cytokines and recruitment of macrophages, neutrophils, and other immune cells, all hallmarks of a strong inflammatory response that can lead to severe complications and multi-organ damage. Mortality in COVID-19 patients is associated with a high prevalence of neutrophil extracellular trap (NET) formation and microthrombosis that are exacerbated by hyperglycemia, diabetes, and old age. SARS-CoV-2 infection in humans and non-human primates have revealed long-term neurological consequences of COVID-19, possibly concomitant with the formation of Lewy bodies in the brain and invasion of the nervous system via the olfactory bulb. In this paper, we review the relevance of the human cathelicidin LL-37 in SARS-CoV-2 infections. LL-37 is an immunomodulatory, host defense peptide with direct anti-SARS-CoV-2 activity, and pleiotropic effects on the inflammatory response, neovascularization, Lewy body formation, and pancreatic islet cell function. The bioactive form of vitamin D and a number of other compounds induce LL-37 expression and one might predict its upregulation, could reduce the prevalence of severe COVID-19. We hypothesize upregulation of LL-37 will act therapeutically, facilitating efficient NET clearance by macrophages, speeding endothelial repair after inflammatory tissue damage, preventing α-synuclein aggregation, and supporting blood-glucose level stabilization by facilitating insulin release and islet β-cell neogenesis. In addition, it has been postulated that LL-37 can directly bind the S1 domain of SARS-CoV-2, mask angiotensin converting enzyme 2 (ACE2) receptors, and limit SARS-CoV-2 infection. Purposeful upregulation of LL-37 could also serve as a preventative and therapeutic strategy for SARS-CoV-2 infections.

High Vitamin D Concentrations Restore the Ability to Express LL37 by M. tuberculosis-Infected Human Macrophages.

Vitamin D has an immunomodulatory function and is involved in eliminating pathogens. Vitamin D deficiencies reported in Type 2 diabetes mellitus (T2DM) patients make them more susceptible to developing tuberculosis (TB). The macrophages are the immune cells that control intracellular pathogens by producing the antimicrobial peptide cathelicidin-LL37. This pathway involves TLR activation by pathogens, vitamin D receptor (VDR) ligation, and the enzyme 1α-hydroxylase Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1). However, it is not clear whether the biological actions of vitamin D are affected by high glucose concentrations. This study aimed to evaluate the vitamin D contribution in the expression of VDR and CYP27B1, involved in the conversion of an inactive to an active form of vitamin D in the infected macrophages using M. tuberculosis as an infection model. The expression of LL37 and the nucleus translocation of VDR were evaluated as the readout of the response of vitamin D and determined if those processes are affected by glucose concentrations. Macrophages from healthy donors were cultured under glucose concentrations of 5.5, 15, or 30 mM, stimulated with vitamin D in inactive (25(OH)D3) or active (1,25(OH)2D3) forms, and infected with M. tuberculosis. The vitamin D-dependent induction of LL37 and the expression of VDR and CYP27B1 genes were analyzed by qPCR, and VDR translocation was analyzed in nuclear protein extracts by ELISA. M. tuberculosis downregulated the expression of LL37 regardless of the glucose concentration, whereas VDR and CYP27B1 upregulated it regardless of the glucose concentration. After evaluating two concentrations of vitamin D, 1 nM or 1 μM, the high concentration (1 μM) was necessary to restore the induction of LL37 expression in M. tuberculosis-infected macrophages. High concentrations of the inactive form of vitamin D restore the infected macrophages’ ability to express LL37 regardless of the glucose concentration. This finding supports the idea that vitamin D administration in patients with T2DM could benefit TB control and prevention.

Metformin strengthens uroepithelial immunity against E. coli infection

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease.

The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.

C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

To study the molecular interplay between TLRs and complement representing ancient danger-sensing mechanisms, we examined the regulation of the C3a/anaphylatoxin C3a receptor (C3aR) axis in normal human epidermal keratinocytes (NHEKs) by treatment with different TLR ligands. Protein staining followed by flow cytometry revealed highly constitutive intracellular expression levels of the C3aR in NHEKs. Stimulation with Poly I:C up-regulated C3aR mRNA and intra- and extracellular expression in NHEKs which showed functional relevance by up-regulating CXCL10 and down-regulating C3 expression in response to C3a. mRNA and protein levels of C3 and protease cathepsin L (CTSL) that can cleave C3 were up-regulated by the TLR3 ligand Poly I:C. Enhanced intracellular expression levels of the biologically active C3 fragment (C3a), in response to TLR3 stimulation were also detectable in NHEKs. Cathelicidin antimicrobial peptide LL-37 potentiated Poly I:C-induced C3aR, C3, and CTSL up-regulation. In conclusion, we point to a role of TLR3 to promote up-regulation of C3aR, C3, and CTSL expression levels and generation of C3a. Our data provide evidence that local generation and activation of complement components as described for T cells or myeloid cells represent a scenario which may take place in a similar way in NHEKs.

The potentials of short fragments of human anti-microbial peptide LL-37 as a novel therapeutic modality for diseases.

Human cathelicidin antimicrobial peptide LL-37 (LL-37) is an antimicrobial peptide derived from its precursor protein hCAP18, which is an only cathelicidin in human. LL-37 not only serves as a mediator of innate immune defense against invading microorganisms, but it also plays an essential role in tissue homeostasis, regenerative processes, regulation of proinflammatory responses, and inhibition of cancer progression. Therefore, LL-37 has been considered as a drug lead for diseases. However, high levels of LL-37 may reduce cell viability and promote apoptosis of osteoblasts, vascular smooth muscle cells, periodontal ligament cells, neutrophils, airway epithelial cells and T cells. Recent evidence reveals that LL-37-derived short peptides possess similar biological activities as the whole LL-37 with reduced cytotoxicity. Thus, such small molecules constitute a pool of potential therapeutic agents for diseases.

Brief Report: Increased Cotinine Concentrations are Associated With Reduced Expression of Cathelicidin (LL-37) and NOD-2 in Alveolar Macrophages of PLWH Who Smoke.

There is a strong link between cigarette smoking and pulmonary complications among people living with HIV. However, the effects of smoking on the local lung immune environment in this population remain unclear. Bronchoalveolar lavage and saliva were collected from HIV-infected smokers involved in a prospective study investigating alveolar macrophage expression of host defense molecules. Salivary cotinine concentrations were inversely related to expression of the immune cell receptor nucleotide-binding oligomerization domain-2 and the cathelicidin antimicrobial peptide LL-37. The negative correlation between salivary cotinine and LL-37 was particularly strong. Our study provides insight into how nicotine may adversely affect lung innate immunity in HIV.

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.

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.

MP07-06 CHRONIC LL-37 INDUCED CYSTITIS PRODUCES PAIN INDEPENDENT OF INFLAMMATION AND MODIFIED GLYCOSAMINOGLYCANS ARE POTENT NON-OPIOID ANALGESICS

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis (MP07)1 Apr 2020MP07-06 CHRONIC LL-37 INDUCED CYSTITIS PRODUCES PAIN INDEPENDENT OF INFLAMMATION AND MODIFIED GLYCOSAMINOGLYCANS ARE POTENT NON-OPIOID ANALGESICS Austin Schults*, Wanjian Jia, M. Martin Jensen, Glenn Prestwich, and Siam Oottamasathien Austin Schults*Austin Schults* More articles by this author , Wanjian JiaWanjian Jia More articles by this author , M. Martin JensenM. Martin Jensen More articles by this author , Glenn PrestwichGlenn Prestwich More articles by this author , and Siam OottamasathienSiam Oottamasathien More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000827.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Interstitial cystitis/painful bladder syndrome (IC/PBS) can be a debilitating condition that dramatically impacts the quality of life for patients. The primary goal of patients with IC/PBS is to provide symptomatic relief. The immunomodulatory human cathelicidin (antimicrobial peptide) LL-37 induces profound acute bladder inflammation and pain. Our objectives were to evaluate a chronic pain profile associated with an LL-37-induced murine model of IC/PBS, along with the analgesic effects of modified glycosaminoglycan ether compounds. We hypothesized that chronic pain could be produced that is independent of inflammation and that a semi-synthetic glycosaminoglycan ether (SAGE GM-0111) could be a potent non-opioid analgesic in the setting of chronic LL-37-induced cystitis. METHODS: Bladder instillation of 150μL of 80 µM LL-37 for 1-hr, biweekly for four weeks induced the chronic IC/PBS model. SAGE analgesic effects were tested by instilling SAGE GM-0111. Pain responses to suprapubic stimulation were assessed using von Frey filaments (0.04gm to 4.0gm) before LL-37 instillation and before sacrifice. Inflammation and fibrosis were evaluated using tissue myeloperoxidase (MPO) concentration, collagen content, gross inspection, and microscopic histologic examination. RESULTS: SAGE GM-0111 reduced pain in treated animals significantly (54 ± 32% response rate) in animals exposed to LL-37 insult (96 ± 7% response rate, p<0.001) at 1g of stimulation. Collagen content, gross anatomy, histology with H&E and Masson’s trichrome staining indicated that fibrosis did not occur over the duration of the study. At the time of sacrifice there was no significant variation in tissue inflammation between groups. CONCLUSIONS: LL-37-induced cystitis yielded pain to be independent of inflammation. These findings further substantiate the physiological applicability of the LL-37-induced cystitis model to mimic human IC/PBS pathophysiology. Finally, our SAGE compound demonstrated potent non-opioid analgesic effects within this surrogate model. Source of Funding: NIH NIDDK R01 DK100868 (SO), Primary Children’s Hospital Integrated Science Award (SO), NIH K12 UL1RR025764 (SO), National Science Foundation Graduate Research Fellowship 1256065 (MMJ) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e97-e97 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Austin Schults* More articles by this author Wanjian Jia More articles by this author M. Martin Jensen More articles by this author Glenn Prestwich More articles by this author Siam Oottamasathien More articles by this author Expand All Advertisement PDF downloadLoading ...

Engineered Human Cathelicidin Antimicrobial Peptides Inhibit Ebola Virus Infection.

SummaryThe 2014–2016 West Africa Ebola virus (EBOV) outbreak coupled with the most recent outbreaks in Central Africa underscore the need to develop effective treatment strategies against EBOV. Although several therapeutic options have shown great potential, developing a wider breadth of countermeasures would increase our efforts to combat the highly lethal EBOV. Here we show that human cathelicidin antimicrobial peptide (AMP) LL-37 and engineered LL-37 AMPs inhibit the infection of recombinant virus pseudotyped with EBOV glycoprotein (GP) and the wild-type EBOV. These AMPs target EBOV infection at the endosomal cell-entry step by impairing cathepsin B-mediated processing of EBOV GP. Furthermore, two engineered AMPs containing D-amino acids are particularly potent in blocking EBOV infection in comparison with other AMPs, most likely owing to their resistance to intracellular enzymatic degradation. Our results identify AMPs as a novel class of anti-EBOV therapeutics and demonstrate the feasibility of engineering AMPs for improved therapeutic efficacy.

Short-Term versus Long-Term Culture of A549 Cells for Evaluating the Effects of Lipopolysaccharide on Oxidative Stress, Surfactant Proteins and Cathelicidin LL-37.

Alveolar epithelial type II (ATII) cells and their proper function are essential for maintaining lung integrity and homeostasis. However, they can be damaged by lipopolysaccharide (LPS) during Gram-negative bacterial infection. Thus, this study evaluated and compared the effects of LPS on short and long-term cultures of A549 cells by determining the cell viability, levels of oxidative stress and antimicrobial peptide cathelicidin LL-37 and changes in the expression of surfactant proteins (SPs). Moreover, we compared A549 cell response to LPS in the presence of different serum concentrations. Additionally, the effect of N-acetylcysteine (NAC) on LPS-induced oxidative stress as a possible treatment was determined. Our results indicate that A549 cells are relatively resistant to LPS and able to maintain integrity even at high LPS concentrations. Their response to endotoxin is partially dependent on serum concentration. NAC failed to lower LPS-induced oxidative stress in A549 cells. Finally, LPS modulates SP gene expression in A549 cells in a time dependent manner and differences between short and long-term cultures were present. Our results support the idea that long-term cultivation of A549 cells could promote a more ATII-like phenotype and thus could be a more suitable model for ATII cells, especially for in vitro studies dealing with surfactant production.

Dynamics of immune and biochemical features of oral fluid in persons with caries receiving vitamin D

The aim of the study was to evaluate the content of the vitamin D metabolite (25(OH)D3) in the blood serum, the level of some indicators of the immune and peroxidic status in the oral fluid in people with carious processes of different intensities while taking the native solution of vitamin D (international non-proprietary name Cholecalciferol). The study included 47 people aged 20 to 22 with caries and low levels of 25(OH)D3 in blood serum. The control group consisted of 13 people with DMFT index of 0.00 [0.00; 0.00] and a normal serum content of 25(OH)D3. The amount of antimicrobial peptide cathelicidin LL-37, secretory immunoglobulin A, lipopolysaccharide binding protein (LBP) was determined in the oral fluid using ELISA reagent kits, and the content of intermediate lipid peroxidation products such as malondialdehyde (TBA-active products) and the level of total antioxidant activity were spectrophotometrically evaluated. Reduced serum concentrations of 25(OH)D3 were recorded predominantly in individuals with a DMFT index of more than 9.0. In patients with caries, at a concentration of 25(OH)D3 in blood serum below 30 ng/ml, a decrease in the values of secretory immunoglobulin A, lipopolysaccharide-binding protein, cathelicidin, and the level of total antioxidant activity was observed, accompanied by an increase in the number of intermediate products of lyoperoxidation in the oral fluid. The course intake of the native AquaDtrim vitamin D solution (Cholecalciferol) led to normalization of the values of certain immune and biochemical parameters (cathelicidin LL-37, secretory immunoglobulin A, LBP of the oral fluid) and restoration of balance in the system «lipid peroxidation - antioxidants». The results indicate the advisability of assessing the level of 25(OH)D3 in the body for the diagnosis of pathological processes in the oral cavity and the use of vitamin D preparations in the prevention and complex therapy of carious process.

Eczema Herpeticum: Clinical and Pathophysiological Aspects.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the world. AD is a complex pathology mainly characterized by an impaired skin barrier, immune response dysfunction, and unbalanced skin microbiota. Moreover, AD patients exhibit an increased risk of developing bacterial and viral infections. One of the most current, and potentially life-threatening, viral infection is caused by herpes simplex virus (HSV), which occurs in about 3% of AD patients under the name of eczema herpeticum (EH). Following a first part dedicated to the clinical features, virological diagnosis, and current treatments of EH, this review will focus on the description of the pathophysiology and, more particularly, the presently known predisposing factors to herpetic complications in AD patients. These factors include those related to impairment of the skin barrier such as deficit in filaggrin and anomalies in tight and adherens junctions. In addition, low production of the antimicrobial peptides cathelicidin LL-37 and human β-defensins; overexpression of cytokines such as interleukin (IL)-4, IL-13, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP); or downregulation of type I to III interferons as well as defect in functions of immune cells such as dendritic, natural killer, and regulatory T cells have been involved. Otherwise, genetic polymorphisms and AD topical calcineurin inhibitor treatments have been associated with an increased risk of EH. Finally, dysbiosis of skin microbiota characterized in AD patients by Staphylococcus aureus colonization and toxin secretion, such as α-toxin, has been described as promoting HSV replication and could therefore contribute to EH.

The cysteine protease ApdS from Streptococcus suis promotes evasion of innate immune defenses by cleaving the antimicrobial peptide cathelicidin LL-37

Streptococcus suis is a globally distributed zoonotic pathogen associated with meningitis and septicemia in humans, posing a serious threat to public health. To successfully invade and disseminate within its host, this bacterium must overcome the innate immune system. The antimicrobial peptide LL-37 impedes invading pathogens by directly perforating bacterial membranes and stimulating the immune function of neutrophils, which are the major effector cells against S. suis However, little is known about the biological relationship between S. suis and LL-37 and how this bacterium adapts to and evades LL-37-mediated immune responses. In this study by using an array of approaches, including enzyme, chemotaxis, cytokine assays, quantitative RT-PCR, and CD spectroscopy, we found that the cysteine protease ApdS from S. suis cleaves LL-37 and thereby plays a key role in the interaction between S. suis and human neutrophils. S. suis infection stimulated LL-37 production in human neutrophils, and S. suis exposure to LL-37 up-regulated ApdS protease expression in the bacterium. We observed that ApdS targets and rapidly cleaves LL-37, impairing its bactericidal activity against S. suis We attributed this effect to the decreased helical content of the secondary structure in the truncated peptide. Moreover, ApdS rescued S. suis from killing by human neutrophils and neutrophil extracellular traps because LL-37 truncation attenuated neutrophil chemotaxis and inhibited the formation of extracellular traps and the production of reactive oxygen species. Altogether, our findings reveal an immunosuppressive strategy of S. suis whereby the bacterium blunts the innate host defenses via ApdS protease-mediated LL-37 cleavage.