Cooperative Function of LL-37 and HNP1 Protects Mammalian Cell Membranes from Lysis.
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ABSTRACT:
SUBMITTER: Drab E
PROVIDER: S-EPMC7822736 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula. Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification. Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10 5 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10 7 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA. Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations, conditions where the antimicrobial agent alone is relatively ineffective.
| S-EPMC10285323 | biostudies-literature
Project description:Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine-sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.
| S-EPMC4036085 | biostudies-literature
Project description:Gene expression in THP-1 cells treated for 6 hours with CNT and GNP conjugated with LL-37, LL-37 spiked as well as free LL-37: Reference (Total RNA Mixture of all samples) vs. treated cells
2017-06-17 | GSE98630 | GEO
Project description:Human cathelicidin LL-37 is an antimicrobial peptide that has a broad spectrum of antimicrobial activities but also acts on host cells to exert immunomodulatory functions. It has been suggested that the increase of LL-37 in atherosclerotic aortas and the dysregulated autophagy of endothelial cells are involved in the pathogenesis of atherosclerosis. In this study, to elucidate the role of LL-37 in atherosclerosis, we investigated the effect of LL-37 on autophagy in endothelial cells using HUVECs. First, LL-37 upregulated LC3-II (an autophagosomal membrane marker) and enhanced the formation of LC3-positive puncta in the cells, suggesting that LL-37 induces autophagy in endothelial cells. Second, LL-37 was associated with p62, which recognizes ubiquitinated proteins and transfers them to autophagosomes, suggesting that LL-37 is ubiquitinated and recognized by p62. Third, the degradation of LL-37 was delayed, and LL-37 induced cell death in atg7 knockdown cells, which was accompanied by the formation of protein aggregates in the cells. Taken together, these observations suggest that LL-37 induces autophagy in endothelial cells but enhances cell death in autophagy-dysfunctional conditions, in which the intracellular degradation of LL-37 is disturbed. Thus, LL-37 may exert an adverse action on autophagy-dysfunctional endothelial cells to induce cell death in the pathogenesis of atherosclerosis.
| S-EPMC9047070 | biostudies-literature
Project description:Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 μm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 μm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.
| S-EPMC6921147 | biostudies-literature
Project description:Rationale: Antimicrobial peptides, such as cathelicidin LL-37/hCAP-18, are important effectors of the innate immune system with direct antibacterial activity. In addition, LL-37 is involved in the regulation of tumor cell growth. However, the molecular mechanisms underlying the functions of LL-37 in promoting lung cancer are not fully understood. Methods: The expression of LL-37 in the tissues and sera of patients with non-small cell lung cancer was determined through immunohistological, immunofluorescence analysis, and enzyme-linked immunosorbent assay. The animal model of wild-type and Cramp knockout mice was employed to evaluate the tumorigenic effect of LL-37 in non-small cell lung cancer. The mechanism of LL-37 involving in the promotion of lung tumor growth was evaluated via microarray analyses, recombinant protein treatment approaches in vitro, tumor immunohistochemical assays, and intervention studies in vivo. Results: LL-37 produced by myeloid cells was frequently upregulated in primary human lung cancer tissues. Moreover, its expression level correlated with poor clinical outcome. LL-37 activated Wnt/?-catenin signaling by inducing the phosphorylation of protein kinase B and subsequent phosphorylation of glycogen synthase kinase 3? mediated by the toll-like receptor-4 expressed in lung tumor cells. LL-37 treatment of tumor cells also decreased the levels of Axin2. In contrast, it elevated those of an RNA-binding protein (tristetraprolin), which may be involved in the mechanism through which LL-37 induces activation of Wnt/?-catenin. Conclusion: LL-37 may be a critical molecular link between tumor-supportive immune cells and tumors, facilitating the progression of lung cancer.
| S-EPMC6531301 | biostudies-literature
Project description:BackgroundPsoriasis (PsO) is a T-cell-associated inflammatory autoimmune dermatitis. Leucine leucine-37 (LL-37) is upregulated in PsO patients and correlated with the area and severity of PsO. However, the exact relation between LL-37 and T cell-associated inflammation is not well understood. It is very important to clarify the relationship between LL-37 and inflammatory response for clinical diagnosis and treatment of PsO. This study investigated the serum levels of LL-37 and inflammatory cytokines, as well as correlations between them in PsO patients, which aimed to provide new ideas for the diagnosis and treatment of PsO.MethodsPsO patients (n = 50) and healthy volunteers (n = 33) were recruited in this study. Skin specimens were stained with hematoxylin and eosin (H&E). The serum levels of LL-37, T-helper type 1 (Th1, IFN-γ), T-helper type 17 (Th17, IL-17), T-helper type 22 (Th22, IL-22), and T-helper type 2 cytokines (Th2, IL-4) were assessed by enzyme-linked immunosorbent assay. Some of the patients were re-recruited after treatment to evaluate LL-37 and cytokines levels.ResultsPathological changes were observed in PsO skin lesions. LL-37, IFN-γ, IL-17, and IL-22 serum levels were much higher in PsO patients than those in healthy volunteers (p < .001), and posttreatment reduction was observed in five patients. However, no remarkable difference in IL-4 level (p > .05) was found. LL-37 level was positively correlated with IFN-γ, IL-17, and IL-22 levels (p < .001) in PsO patients.ConclusionLL-37 expression was significantly associated with inflammatory response, which may provide us new ideas for diagnosing and monitoring disease activity of PsO.
| S-EPMC10013411 | biostudies-literature
Project description:Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ?X174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.
| S-EPMC4255137 | biostudies-literature
Project description:Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.
| S-EPMC4023799 | biostudies-other
Project description:Purpose:Staphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. Methods:In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro. Results:The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. Conclusion:This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.
| S-EPMC6911324 | biostudies-literature