Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity.

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Numerical evaluation of cutting strategies for thin-walled parts.

Static form errors due to in-process deflections is a major concern in flank milling of thin-walled parts. To increase both productivity and part geometric accuracy, there is a need to predict and control these form errors. In this work, a modelling framework for prediction of the cutting force-induced form errors, or thickness errors, during flank milling of a thin-walled workpiece is proposed. The modelled workpiece geometry is continuously updated to account for material removal and the reduced stiffness matrix is calculated for nodes in the engagement zone. The proposed modelling framework is able to predict the resulting thickness errors for a thin-walled plate which is cut on both sides. Several cutting strategies and cut patterns using constant z-level finishing are studied. The modelling framework is used to investigate the effect of different cut patterns, machining allowance, cutting tools and cutting parameters on the resulting thickness errors. The framework is experimentally validated for various cutting sequences and cutting parameters. The predicted thickness errors closely correspond to the experimental results. It is shown from numerical evaluations that the selection of an appropriate cut pattern is crucial in order to reduce the thickness error. Furthermore, it is shown that an increased machining allowance gives a decreased thickness error for thin-walled plates.

The antimicrobial peptide LL-37 triggers release of apoptosis-inducing factor and shows direct effects on mitochondria.

The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 µM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 µM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.

Modeling of the fracture energy on the finite element simulation in Ti6Al4V alloy machining.

One of the main problems that exists when working with Finite Element Methods (FEM) applied to machining processes is the lack of adequate experimental data for simulating the material properties. Moreover, for damage models based on fracture energy, the correct selection of the energy value is critical for the chip formation process. It is usually difficult to obtain the fracture energy values and requires complex tests. In this work, an analysis of the influence of this fracture energy on the cutting force and the chip generation process has been carried out for different sets of cutting parameters. The aim is to present an empirical relationship, that allows selecting the fracture energy based on the cutting force and cutting parameters. The work is based on a FEM model of an orthogonal turning process for Ti6Al4V alloy using Abaqus/Explicit and the fracture energy empirical relation. This work shows that it is necessary to adjust the fracture energy for each combination of cutting conditions, to be able to fit the experimental results. The cutting force and the chip geometry are analyzed, showing how the developed model adapts to the experimental results. It shows that as the cutting speed and the feed increase, the fracture energy value that best adapts to the model decreases. The evolution shows a more pronounced decrease related to the feed increment and high cutting speed.

FlexTaxD: flexible modification of taxonomy databases for improved sequence classification.

SUMMARY: The Flexible Taxonomy Database framework provides a method for modification and merging official and custom taxonomic databases to create improved databases. Using such databases will increase accuracy and precision of existing methods to classify sequence reads. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/FOI-Bioinformatics/flextaxd and installable through Bioconda.

Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad.

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

LL-37-induced caspase-independent apoptosis is associated with plasma membrane permeabilization in human osteoblast-like cells.

The host defense peptide LL-37 is active against both gram-positive and gram-negative bacteria, but it has also been shown to reduce human host cell viability. However, the mechanisms behind LL-37-induced human host cell cytotoxicity are not yet fully understood. Here, we assess if LL-37-evoked attenuation of human osteoblast-like MG63 cell viability is associated with apoptosis, and if the underlying mechanism may involve LL-37-induced plasma membrane permeabilization. MG63 cell viability and plasma membrane permeabilization were investigated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and by measuring lactate dehydrogenase (LDH) release, respectively. Apoptosis was assessed by the terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay and Annexin V flow cytometry, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage were determined by Western blot. LL-37 (4 and 10 µM) reduced both cell number and cell viability, and these effects were associated with a pro-apoptotic effect demonstrated by positive TUNEL staining and Annexin V flow cytometry. LL-37-induced apoptosis was not coupled to either caspase-3 or PARP cleavage, suggesting that LL-37 causes caspase-independent apoptosis in MG63 cells. Both LL-37 and the well-known plasma membrane permeabilizer Triton X-100 reduced cell viability and stimulated LDH release. Triton X-100-treated cells showed positive TUNEL staining, and the detergent accumulated cells in late apoptosis/necrosis. Similar to LL-37, Triton X-100 caused no PARP cleavage. We conclude that LL-37 promotes caspase-independent apoptosis, and that this effect seems coupled to plasma membrane permeabilization in human MG63 cells.

Complete Genome Sequences of Allofrancisella inopinata SYSU YG23 and Allofrancisella frigidaquae SYSU 10HL1970, Isolated from Water from Cooling Systems in China.

Near neighbors to the causative agent of tularemia, Francisella tularensis, isolated from diverse sources, have been reported in recent years. In this announcement, we present the complete sequences of circular chromosomes of one of the closest neighboring genera of Francisella (i.e., the type strains of Allofrancisella inopinata and Allofrancisella frigidaquae).

A whole-genome sequenced control population in northern Sweden reveals subregional genetic differences.

The number of national reference populations that are whole-genome sequenced are rapidly increasing. Partly driving this development is the fact that genetic disease studies benefit from knowing the genetic variation typical for the geographical area of interest. A whole-genome sequenced Swedish national reference population (n = 1000) has been recently published but with few samples from northern Sweden. In the present study we have whole-genome sequenced a control population (n = 300) (ACpop) from Västerbotten County, a sparsely populated region in northern Sweden previously shown to be genetically different from southern Sweden. The aggregated variant frequencies within ACpop are publicly available (DOI 10.17044/NBIS/G000005) to function as a basic resource in clinical genetics and for genetic studies. Our analysis of ACpop, representing approximately 0.11% of the population in Västerbotten, indicates the presence of a genetic substructure within the county. Furthermore, a demographic analysis showed that the population from which samples were drawn was to a large extent geographically stationary, a finding that was corroborated in the genetic analysis down to the level of municipalities. Including ACpop in the reference population when imputing unknown variants in a Västerbotten cohort resulted in a strong increase in the number of high-confidence imputed variants (up to 81% for variants with minor allele frequency < 5%). ACpop was initially designed for cancer disease studies, but the genetic structure within the cohort will be of general interest for all genetic disease studies in northern Sweden.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial cells of human whole saliva.

The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.

doepipeline: a systematic approach to optimizing multi-level and multi-step data processing workflows.

BACKGROUND: Selecting the proper parameter settings for bioinformatic software tools is challenging. Not only will each parameter have an individual effect on the outcome, but there are also potential interaction effects between parameters. Both of these effects may be difficult to predict. To make the situation even more complex, multiple tools may be run in a sequential pipeline where the final output depends on the parameter configuration for each tool in the pipeline. Because of the complexity and difficulty of predicting outcomes, in practice parameters are often left at default settings or set based on personal or peer experience obtained in a trial and error fashion. To allow for the reliable and efficient selection of parameters for bioinformatic pipelines, a systematic approach is needed. RESULTS: We present doepipeline, a novel approach to optimizing bioinformatic software parameters, based on core concepts of the Design of Experiments methodology and recent advances in subset designs. Optimal parameter settings are first approximated in a screening phase using a subset design that efficiently spans the entire search space, then optimized in the subsequent phase using response surface designs and OLS modeling. Doepipeline was used to optimize parameters in four use cases; 1) de-novo assembly, 2) scaffolding of a fragmented genome assembly, 3) k-mer taxonomic classification of Oxford Nanopore Technologies MinION reads, and 4) genetic variant calling. In all four cases, doepipeline found parameter settings that produced a better outcome with respect to the characteristic measured when compared to using default values. Our approach is implemented and available in the Python package doepipeline. CONCLUSIONS: Our proposed methodology provides a systematic and robust framework for optimizing software parameter settings, in contrast to labor- and time-intensive manual parameter tweaking. Implementation in doepipeline makes our methodology accessible and user-friendly, and allows for automatic optimization of tools in a wide range of cases. The source code of doepipeline is available at https://github.com/clicumu/doepipeline and it can be installed through conda-forge.

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

The host defense peptide LL-37 is internalized by human periodontal ligament cells and prevents LPS-induced MCP-1 production.

OBJECTIVE: The human host defense peptide LL-37 both shows antimicrobial effects and modulates host cell properties. Here, we assess the effects of synthesized LL-37 on lipopolysaccharide (LPS)-induced inflammation in human periodontal ligament (PDL) cells and investigates underlying mechanisms. BACKGROUND: LL-37 has been detected in the periodontal tissues, but its functional importance for PDL cell innate immune responses is not known. METHODS: Human PDL cells were obtained from premolars extracted on orthodontic indications. Cellular pro-inflammatory monocyte chemoattractant protein-1 (MCP-1) mRNA expression was determined using quantitative real-time RT-PCR. MCP-1 protein production was assessed by western blot and ELISA. Internalization of LL-37 by PDL cells was visualized by immunocytochemistry. Nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activity was assessed by western blot of phosphorylated p65, phosphorylated p105, and IκBα proteins. Binding of LL-37 to PDL cell DNA was determined by isolation and purification of DNA and dot blot for LL-37 immunoreactivity. RESULTS: Treatment with LL-37 (1 µmol/L) for 24 hours prevented LPS-induced stimulation of MCP-1 expression analyzed both on transcript and on protein levels. Stimulation with LL-37 (1 µmol/L) for 24 hours had no effect on toll-like receptor (TLR)2 and TLR4 transcript expression, suggesting that LL-37 acts downstream of the TLRs. Preincubation with LL-37 for 60 minutes followed by stimulation with LPS for 24 hours in the absence of LL-37 completely prevented LPS-evoked MCP-1 transcript expression, implying that LL-37 acts intracellularly and not via binding and neutralization of LPS. In PDL cells stimulated with LL-37 for 60 minutes, the peptide was internalized as demonstrated by immunocytochemistry, suggesting an intracellular mechanism of action. LL-37 immunoreactivity was observed both in the cytosol and in the nucleus. Downregulation of LPS-induced MCP-1 by LL-37 was not mediated by reduction in NF-κB activity as shown by unaltered expression of phosphorylated p65, phosphorylated p105, and IκBα NF-κB proteins in the presence of LL-37. Immunoreactivity for LL-37 was observed in PDL cell DNA treated with but not without 0.1 and 1 µmol/L LL-37 for 60 minutes in vitro. CONCLUSION: LL-37 abolishes LPS-induced MCP-1 production in human PDL cells through an intracellular, NF-κB-independent mechanism which probably involves direct interaction between LL-37 and DNA.

A geographically matched control population efficiently limits the number of candidate disease-causing variants in an unbiased whole-genome analysis.

Whole-genome sequencing is a promising approach for human autosomal dominant disease studies. However, the vast number of genetic variants observed by this method constitutes a challenge when trying to identify the causal variants. This is often handled by restricting disease studies to the most damaging variants, e.g. those found in coding regions, and overlooking the remaining genetic variation. Such a biased approach explains in part why the genetic causes of many families with dominantly inherited diseases, in spite of being included in whole-genome sequencing studies, are left unsolved today. Here we explore the use of a geographically matched control population to minimize the number of candidate disease-causing variants without excluding variants based on assumptions on genomic position or functional predictions. To exemplify the benefit of the geographically matched control population we apply a typical disease variant filtering strategy in a family with an autosomal dominant form of colorectal cancer. With the use of the geographically matched control population we end up with 26 candidate variants genome wide. This is in contrast to the tens of thousands of candidates left when only making use of available public variant datasets. The effect of the local control population is dual, it (1) reduces the total number of candidate variants shared between affected individuals, and more importantly (2) increases the rate by which the number of candidate variants are reduced as additional affected family members are included in the filtering strategy. We demonstrate that the application of a geographically matched control population effectively limits the number of candidate disease-causing variants and may provide the means by which variants suitable for functional studies are identified genome wide.

Prompt apoptotic response to high glucose in SGLT-expressing renal cells.

It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet, little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure. In the present ex vivo study of rat renal cells, we compared the apoptotic response to a moderate increase in glucose concentration. We studied cell types that commonly are targeted in diabetic kidney disease (DKD): proximal tubule cells, which express Na+-dependent glucose transporter (SGLT)2, mesangial cells, which express SGLT1, and podocytes, which lack SGLT and take up glucose via insulin-dependent glucose transporter 4. Proximal tubule cells and mesangial cells responded within 4-8 h of exposure to 15 mM glucose with translocation of the apoptotic protein Bax to mitochondria and an increased apoptotic index. SGLT downregulation and exposure to SGLT inhibitors abolished the apoptotic response. The onset of overt DKD generally coincides with the onset of albuminuria. Albumin had an additive effect on the apoptotic response. Ouabain, which interferes with the apoptotic onset, rescued from the apoptotic response. Insulin-supplemented podocytes remained resistant to 15 and 30 mM glucose for at least 24 h. Our study points to a previously unappreciated role of SGLT-dependent glucose uptake as a risk factor for diabetic complications and highlights the importance of therapeutic approaches that specifically target the different cell types in DKD.

Sesquiterpene lactones from Ambrosia arborescens Mill. inhibit pro-inflammatory cytokine expression and modulate NF-κB signaling in human skin cells.

BACKGROUND: Ambrosia arborescens has been used in Andean traditional medicine to reduce problems associated with various inflammatory diseases and conditions, although the underlying mechanism is unknown. HYPOTHESIS/PURPOSE: The sesquiterpene lactones (SLs) coronopilin and damsin, which are major secondary metabolites of A. arborescens, have anti-inflammatory activity by attenuation of IL-6 and MCP-1 expression and inhibition of NF-κB in human dermal fibroblasts (HDFa) and human keratinocytes (HaCaT). STUDY DESIGN: In order to confirm a high concentration of damsin and coronopilin in the plant material, a quantitative method was developed. The effect of the pure compounds on cytokine and NF-κB expression was examined, as well as their effects on HDFa and HaCaT cell morphology and viability. METHODS: Coronopilin and damsin were quantified by HPLC-DAD analysis, from EtOAc extracts of the aerial parts of A. arborescens. Cell morphology was investigated by phase-contrast microscopy and cell viability by the MTT assay. IL-6 and MCP-1 cytokine gene expression was assessed by quantitative real-time RT-PCR in LPS stimulated cells. The NF-κB pathway was studied through western blotting of the phosphorylated forms of p65 and p50/p105, as well as the non-phosphorylated IκB. Dexamethasone was used as positive control. RESULTS: Dry aerial parts contained 12.3  mg/g and 13.4  mg/g of coronopilin and damsin, respectively. Treatment with either compound (1-10 µM) for 24  h attenuated LPS-induced mRNA expression of the pro-inflammatory cytokine IL-6 and the chemokine MCP-1 in HDFa cells. The down-regulation of MCP-1 mRNA induced by coronopilin and damsin was confirmed on the protein level. Damsin reduced phosphorylated p65 and p105 subunits in HDFa cells. Neither coronopilin nor damsin affected HDFa cell morphology and viability within the used concentration range (1-10 µM). Also, in HaCaT cells, treatment with damsin (1-10 µM) for 24  h inhibited the MCP-1 expression, and damsin thereby attenuated cytokine expression both in HDFa and HaCaT cells. CONCLUSION: We show that coronopilin and damsin from A. arborescens inhibit pro-inflammatory IL-6 and MCP-1 expression in human skin cells via NF-κB inhibition, suggesting that they may be useful for antagonizing inflammatory conditions of the human skin.

The host defense peptide LL-37 triggers release of nucleic acids from human mast cells.

The human host defense peptide LL-37 possesses antimicrobial activity but also affects host cell function and viability. Mast cells are involved in innate immunity but no data have been presented on effects of LL-37 on human mast cell viability and export of nucleic acids. Here, we demonstrated by immunofluorescence microscopy that synthesized LL-37 was internalized by human LAD2 mast cells and detected both in cytoplasm and nucleus. Treatment with high (4 and 10 µM) but not low (1 µM) concentrations of LL-37 for 4 h reduced cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Stimulation with 10 µM LL-37 for 4 h enhanced export of nucleic acids, total protein and lactate dehydrogenase (LDH), suggesting that both nuclear and plasma membranes are permeabilized by LL-37. Although LL-37 triggered release of nucleic acids, no extracellular trap-like structures were observed by laser scanning confocal microscopy of cells incubated with the plasma membrane impermeable nucleic acid fluorophore SYTOX-Green, indicating that LL-37 promotes export of nucleic acids but not formation of extracellular traps. On the other hand, phorbol-12-myristate-13-acetate (PMA), which is a well-known inducer of extracellular traps, stimulated export of nucleic acids and also formation of extracellular trap-like structures. However, PMA had no effect on export of either total protein or LDH. Hence, LL-37 and PMA seem to stimulate export of nucleic acids from LAD2 mast cells through different pathways. In conclusion, we demonstrate that LL-37 triggers release of nucleic acids from human mast cells but not the formation of extracellular trap-like structures.

LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis.

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.

The host defense peptide LL-37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils.

The human host defense peptide, LL-37, is an important player in the first line of defense against invading microorganisms. LL-37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL-37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL-37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL-37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL-37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL-37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL-37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL-37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL-37. For the first time, we demonstrate hCAP18/LL-37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL-37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL-37 content of whole saliva.