Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Higher levels of LL37 expression and NET release are determined in patients with SVV. (A) Telomere length of SVV and HCs by real-time Q-PCR; (B) levels of LL37 in serum from the disease group and healthy group; (C) levels of LL37 in urine from the disease group and healthy group; (D) levels of NET remnants in serum from patients with SVV or HCs; (E) levels of cf-DNA in serum from patients with SVV or HCs (A-E). N=70 for each group. The error bars of the graph represent the SEM. Unpaired Student’s t-test was conducted to analyze the statistical significance of two independent experiments; (F) an Alexa 488-labeled antibody against H2A-H2B-DNA complexes was used to visualize NETs in green, and DAPI was used to stain the nuclei in blue. Pictures were merged to form an overlay image; (G) H2A-H2B-DNA complexes, LL37, and nuclei are stained green, red, and blue, respectively. Pictures were merged to form an overlay image. A.U., arbitrary unit.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: Expressing, Labeling, Staining

Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Immune aging of neutrophils and inflammatory medium release are determined in situ in individuals with SVV. (A) Neutrophils (polymorphic nuclear) infiltrated the kidney tissue from patients with SVV. Telomere dysfunction was found by the co-localization of γH2AX (purple) and telomeres (green) in the nucleus (DNA in blue). Immunostaining of LL37 showed perinuclear LL37 (red) in neutrophils that suffered DNA dysfunction; (B) H2A-H2B-DNA complexes, LL37, and nuclei are stained green, red, and blue, respectively. Pictures were merged to form an overlay image. LL37 assembled around the polymorphic nucleus in a background of NET deposition in the kidney tissue of SVV patients.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: In Situ, Immunostaining, Staining

Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Human neutrophils from SVV produce more LL37-mediated-NETs. (A) The percentage of spontaneously netting neutrophils from healthy controls (HCs) and SVV patients and the percentage of netting neutrophils after irradiation; (B) degree of NET release of neutrophils from HCs and SVV in the presence or absence of aprotinin; (C) degree of NET release of neutrophils after irradiation in the presence or absence of aprotinin. The graph is a minimum of 15 images derived from 3 independent experiments. The error bars of the graph represent SEM. Unpaired Student’s t-test was conducted to analyze the statistical significance of two independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05; NS, not significant.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: Irradiation, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Risk Factors Associated with the Development of Thrombotic Microangiopathy in Patients with Dermatomyositis

doi: 10.3390/ijms27010315

Figure Lengend Snippet: Representative immunofluorescence confocal image of a muscle biopsy showing neutrophil extracellular traps (NETs) infiltrating the muscle tissue of dermatomyositis patient. Blue represents DNA (Column 1a , 2a , 3a ), green represents citrullinated histone H3 ( 1b , 2b , 3b ), and red represents LL37 ( 1c , 2c , 3c ). Right column images depict merged images for each tissue ( 1d , 2d , 3d ). Original magnification, ×10. Scale bar 100 µm in the first two rows and 50 µm in the third one.

Article Snippet: Then, the slides were incubated overnight at 4 °C with rabbit anti–human citrullinated histone H3 (Abcam, Cambridge, UK; dilution 1:750), and mouse anti–human LL37 (Santa Cruz Biotechnology, Dallas, TX, USA; 1: 100) diluted in 5% BSA.

Techniques: Immunofluorescence

Journal: The Journal of investigative dermatology

Article Title: SARS-CoV-2-Induced Vasculitic Skin Lesions Are Associated with Massive Spike Protein Depositions in Autophagosomes.

doi: 10.1016/j.jid.2023.07.018

Figure Lengend Snippet: Figure 4. Dermal microvascular cells in COVID-19. Representative serial sections of one COVID-19 skin sample with diverse stainings. (a) H&E overview, square marking the same vessel seen in b, c, and d. (b) LC3B (red), LC3C (green), SARS-CoV-2 spikes (blue). (c) IgG (red) and VEGF (blue). (d) MPO (green), LL37 (red), and CD31 (blue). DAPI (gray) was used for nuclear staining (in bed). Bar in a ¼ 10 mm b ¼ 50 mm. MPO, myeloperoxidase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: The following primary antibodies were used: rabbit anti-LC3B (Novus Biologicals, Centennial, CO), goat anti-LC3C (Abcam, Cambridge, United Kingdom), mouse antieSARS-CoV-2 spike (GeneTex, Irvine, CA), rabbit anti-VEGF (Abcam), goat anti- myeloperoxidase (R&D Systems, Minneapolis, MN), rabbit anti- LL37 (Novus Biologicals), mouse anti-CD31 (Dako, Waltham, MA), or mouse anti-CD163 (DCS Diagnostics, Hamburg, Germany).

Techniques: Staining

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: NETs foster BC lung metastasis by inducing the apoptosis of CD8 + T cells through cathelicidin. (A) Representative immunofluorescence images showing the colocalization of NETs with CD8 + T cells (CD8), CD4 + T cells (CD4), macrophages (F4/80), fibroblasts (α-Sma), or endothelial cells (CD31) at the premetastatic stage [4T1-LM3 (BALB/c) model, n = 5]. The colocalization was assessed by analyzing 10 fields per mouse. The bar graph on the right quantifies the extent of colocalization of NETs with each cell type in the lung tissue. (B) 4T1-LM3 (BALB/c) mouse model ( n = 6) was treated with anti-IgG antibody (control), anti-Ly6g antibody, or PADI4 inhibitor (GSK484). CD8 + T cells were then isolated from the macrometastatic lung of these mice and incubated with the cell death dye Zombie Aqua. The proportion of dead CD8 + T cells was analyzed by FACS. The bar graph quantifies the proportion of dead CD8 + T cells in each treatment group. (C) Representative immunofluorescence staining images showing CD8 + T cells in metastatic lungs from mice in the indicated treatment. Corresponding quantifications are shown on the right ( n = 6). (D and E) CD8 + T cells were treated with vehicle (cell-free culture medium), CM-Neu, NETs (5 μg/ml), or NETs (10 μg/ml). The apoptosis [ n = 4, (D)] and viability of CD8 + T cells [ n = 3, (E)] were quantified. (F) CD8 + T cells were isolated from the spleens of BALB/c mice and treated with or without NETs (10 μg/ml) for 48 h prior to RNA-seq analysis. KEGG pathway analysis was subsequently performed on the differentially expressed genes (DEGs) with an absolute log 2 fold change > 0.58. (G) Heatmap showing the expression of apoptosis-related DEGs identified in CD8 + T cells following treatment with or without NETs ( n = 3). (H) Representative immunofluorescence micrographs showing NET formation in mouse and human neutrophils after stimulation with PMA (200 nM). LL37(green) and Cramp (green) are localized on the DNA scaffold of NETs. (I and J) CD8 + T cells were isolated at the macrometastatic stage from 3 groups of mice, including WT mice, Cramp -deficient mice, and WT mice injected with E0771-LM3 cells and treated with the PADI4 inhibitor (GSK484) and Cramp. (I) CD8 + T cells were incubated with cell death dye Zombie Aqua, and the proportion of dead cells was assessed by FACS. (J) Representative immunofluorescence micrographs showing CD8 + T cells in macrometastatic lung tissues of each group. The data with error bars are presented as the mean ± SD; statistical significance was determined by one-way ANOVA test (A to E, I, and J). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; Bax , bcl2-associated x protein; Bcl2 , B cell lymphoma 2; Bcl2l1 , bcl2-like 1; Bid , bh3 interacting-domain death agonist; Casp3 , caspase 3; CD8, cluster of differentiation 8; CM-Neu, neutrophil-derived conditioned medium; Cramp, cathelicidin antimicrobial peptide; Ctsd , cathepsin d; Ctsl , cathepsin l; Cycs , cytochrome c; Dab2ip , dab2 interacting protein; DAPI, 4',6-diamidino-2-phenylindole; E0771-LM3, E0771-lung metastasis 3; FACS, fluorescence-activated cell sorting; FPKM, fragments per kilobase million; H3cit; citrullinated histone H3; HPF, high power field; KEGG, Kyoto Encyclopedia of Genes and Genomes; Ly6g, lymphocyte antigen 6 complex locus g; Mcl1 , myeloid cell leukemia 1; MPO, myeloperoxidase; PADI4, peptidyl arginine deiminase 4; PerCP/Cy5.5, peridinin chlorophyll protein-cyanine5.5; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; Puma , p53 up-regulated modulator of apoptosis; RNA-seq, RNA sequencing; SD, standard deviation; α-Sma, α-smooth muscle actin.

Article Snippet: Cathelicidin expression in CD8 + T cells was then detected by immunofluorescence staining using rabbit anti-cathelicidin antibody (1:100, 12009-1-AP, Proteintech), and fluorescent signals were captured using Leica DMi8 confocal microscopy.

Techniques: Immunofluorescence, Control, Isolation, Incubation, Staining, RNA Sequencing, Expressing, Injection, Derivative Assay, Fluorescence, FACS, Standard Deviation

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Cathelicidin binding to ANT1 induces mPTP opening and promotes apoptosis in CD8 + T cell. (A) CD8 + T cells isolated from WT mice were treated with NETs derived from WT or Cramp −/− mice, or with Cramp, and then stained with JC-1. ΔΨm was determined by flow cytometry ( n = 4). The bar graph on the right shows the decreased ΔΨm of CD8 + T cells. (B) Representative immunofluorescence images showing colocalization of Cramp (green) with mitochondria (red) in CD8 + T cells by confocal laser microscopy. (C and D) CD8 + T cells were treated with vehicle (cell-free culture medium), NETs derived from WT or Cramp −/− mice, or Cramp. Cytoplasmic and mitochondrial ROS were measured using DCFH-DA and MitoSOX red probes [ n = 5, (C)]. Representative transmission electron microscopic images of mitochondrial ultrastructure in CD8 + T cells under the indicated treatments (D). Red arrows point to mitochondria exhibiting swelling, disorganized cristae, and loss of membrane integrity. (E) Mouse or human CD8 + T cells were treated with Cramp or LL37. The mPTP opening of mitochondria was measured by calcein loading/CoCl 2 quenching. (F) Protein–protein interactions were assessed in vitro using a pull-down assay. Recombinant proteins Ant1 (or Vdac1) and biotin-labeled Cramp (or a cecropin-like antibacterial peptide from Helicobacter pylori ) were used for this purpose. (G) Co-IP was used to check the interaction between mitochondrial protein Ant1 and Vdac1 in mouse CD8 + T cells under the treatment with different concentrations of NETs derived from WT and Cramp − / − mice ( n = 3). (H and I) Mouse CD8 + T cells were treated with Cramp or Cramp combined with different concentrations of BKA ( n = 4). (H) The interaction between mitochondrial protein Ant1 and Vdac1 in CD8 + T cells was assessed by Co-IP. (I) The ΔΨm of mitochondria in CD8 + T cells was assessed by flow cytometry after staining with JC-1. (J) CD8 + T cells were treated as in (I), and CD8 + T cell apoptosis was quantified by flow cytometry ( n = 4). The bar graph on the right shows the apoptosis of CD8 + T cells. The data with error bars are presented as the mean ± SD; statistical significance was determined by one-way ANOVA test (A, C, I, and J). ANOVA, analysis of variance; Ant1, adenine nucleotide translocator 1; BKA, bongkrekic acid; CD8, cluster of differentiation 8; Co-IP, coimmunoprecipitation; Cramp, cathelicidin antimicrobial peptide; Cyto, cytoplasm; DAPI, 4’,6-diamidino-2-phenylindole; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; Hp, Helicobacter pylori ; kDa, kilodalton; Mito, mitochondrial; mPTP, mitochondrial permeability transition pore; NETs, neutrophil extracellular traps; PI, propidium iodide; ROS, reactive oxygen species; Vdac1, voltage dependent anion channel 1; WT, wild type; SD, standard deviation; ΔΨm, mitochondrial membrane potential.

Article Snippet: Cathelicidin expression in CD8 + T cells was then detected by immunofluorescence staining using rabbit anti-cathelicidin antibody (1:100, 12009-1-AP, Proteintech), and fluorescent signals were captured using Leica DMi8 confocal microscopy.

Techniques: Binding Assay, Isolation, Derivative Assay, Staining, Flow Cytometry, Immunofluorescence, Microscopy, Transmission Assay, Membrane, Protein-Protein interactions, In Vitro, Pull Down Assay, Recombinant, Labeling, Co-Immunoprecipitation Assay, Permeability, Standard Deviation

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Prognostic significance of NETs in human BC. (A) Representative FACS plot showing the ratio of human CD84 high and CD84 low neutrophils in healthy individuals ( n = 50) and patients with BC at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. To define the CD84 high and CD84 low subsets in humans, we first established the positive gating threshold using FMO controls. Subsequently, the boundary between “high” and “low” subsets was determined based on a clear inflection point observed in the fluorescence intensity histogram. Statistical significance was determined by comparing with the healthy group. The bar graph on the right quantifies the ratio of human CD84 high and CD84 low neutrophils. (B) Representative immunofluorescence micrographs showing NET formation of CD84 high and CD84 low neutrophils, which were sorted by FACS after treatment with PMA for 2 h ( n = 6). NETs were stained with antibodies against MPO (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies the formation of NETs. (C) Plasma NET levels in healthy individuals ( n = 50) and BC patients at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. (D) Kaplan–Meier survival curves showing the overall survival (OS) of BC patients with low (NETs < 344.91 pg/ml; n = 83) or high (NETs ≥ 344.91 pg/ml; n = 77) concentrations of plasma NETs. BC patients were stratified into high and low NET groups using the mean plasma NET level of the entire cohort as the cutoff. (E) Receiver operator characteristic (ROC) curve analysis of plasma NET levels for predicting BC patients’ lung metastases ( n = 160). The area under the curve (AUC) value reflects the model’s power to distinguish between BC patients with and without lung metastasis within 6 years after diagnosis. Higher AUC values (approaching 1) denote superior differentiation accuracy at this time point. (F) Correlation between plasma NET levels and CD8 + T cell proportion in healthy individuals and patients with BC ( n = 210). (G) Kaplan–Meier analysis showing the recurrence-free survival of BC patients with high or low levels of LL37 ( n = 4,929). Data were obtained from the Kaplan–Meier plotter database, which does not provide detailed numerical thresholds for LL37 level classification. (H) Mechanism scheme of Ly6g high and Ly6g low neutrophils in promoting pulmonary metastasis of BC. Briefly, Ly6g high neutrophils accumulated in the premetastatic stage and induced CD8 + T cell apoptosis through NETosis. The NET-derived cathelicidin directly bound with Ant1, an mPTP protein in CD8 + T cells, leading to conformational changes in the Ant1 and subsequent Ant1–Vdac1 complex formation, which resulted in mPTP opening, loss of ΔΨm, and uncoupling of mitochondrial electron transport chain in CD8 + T cells. Ly6g low neutrophils bearing MDSC-like transcriptional signatures exhibit a superior capacity to inhibit the proliferation and effector functions of CD8 + T cells. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (A), Student’s t test (B), 1-way ANOVA test (C), and 2-sided log-rank test (D and G). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; APC, allophycocyanin; BC, breast cancer; CD8, cluster of differentiation 8; CD84, cluster of differentiation 84; CI, confidence interval; DAPI, 4',6-diamidino-2-phenylindole; E0771-LM3, E0771-lung metastasis 3; FACS, fluorescence-activated cell sorting; FMO, fluorescence-minus-one; H3cit, citrullinated histone H3; HR, hazard ratio; Interferon-γ, IFN-γ; Il-1β, interleukin-1β; Ly6g, lymphocyte antigen 6 complex locus g; MDSC, myeloid-derived suppressor cell; MPO, myeloperoxidase; mPTP, mitochondrial permeability transition pore; NETs, neutrophil extracellular traps; PADI4, peptidyl arginine deiminase 4; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; RFS, recurrence-free survival; ROS, reactive oxygen species; Vdac1, voltage-dependent anion channel 1; SD, standard deviation; ΔΨm, mitochondrial membrane potential.

Article Snippet: Cathelicidin expression in CD8 + T cells was then detected by immunofluorescence staining using rabbit anti-cathelicidin antibody (1:100, 12009-1-AP, Proteintech), and fluorescent signals were captured using Leica DMi8 confocal microscopy.

Techniques: Fluorescence, Immunofluorescence, Staining, Clinical Proteomics, Biomarker Discovery, Derivative Assay, FACS, Permeability, Standard Deviation, Membrane

Journal: Annals of Dermatology

Article Title: Citron Essential Oils Alleviate the Mediators Related to Rosacea Pathophysiology in Epidermal Keratinocytes

doi: 10.5021/ad.2018.30.6.653

Figure Lengend Snippet: (A) Messenger RNA levels of kallikrein 5 (KLK5), LL-37, and vitamin D receptor (VDR) induced by vitamin D3 decreased after treatment with citron essential oils in normal human epidermal keratinocytes (NHEKs); semi-quantitative reverse-transcription-polymerase chain reaction. (B) Protein levels of KLK5 and LL-37 induced by VD3 in NHEKs decreased after treatment with citron seed and unripe citron essential oils; enzyme-linked immunosorbent assay. Bars indicate standard deviations. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. * p <0.05, ** p <0.01.

Article Snippet: The cell suspensions were fixed with paraformaldehyde, blocked with 5% goat serum, and incubated with rabbit polyclonal anti-cathelicidin (LL-37) antibody (Abcam, Cambridge, MA USA), mouse monoclonal anti-VEGF antibody (Novusbio Biological, Littleton, CO, USA), or rabbit polyclonal anti-VR1 (TRPV1) antibody (Abcam).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Annals of Dermatology

Article Title: Citron Essential Oils Alleviate the Mediators Related to Rosacea Pathophysiology in Epidermal Keratinocytes

doi: 10.5021/ad.2018.30.6.653

Figure Lengend Snippet: Expression of LL-37 induced by vitamin D 3 (VD 3 ) in normal human epidermal keratinocytes was suppressed after treatment with 0.02% citron essential oils; immunocytochemistry, 200×. DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The cell suspensions were fixed with paraformaldehyde, blocked with 5% goat serum, and incubated with rabbit polyclonal anti-cathelicidin (LL-37) antibody (Abcam, Cambridge, MA USA), mouse monoclonal anti-VEGF antibody (Novusbio Biological, Littleton, CO, USA), or rabbit polyclonal anti-VR1 (TRPV1) antibody (Abcam).

Techniques: Expressing, Immunocytochemistry

Journal: Annals of Dermatology

Article Title: Citron Essential Oils Alleviate the Mediators Related to Rosacea Pathophysiology in Epidermal Keratinocytes

doi: 10.5021/ad.2018.30.6.653

Figure Lengend Snippet: (A) Messenger RNA levels of vascular endothelial growth factor (VEGF) and transient receptor potential vanilloid 1 (TRPV1) induced by interleukin 33 (IL-33) and LL-37 decreased after treatment with citron essential oils in normal human epidermal keratinocytes (NHEKs); real-time polymerase chain reaction. (B) Protein levels of VEGF and TRPV1 induced by IL-33 and LL-37 in NHEKs decreased after treatment with citron essential oils; enzyme-linked immunosorbent assay. Bars indicate standard deviations. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. * p <0.05, ** p <0.01.

Article Snippet: The cell suspensions were fixed with paraformaldehyde, blocked with 5% goat serum, and incubated with rabbit polyclonal anti-cathelicidin (LL-37) antibody (Abcam, Cambridge, MA USA), mouse monoclonal anti-VEGF antibody (Novusbio Biological, Littleton, CO, USA), or rabbit polyclonal anti-VR1 (TRPV1) antibody (Abcam).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay