Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Human polymorphonuclear neutrophils (PMN) decrease ITPR2 levels in primary human hepatocytes (HHC). HepG2 cells were co-cultured with PMN from 6 different healthy volunteers in a 1:1 ratio number for 18-24 hours. Following co-culture, hepatocytes were assessed for ITPR2 levels by immunoblotting. Representatives blots ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=6 (****p<0.0001, unpaired t-test). (b) PMN decrease ITPR2 levels in HepG2 cells. Cells were co-cultured with PMN from 9 different volunteers in a 1:1 ratio number for 18-24 hours. Following co-culture, cells were assessed for ITPR2 levels by immunoblotting. Representatives blots ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=9 (****p<0.0001, unpaired t-test). (c) PMN decrease the amplitude of Ca 2+ signals evoked by adenosine triphosphate (ATP) in HepG2 cells. HepG2 cells pre-incubated for 20 hours with or without PMN were loaded with Fluo-4/AM (6 μM) for 30 minutes and then stimulated with ATP (20 μM). Left: Representative fluorescence intensity tracings from measurements in selected regions of interest of the cells. Right: Quantification from 9 coverslips of HepG2 cells (317 total cells) and 8 coverslips of HepG2+PMN (279 total cells), from 3 independent experiments for each condition. Graph shows mean ± SD (****p<0.0001, unpaired t-test). (d) PMN decrease proliferation of HepG2 cells. EdU (5-ethynyl-2’-deoxyuridine) proliferation assay was performed in HepG2 cells co-cultured with or without PMN in a 1:1 ratio number for 12 hours. Left: Representative image of cells with EdU-Alexa Fluor 488 labeling (green), indicative of proliferation, and Hoechst 33342 labeling of nuclei (blue). Scale bar, 50 μm. Right: Quantitative analysis of EdU-positive cells (%, n=5-6 coverslips for each condition in 3 independent experiments. Data are mean ± SD (****p < 0.0001, unpaired t-test). (e) PMN do not induce death of HepG2 cells. Live/dead cell analysis of HepG2 cells using double staining of calcein-AM (green) and Ethidium Homodimer-1 (EthD-1, red) with or without co-incubation with PMN for 20 hours. For positive control of dead cells, HepG2 cells were treated with 70% ethanol for 10 minutes. Representative image shows HepG2 cells stained with calcein-AM and EthD-1. Scale bar, 50 μm. Lower right panel shows the quantification of dead cells (%). Data are mean ± SD, n=5-10 fields in 3 cover slips per each condition in 3 independent experiments (N.S., not significant, unpaired t-test). (f) ITPR2 levels in HepG2 cells recover after PMN are removed. After co-culturing HepG2 cells with PMN for 20 hours, cells were washed with phosphate-buffered saline until PMN were no longer visible, then cultured for 2 hours and were collected (labeled as After removal of PMN). The ITPR2 levels was compared to that of HepG2 cells co-cultured with PMN for 22 hours or control (labeled as + PMN or HepG2 respectively). Representative blots ( left ) and quantification ( right ) are shown. Data are mean ± SD. n=3-5 (*p<0.05, unpaired t-test).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Incubation, Fluorescence, Proliferation Assay, Labeling, Cell Analysis, Double Staining, Positive Control, Staining, Saline, Control

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: Human polymorphonuclear neutrophils (PMN) do not induce cell death in HepG2 cells, even after 40 hours of exposure. Live/dead cell analysis of HepG2 cells using double staining with calcein AM (green) and ethidium Homodimer-1 (EthD-1, red) with or without PMN for 40 hours. For positive control of dead cells, HepG2 cells were treated with 70% ethanol for 10 minutes. The representative image ( top , scale bar: 50 μm) and quantification of dead cells (%) are shown ( right ). Data are mean ± SD, n=5-10 fields in 3 coverslips per each condition in 3 independent experiments. (N.S., no significant difference by unpaired t-test).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Cell Analysis, Double Staining, Positive Control

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Conditioned medium (C.M.) from human polymorphonuclear neutrophils (PMN) do not decrease ITPR2 levels in HepG2 cells. Cells were cultured for 18-24h with C.M. derived from 16 hours incubation with PMN, and ITPR2 levels were assessed by immunoblotting. Representative blots ( top ), and quantification ( bottom ) are shown. Data are mean ± SD, n=3. (N.S., no significant difference, ***p<0.001 by one-way ANOVA). (b) PMN do not decrease ITPR2 levels in HepG2 cells that are separated by a semipermeable membrane. PMN were placed in the upper compartment of 3 μM pore transwell system (T.W.) and co-cultured with HepG2 cells cultured in the lower compartment. After 18–24 hours of co-culture, HepG2 cells were assessed for ITPR2 levels by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3 (N.S., no significant difference, *p<0.05 by one-way ANOVA). (c) ‘Naked’ PMN partially lose the ability to decrease ITPR2 in HepG2 cells. Naked PMN were prepared by removing cell surface glycosylphosphatidylinositol (GPI)-anchored proteins by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). After co-culturing naked PMN with HepG2 for 18-24 hours, the ITPR2 levels in HepG2 cells was assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=6 (**p<0.01, one-way ANOVA). (d) Functional blocking treatment by anti-integrin alpha-2 (ITGA2) or anti-integrin alpha M (ITGAM) antibodies do not prevent the decrease in ITPR2 of HepG2 cells by PMN. HepG2 cells were pre-incubated with anti-ITGA2 or ITGAM antibodies for 2 hours and then co-incubated with PMN. These are candidate integrins identified by bioinformatic analysis of RNA-seq from HepG2 cells after co-culturing with PMN. After co-culture, HepG2 cells were assessed for ITPR2 levels by immunoblotting. Representative blots ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=3 (N.S., no significant difference by one-way ANOVA). (e) The components released in neutrophil extracellular traps (neutrophil elastase, myeloperoxidase (MPO) or high mobility group box-1 (HMGB1)) do not decrease ITPR2 levels in HepG2 cells. Cells were co-incubated with varying concentrations of elastase, MPO or HMGB1 for 20 hours and then assessed for ITPR2 levels by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3-4 (N.S., no significant difference by one-way ANOVA). (f) Flowchart illustrating the experimental procedure to fractionate PMN is shown. (g) The granule fraction but not the cytoplasm/membrane fraction from PMN decreases ITPR2 in HepG2 cells. After incubation for 20 hours with the indicated fractions, HepG2 cells were assessed for ITPR2 levels by immunoblotting. Representative blots ( left ), and quantification ( right ) are shown. Data are mean ± SD, n=4 (N.S., no significant difference; ****p<0.0001 by one-way ANOVA). (h) The granule fraction decreases ITPR2 in HepG2 cells in a concentration-dependent fashion that follows first-order kinetics, similar to what is observed with intact PMN. HepG2 cells were co-cultured with PMN ( left ) or the granule fractions ( right ) extracted from the indicated number of PMN from 5 different healthy control subjects for 20 hours. Following co-culture, HepG2 cells were assessed for ITPR2 levels by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Note both data sets are linear on a log-log scale. Shown are mean ± SEM, n=5 (N.S., not significantly different; *p<0.05; ***p<0.001; ****p<0.0001 by one-way ANOVA).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Cell Culture, Derivative Assay, Incubation, Western Blot, Membrane, Co-Culture Assay, Functional Assay, Blocking Assay, RNA Sequencing, Concentration Assay, Control

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) The granule fraction of human polymorphonuclear neutrophils (PMN) contains MPO and elastase. Immunoblots for myeloperoxidase (MPO) and elastase of granule fraction lysates extracted from PMN from three volunteers (#1-3) are shown. The rightmost lane shows the lysate of PMN as a positive control. (b) Neither the plasma membrane nor the nucleus of PMN decreases ITPR2 in HepG2 cells. Plasma membranes or nuclei were extracted from PMN using the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit. These fractions were administered to HepG2 cells for 20 hours and then the levels of ITPR2 were assessed by immunoblotting. Representative blots ( left ), and quantification ( right ) are shown. Data are mean ± SD, n=4-5. (N.S., no significant difference, ****p<0.0001 by one-way ANOVA).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Western Blot, Positive Control, Clinical Proteomics, Membrane, Isolation, Cell Fractionation

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) The levels of ITPR2 in HepG2 cells are decreased 1 hour after administration of human polymorphonuclear neutrophils (PMN) and remains low after 4 and 20 hours. After co-culturing PMN with HepG2 cells for the indicated times, the amount of ITPR2 in HepG2 cells was assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3 (***p<0.001, ****p<0.0001, by one-way ANOVA). (b) ITPR2 mRNA in HepG2 cells is increased 1 hour after co-culture with PMN. Reverse transcription quantitative PCR (RT-qPCR) was performed to assess ITPR2 mRNA levels in HepG2 cells after co-culture with PMN for the indicated times. Quantitative analysis is shown. Data are mean ± SD, n=3 (**p<0.01, ****p<0.0001 by unpaired t-test). (c) The neutrophil-induced decrease in ITPR2 in HepG2 cells is not due to classical proteolytic mechanisms. HepG2 cells were pre-incubated with MG132 (proteasome inhibitor, 50 μM), bafilomycin A1 (Baf, autophagy inhibitor, 50nM), or ALLN (Ac-Leu-Leu-Nle-Aldehyde, calpain inhibitor, 50 μM) for 1 hour and followed for 1 hour after co-culture with PMN. The ITPR2 levels in HepG2 cells were assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3-4 (N.S., no significant difference by one-way ANOVA). (d) PMN transfer myeloperoxidase (MPO, green ) and elastase ( red ) to HepG2 cells. Cells were co-cultured with PMN for 1 hour, washed with phosphate-buffered saline until PMN were not visible, then stained with anti-MPO and anti-elastase antibodies. Representative serial optical sections obtained by confocal immunofluorescence imaging (Z-stack) of HepG2 cells is shown. Hoechst 33342 is used to stain nuclei (blue). Scale bar, 20 μm. (e) Immunoblotting confirms that MPO and elastase are detected in HepG2 cells after co-incubated with PMN. After HepG2 cells were co-cultured with PMN for 1 hour, cells were washed with phosphate-buffered saline until no PMN were visible. Then, HepG2 cells were assessed for ITPR2, MPO and elastase by immunoblotting. Representative blots ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=3 (****p<0.0001, unpaired t-test). (f) ITPR2 is degraded by elastase but not by MPO. HepG2 cells (3.0 x 10 6 ) were homogenized in 100 μL of phosphate-buffered saline by sonication, followed by addition of MPO or neutrophil elastase at the concentrations indicated. Five minutes later, ITPR2 levels were assessed by immunoblotting. Note that there are multiple lower molecular weight bands in homogenate treated with the lower elastase concentration, but digestion is complete in homogenates treated with the higher concentration. Representative blot ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=3 (N.S., no significant difference, **p<0.01 by one-way ANOVA). (g) The serin protease inhibitor AEBSF prevents the loss of ITPR2 in HepG2 cells induced by PMN. After co-culturing PMN with HepG2 cells with or without 10 μM AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride) for 1 hour, the ITPR2 levels in HepG2 cells was assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3. (N.S., no significant difference, ***p<0.001 by one-way ANOVA). (h) The MPO inhibitor PF1355 does not prevent the PMN-induced loss of ITPR2 in HepG2 cells. After co-culturing PMN with HepG2 cells with or without 10 μM PF-1355 (2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide) for 1 hour, the ITPR2 levels in HepG2 cells was assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3. (N.S., no significant difference, ***p<0.001 by one-way ANOVA). (i) The target proteins in hepatocytes by neutrophil elastase are not non-specific. After co-culturing PMN with HepG2 cells for 1 hour, the amounts of ITPR1, ITPR2, ITPR3, SERCA2, calnexin, SEC61B in the HepG2 cells were assessed by immunoblotting. The ITPRs and SERCA2 were degraded but calnexin and SEC61B were not. Representative blots ( left ) and quantitative analysis ( right ) are shown. ITPR1, ITPR2 and ITPR3 have similar molecular weights, so these samples were blotted onto different membranes; GAPDH is checked at the same time. Data are mean ± SD, n=3. (N.S., no significant difference; **p<0.01; ****p<0.0001 by unpaired t-test).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Western Blot, Co-Culture Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, Cell Culture, Saline, Staining, Immunofluorescence, Imaging, Sonication, Molecular Weight, Concentration Assay, Protease Inhibitor

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Human polymorphonuclear neutrophils (PMN) decrease the amplitude of calcium signals in HepG2 cells. Cells were pre-incubated for 1 hour with or without PMN, then loaded with 6 μM Fluo-4/AM for 30 minutes and stimulated with 20 μM adenosine triphosphate (ATP). Representative time course of amplitude of calcium signal (% of baseline) are shown measured in selected regions of interest of the cells using ImageJ. The right graph was results of quantification (6 coverslip of HepG2, n=330; 6 cover slips of HepG2 + PMN n=283, from 2 independent experiments, graphs are mean ± SD, ****p <0.0001, unpaired t-test). (b) After removal of PMN, calcium signals in HepG2 cells improve over time. HepG2 cells were loaded with 6 μM Fluo-4/AM for 30 minutes and stimulated with 20 μM ATP. The amplitude of calcium signals (% of baseline) were measured in selected regions of interest of the cells using ImageJ at 3 time points: when HepG2 cells were co-cultured with PMN for 1 hour, when PMN were completely washed and cultured for 1 or 19 hours (labeled as 2h and 20h). The graph shows the results of quantification (5-7 coverslip per each, cell number are n=266, 208, 135 from 2 independent experiments, graphs are mean ± SD, ****p<0.0001, one-way ANOVA).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Incubation, Cell Culture, Labeling

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Longer exposure of the representative immunoblot in shows that the ITPR2 bands are laddered. (b) Human polymorphonuclear neutrophils (PMN)-induced loss of ITPR2 in HepG2 cells is not prevented by a trypsin inhibitor or a caspase 3 inhibitor. HepG2 cells were co-cultured with PMN for 20 hours with/without a caspase 3 inhibitor (50μM) or a trypsin inhibitor (AdaAhx3L3VS, 50nM). After co-culture with PMN, the ITPR2 levels in the HepG2 cells was assessed by immunoblotting. Representative blots of HepG2 cells co-cultured with 0.5 million and 1 million PMN are shown ( top ). Quantitative analysis ( bottom ) is shown only for HepG2 cells co-cultured with 1 million PMN. Data are mean ± SD, n=3 (N.S., no significant difference by one-way ANOVA). (c) Granule proteins are transferred from neutrophils into primary human hepatocytes (HHC). After co-culture with PMN for 20 hours, HHC were assessed for ITPR2 and myeloperoxidase (MPO) by immunoblotting. Representative blots ( top ) and quantification for MPO ( bottom ) is shown. Data are mean ± SD, n=7 from 3 independent experiments (****p<0.0001 by one-way ANOVA). (d) HepG2 cells take up components of the granule fraction from neutrophils. HepG2 cells were incubated with neutrophil-derived granule fraction stained with CellMask Orange fluorescent lipophilic membrane dye for 1 hour, washed, then observed by confocal microscopy. A representative images shows red granules incorporated in HepG2 cells ( top , scale bar: 20 μm). In addition, HepG2 cells were incubated with non-stained granule fraction for 1 hour and observed by confocal microscopy after immunostaining for anti-MPO antibody ( green ). A representative images of HepG2 cells are shown ( bottom , scale bar, 20 μm). (e) MPO inhibitor-2 (5-Fluoro-1H-indol-3-yl butanamide) does not prevent the decrease in ITPR2 in HepG2 cells co-cultured with PMN. After co-culturing with PMN with or without 5 μM MPO inhibitor-2 for 1 hour, the ITPR2 levels in HepG2 cells were assessed by immunoblotting. Representative blots ( top ) and quantification ( bottom ) are shown. Data are mean ± SD, n=3 (N.S., no significant difference by one-way ANOVA).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Western Blot, Cell Culture, Co-Culture Assay, Incubation, Derivative Assay, Staining, Membrane, Confocal Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Heat maps are shown for two experiments, comparing RNA-seq data of HepG2 cells treated with human polymorphonuclear neutrophils (PMN) or extracted granule fraction (both from three different volunteers) for 20 hours and respective controls (HepG2). The left map shows the PMN treatment, and the right one shows the granule fraction treatment. (b) The Venn diagram shows the number of genes that were significantly up- or down-regulated (false discovery rate (FDR) < 0.05, log fold change < −0.5 or >0.5) for HepG2 cells treated with neutrophil or granule fraction compared to the respective controls. A list of genes commonly up- or down-regulated in the two RNA-seqs is shown in the bottom. 10 genes were extracted, including Serpin E2 and Serpin A3. (c) The mRNA levels of serpin E2 and serpin A3 in HepG2 cells are significantly elevated after co-culture with PMN for 1 hour. Reverse transcription quantitative PCR (RT-qPCR) was performed to assess the mRNA levels of serpin E2 and serpin A3 in HepG2 cells after co-culture with PMN for 1 hour. Data are mean ± SD, n=3. (*p<0.05, unpaired t-test). (d) Degradation of ITPR2 by neutrophil elastase is inhibited by serpin E2 and serpin A3. HepG2 cells (1.0 x 10 6 ) were homogenized by sonication in 100 μL phosphate-buffered saline followed by addition of neutrophil elastase (0.02 μg/mL) with or without serpin E2 or serpin A3 (20 μg/mL). 5 minutes later, ITPR2 levels were assessed by immunoblotting. Representative blots ( left ) and quantification ( right ) are shown. Data are mean ± SD, n=3. (N.S., no significant difference, **p<0.01 by one-way ANOVA).

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: RNA Sequencing, Co-Culture Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sonication, Saline, Western Blot

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: Volcano plots of up- or down-regulated differentially expressed genes from RNA-seq for HepG2 cells treated with neutrophils (HepG2 + PMN, left ) or extracted granule fraction (HepG2 + granules, right ) for 20 hours, compared to control respectively (triplicate per each). Grey dots represent genes which are not differentially expressed, red dots (UP) represent the upregulated genes, and the blue dots (DOWN) represent the downregulated genes. The threshold for the analysis was set at false discovery rate (FDR)<0.05 and log Fold change < −0.5 or >0.5 (vs control). Serpin E2 and Serpin A3 are included as significantly decreased genes in both analyses.

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: RNA Sequencing, Control

Journal: bioRxiv

Article Title: Neutrophils regulate ITPR2 levels in epithelia by direct injection of elastase

doi: 10.1101/2022.09.13.506588

Figure Lengend Snippet: (a) Neutrophils transfer elastase and eliminate ITPR2 in cells derived from colon, lung, and pancreas. Human polymorphonuclear neutrophils (PMN) were co-cultured with HCT166 (colon), A549 (lung), and PANC-1 (pancreas) cells for 1 hour. After co-culture, ITPR2, myeloperoxidase (MPO) and elastase in these cells were assessed by immunoblotting. Representative blots ( left ) and quantification for ITPR2 ( right ) are shown. Data are mean ± SD, n=3 (***p<0.001, unpaired t-test). (b) ITPR2 staining in hepatocytes is reduced in liver biopsy specimens from patients with alcoholic hepatitis (AH) relative to histologically normal controls. Representative immunochemistry images ( left ) and quantitative analysis of ITPR2 staining ( right ) are shown. The brown-colored area is stained with anti-ITPR2 antibody. The yellow areas are under high magnification. Scale bar, 50 μm (white), 10 μm (yellow). Data are mean ± SD; 3 fields were quantified in each biopsy specimen, which included 8 controls and 9 from patients with AH (***p<0.0001 using one-tailed unpaired t-test). (c) Elastase-positive neutrophils are much more prevalent in liver biopsy tissue from AH patients than in normal controls. Control and AH liver tissue were immunohistochemically stained with anti-neutrophil elastase antibodies. Scale bar, 50 μm. The number of elastase-positive cells was markedly increased in liver tissue from AH patients. Representative immunohistochemical staining ( left ) and the number of elastase-positive cells in each field using 20 x magnification ( right ) is shown. Cells were counted in 5-7 areas in each specimen (n=8 control and n=9 AH specimens; ****p<0.0001 by unpaired t-test). (d) Neutrophil elastase is present in hepatocytes that are in proximity to neutrophils. Shown is a representative image of immunochemical staining for elastase in a liver biopsy from a patient with AH. Asterisks indicate neutrophils and arrowheads denote elastase staining in hepatocytes (60x magnification, scale bar, 20 μm). (e) Sequence of events in neutrophil-hepatocyte interactions. Left: neutrophils are in proximity to hepatocytes; Center: neutrophils directly contact hepatocytes to transfer granule contents into the hepatocytes. The neutrophil elastase degrades ITPR2; Right: The neutrophil moves away from the hepatocyte, which recovers by producing serpin E2 and A3 to block the elastase.

Article Snippet: For co-immunolabelling with primary antibodies, rabbit anti-myeloperoxidase (MPO) antibody 1(Abcam, 1:100 dilution, ab9535) and mouse anti-neutrophil elastase antibody (R&D systems, 1:100 dilution, MAB91671) were incubated for 8 hours, at 4°C, followed by a 1 hour incubation at room temperature with goat anti-mouse Alexa 564 and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:500; Invitrogen, A-11030 and A-11008 respectively), respectively.

Techniques: Derivative Assay, Cell Culture, Co-Culture Assay, Western Blot, Staining, One-tailed Test, Control, Immunohistochemical staining, Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: Cathelicidin senses enteric pathogen Salmonella typhimurium/ LPS for colonic chemokine generation: a new innate immune role for a host defense peptide

doi: 10.1101/389726

Figure Lengend Snippet: (A-D) C57BL/6 Cramp +/+ and Cramp −/− mice were orally infected with C. rodentium (1 x 10 8 CFU in 200 μL of PBS) for 7 d. (A) Experimental design depicting C. rodentium infection of mice. (B i ) Hematoxylin-eosin (H&E) staining of mice colonic tissues. Red arrows denote crypt length. ( B ii-iii ) Histograms depicting colon length ( B ii ) or crypt length ( B iii ) in mice at 20X magnification (only for B iii ). (C) Immunofluorescence staining of mouse colonic tissue with anti-Ly6G antibody (5 μg/mL). Fluorescence was calculated using ImageJ 1.50i software and represented as mean fluorescence intensity (MFI). ( B and C ) Images are representative of n=4 mice. ( D and E ) Bar graphs of MPO activity (D) and KC protein expression (E) in murine colonic mucosa after 7 d pi with C. rodentium. (F) Bar graph of fold change in fecal shedding of C. rodentium per gram of feces at 7 d pi. Data are shown as means ± SEM (n=4/group). P < 0.05 (One-way ANOVA post hoc Bonferroni correction for multiple group comparison or two-tailed Student’s t -test for two groups) was considered significant.

Article Snippet: After rinsing with ice cold PBS-Tw, slides were incubated with primary Alexa647 tagged-anti-Lymphocyte antigen 6 complex locus G6D (Ly6G) antibody (5 μg/mL; MAB91671; R&D Systems) diluted in PBS (16 h, 4°C).

Techniques: Infection, Staining, Immunofluorescence, Fluorescence, Software, Activity Assay, Expressing, Comparison, Two Tailed Test

Journal: Burns & Trauma

Article Title: Neutrophil-derived heparin binding protein triggers vascular leakage and synergizes with myeloperoxidase at the early stage of severe burns (With video)

doi: 10.1093/burnst/tkab030

Figure Lengend Snippet: Systemic edema, abnormal neutrophil function and increased glycocalyx decomposition products in the early stage of severe burn patients. (a) The volume of fluid resuscitation and urine volume per hour after admission were closely monitored. The data on the first, second, third, fifth and seventh 24 h were selected for analysis. (b) Fifteen patients with TBSA of more than 30% and second- to third-degree burns were included in this study. The blood sample for testing was taken within 24 h of the injury. Each sample has undergone blood routine examination and ELISA test of HBP, MPO, NE and MMP9 to ensure that the data can be analyzed for correlation. n = 15 in each group. (c) Under agarose neutrophil chemotaxis model was used to detect the chemotaxis of neutrophils in different patients. Representative pictures from 15 independent experiments were selected for the present. n = 15. (d) Neutrophils were isolated from whole blood and incubated at 37°C for 30 min with or without 10 nM PMA. Then the ROS fluorescent probe was added and incubated at 37°C for 15 min. The continuous generation of ROS can be terminated by placing the sample into ice. The concentration of neutrophils was maintained at 5 × 10 6 /ml. n = 15. (e) Neutrophils were isolated from whole blood and cultured in a carbon dioxide incubator for 24 h. The percentage of annexin-V positive cells was detected by apoptosis detection kit. n = 12. (f) Each sample has undergone ELISA test of HA, HS and SDC-1 to ensure that the data can be analyzed for correlation. n = 9 in each group. All samples in are from healthy volunteers and burn patients. Data are mean ± SD; independent sample t -test was used to prove the difference between two groups. * * * * p < 0.0001, * * * p < 0.001, * * p < 0.01, * p < 0.05, compared with control group. fmlp formyl-methionyl-leucyl-phenylalanine, HA hyaluronic acid, HBP heparin binding protein, HS heparan sulfate, LPS lipopolysaccharide, MMP9 matrix metalloprotein-9, MPO myeloperoxidase, PMN polymorphonuclear leukocyte, SDC-1 syndecan-1

Article Snippet: The following kits were used: human HBP ELISA kit (Thermo Fisher, USA), human MPO ELISA Kit (R&D Systems, USA), human elastase ELISA kit (R&D Systems, USA), human MMP9 ELISA kit (R&D Systems, USA), human hyaluronic acid ELISA kit (Biorbyt, UK), human heparan sulfate ELISA kit (Biorbyt, UK), human SDC-1 ELISA kit (Biorbyt, UK), rat HA ELISA kit (Cusabio, China), rat HS ELISA kit (Cusabio, China), and rat SDC-1 ELISA kit (Cusabio, China).

Techniques: Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Isolation, Incubation, Concentration Assay, Cell Culture, Control, Binding Assay

Journal: The European respiratory journal

Article Title: SPLUNC1: A Novel Marker of Cystic Fibrosis Exacerbations

doi: 10.1183/13993003.00507-2020

Figure Lengend Snippet: A) SPLUNC1 densitometry showing degradation by human neutrophil elastase (NE) relative to PBS control, at specified concentrations over 3 hours at 37°C. B) SPLUNC1 densitometry showing degradation by Elastase B (LasB) from P. aeruginosa relative to PBS control at specified concentrations over 8 hours at 37°C. C) NE densitometry in sputum from healthy controls (HC), Stable CF patients (CF Stable), and AE patients (AE) assessed by WB. D) Representative WB showing endogenous expression of SPLUNC1 (20–25 kD) and NE (25–30 kD) in HC and CF sputum samples from the same individuals. Membranes were probed for NE prior to stripping and re-probing for SPLUNC1. E) NE Activity in CF sputum: AMC formation from florigenic NE substrate MAA-3133 following 6 h incubation with HC, CF stable, and AE sputum at 37°C. For experiments in A and B: n = 4–5, 2 individual experiments; Mann-Whitney Test; + = Mean; Bar inside box: Median; Whiskers: Minimum/Maximum; * p < 0.05; ** = p < 0.01; *** = p < 0.005; ns: not statistically significant; HC: Healthy Control; ST: Stable CF; AE: CF exacerbation; m: marker; +ctl: recombinant protein positive control; OD: optic density.

Article Snippet: Western Blot Western blots were performed as previously reported, using human neutrophil elastase (hELA2), mouse monoclonal anti-hELA2 IgG (R&D systems, cat# MAB-91671-100) and mouse polyclonal anti-human SPLUNC1 IgG 28 .

Techniques: Control, Expressing, Stripping Membranes, Activity Assay, Incubation, MANN-WHITNEY, Marker, Recombinant, Positive Control

Journal: Archives of Rheumatology

Article Title: Alarming serum antiprotease levels in axial spondyloarthritis

doi: 10.46497/ArchRheumatol.2024.10466

Figure Lengend Snippet: Comparison of demographic, clinical, and laboratory characteristics of patients with AxSpA and healthy controls

Article Snippet: Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used for measuring serum elafin and SLPI levels (Human Elafin ELISA Kit, catalog no: E4762Hu; Human Secretory Leukocyte Protease Inhibitor ELISA Kit, catalog no: E0880Hu; BT LAB Bioassay Technology Laboratory, Shanghai, China).

Techniques: Comparison, Standard Deviation, Protease Inhibitor, Activity Assay, Functional Assay