Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Inhibition of C3b and C4b deposition by r Em CRT and its functional binding regions measured by ELISA. ( A ) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of r Em CRT, r Em CRT-S, or r Em CRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b ( a ) and C4b ( b ) was detected with anti-C3 or C4 monoclonal antibodies. ( B ) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD 450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added r Em CRT).

Article Snippet: After washing with 1 × TBST containing 5 mM CaCl 2 , the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 μL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Microfluidic devices for measuring neutrophil chemotaxis, phagocytosis, and swarming behaviors. (A) Microscopic image of a segment of the chemotaxis microfluidic device (x10 Brightfield, Nikon TiE) showing neutrophil chemotaxis towards C5a and C3a during increasing mechanical restriction. Each chemoattractant chamber is connected to the cell loading channel through 3 tapered channels. (B) Microscopic image of a segment of the phagocytosis microfluidic device (x10 Brightfield/Fluorescent, Nikon TiE) showing neutrophils (blue nuclei) from the outer chamber migrating towards the central reservoir through a migration channel to phagocytose S. aureus particles (green) in plasma at T 20 minutes. (C) Microscopic images obtained at three different time points (T 0 , T 10 , and T 30 minutes) showing the formation of a neutrophil swarm (blue nuclei) over a cluster of Texas red-labeled zymosan A S. cerevisiae bioparticles (Red).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Migration, Clinical Proteomics, Labeling

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Chemotaxis of isolated neutrophils towards C5a and C3a and the effect of AVA on chemotaxis. (A) The percentage of neutrophils migrating directionally towards increasing concentrations of C5a and C3a compared to media alone (HBSS with 0.5%BSA, N=3, ****p < 0.0001; One-way ANOVA with Dunnett’s test) (B) C3a inhibits neutrophil chemotaxis towards C5a, C5a effect is dependent on concentration, and there is no interaction effect between C3 and C5a. (N=3 donors, p < 0.005, Repeated measures, two way ANOVA, with Sidak’s correction for multiple comparisons). (C) Percentage of neutrophils migrating directionally towards C5a (100 nM) after treatment of neutrophils with C5aR1 antagonist AVA (N=3, 4 or 5, each dot on the plot represents one experiment, ****p < 0.01 for comparisons to C5a control; ns represents not statistical significant. One way ANOVA with Dunnett’s multiple comparison test with single pooled variance).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Isolation, Concentration Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Atypical membrane elasticity of neutrophils and migration patterns when exposed to high C5a dose. (A) Percentage of neutrophil migration patterns during chemotaxis towards C5a, C3a, and fMLP. (N=3). (B) Microscopic images of elongated neutrophils when exposed to C5a 1µM inside the end chambers. Scale = 100 µm. (C) Percentages of unique reversed migration phenotype of neutrophils observed when exposed to high C5a concentration. (N=3, *p < 0.05; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Membrane, Migration, Chemotaxis Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Effect of C5 complement inhibitors on neutrophil chemotaxis towards endogenous complement activation with CVF. (A) Effect of AVA (250 nM) on endogenous complement activation by increasing CVF doses (N=3 donors, *p < 0.05, **p < 0.01; paired two-tailed t -test). (B) Effect of C5 inhibition with ECU (3 µM) and RA101295 (3 µM) on endogenous complement activation by CVF (N=3, **p < 0.01; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Activation Assay, Two Tailed Test, Inhibition

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Schematic representation of complement activation pathways, terminal inhibition strategies, and their effects on neutrophil functional behaviors. The classical, lectin, and alternative pathways converge on the cleavage of the central components C3 into C3a and C3b peptides. C3a acts as an anti-inflammatory mediator towards neutrophils, while C3b enhances phagocytosis through opsonization and forms C5 convertases, which cleaves C5 into C5a and C5b. C5a is a potent pro-inflammatory mediator and chemoattractant, while C5b forms the membrane attack complex (MAC), leading to cell and bacteria lysis. ECU and RA101295 are C5-inhibitors, which block the production of C5a and C5b peptides, while AVA blocks C5a mediated chemotaxis and pro-inflammatory effects.

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Activation Assay, Inhibition, Functional Assay, Membrane, Bacteria, Lysis, Blocking Assay, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: (A) NHERF1 and NHERF2 expression was determined in human mast cell lines (HMC-1, LAD2) and CD34 + primary human mast cells (CD34 + MC) by RT-PCR. Transient transfectants were generated in HEK293 cells expressing HA-tagged C3aR or β2 adrenergic receptor (β2-AR) and (B) Flag-tagged NHERF1 or (C) NHERF2. Cells were exposed to buffer or C3a (100 nM, 37°C for 5 min) as indicated, lysed, immunoprecipitated with anti-HA-antibody, resolved by 10% SDS-PAGE, and transferred onto nitrocellulose membrane. Blots were then probed with anti-Flag antibody to detect NHERF binding to the receptor (top panel) or anti-HA antibody to examine receptor expression (middle panel). Western blotting was performed with anti-Flag antibody on the lysate samples (input) to determine NHERF expression levels (bottom panel). A representative blot from three independent experiments is shown.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Immunoprecipitation, SDS Page, Binding Assay, Western Blot

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: (A) shRNA control, (B) NHERF1, and (C) NHERF2 KD HMC-1 cells were loaded with Indo-1(1 µM), stimulated with C3a (100 nM) for 5 min and intracellular Ca 2+ mobilization was determined (solid lines). The cells were washed three times with ice-cold buffer, resuspended in warm buffer and exposed to a second stimulation of C3a (100 nM) and intracellular Ca 2+ mobilization was again determined (broken lines). Representative traces from at least three separate experiments are shown.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: shRNA

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: (A) shRNA control HMC-1 cells (B) NHERF1 knockdown (KD) and (C) NHERF2 KD cells were exposed to buffer or C3a (100 nM) for 5 min. Cells were washed with ice-cold FACS buffer, incubated with a mouse anti-C3aR antibody or an isotype control antibody followed by PE-labeled donkey anti-mouse IgG antibody and analyzed by flow cytometry. Representative histograms plots from an internalization experiment following exposure to buffer (red line) or C3a (blue line) in shRNA control and NHERF KD cells are shown. (D) shRNA control, NHERF1 KD and NHERF2 KD cells were exposed to C3a for different time periods and receptor internalization was determined as described above. Internalization is expressed as the percentage loss of C3aR following exposure to C3a. Data represent the mean ± SEM from three experiments.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: shRNA, Incubation, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: (A) shRNA control and NHERF KD HMC-1 cells were stimulated with C3a (100 nM) for indicated time intervals. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP antibody. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. The blots were also stripped and reprobed with anti-phopho-Akt and anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. A representative immunoblot from three similar experiments is shown. (B) shRNA control and NHERF KD HMC-1 cells were allowed to chemotax to C3a (10 nM, 37°C for 3 h). Data is represented as the total number of chemotaxed cells in the lower chamber. Bar graphs represent the mean ± SEM from three independent experiments.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: shRNA, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: LAD2 or CD34 + primary human mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against NHERF1 or NHERF2. Western blotting was performed to assess NHERF expression levels in shRNA control and NHERF KD (A) LAD2 and (C) CD34 + primary human mast cells. A representative blot from three independent experiments is shown. shRNA control or NHERF KD (B) LAD2 mast cells or (D) CD34 + mast cells were stimulated with indicated concentrations of C3a or CST (100 nM) and percent degranulation (β-hexosaminidase release) was determined. LAD2 cells were also stimulated by NP-specific IgE/NP-BSA (Ag) as a non-GPCR control. Data are mean ± SEM of three experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p<0.001.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: Stable Transfection, Transduction, shRNA, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

doi: 10.1371/journal.pone.0051355

Figure Lengend Snippet: shRNA control, NHERF1 KD or NHERF2 KD HMC-1 cells were transiently transfected with NF-κB luciferase reporter gene construct along with C3aR. (A) Cells were stimulated with C3a (100 nM for 6 hr) and NF-κB-dependent transcriptional activity was determined by luciferase activity assay. Data is presented as relative luciferase activity normalized to Renilla luciferase activity. (B) Control or NHERF KD cells were stimulated with C3a (100 nM for 6 h) and CCL4 production was determined from the supernatant by ELISA. Data shown are mean ± SEM of three experiments performed in triplicate. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p<0.001.

Article Snippet: Purified C3a was obtained from Complement Tech, Inc. (Tyler, TX).

Techniques: shRNA, Transfection, Luciferase, Construct, Activity Assay, Enzyme-linked Immunosorbent Assay