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: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Excluded variables of multivariate logistic regression analyses.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques:

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: NDRG2 reshapes the pathological structure of astrocytes by inhibiting NF-κB/C3 signaling. (a) GSEA of proteome indicated complement and coagulation cascade signaling were upregulated in diabetic mice (∗∗∗ p = 0.0002 between vehicle and STZ) and downregulated after exercise (∗∗ p = 0.0035 between STZ and STZ + Run). The cumulative enrichment scores were normalized (NES) ( n = 3/group). (b) Heatmaps showing the fold changes of significantly altered proteins in complement and coagulation cascades ( n = 3/group). (c – d) Representative immunofluorescent staining of intact C3 and its cleaved products in the hippocampus. Red: C3, green: GFAP, blue: DAPI. Scale bar, 50 μm. (∗∗∗ p = 0.0006 between vehicle and STZ, ∗∗∗ p = 0.0006 between STZ and STZ + Run) (e – f) Representative immunoblots of complement C3 in the hippocampus were increased in the STZ group compared to vehicle mice (∗∗∗∗ p < 0.0001 between vehicle and STZ), which was downregulated after exercise ( n = 6/group) (∗∗∗ p = 0.0008 between STZ and STZ + Run). (g – k) Representative immunoblots of astrocytic NDRG2 ( g, i ), complement C3 ( g, h ), NF-κB, and p–NF–κB ( j, k ) after NDRG2 loss of function ( n = 6/group) (∗ p = 0.0120 (h) , ∗∗∗∗ p < 0.0001 (i) , ∗∗ p = 0.0012 (k) between STZ + Run + acNDRG2 KO and STZ + Run + control). (l – n) Representative immunoblots of astrocytic NDRG2 ( l ), complement C3 ( m ), NF-κB, and p–NF–κB ( n ) in the hippocampus after NDRG2 gain of function ( n = 6/group) (∗∗ p = 0.0064 ( l ), ∗∗∗ p = 0.0001 ( m ), ∗∗ p = 0.0029 ( n ) between vehicle + AAV-Ctrl and STZ + AAV-Ctrl) (∗∗∗∗ p < 0.0001 ( l ), ∗ p = 0.0264 (m) , ∗∗ p = 0.0040 (n) between STZ + AAV-NDRG2 and STZ + AAV-Ctrl). Data are presented as mean ± SEM. GSEA analysis was performed in a . One-way ANOVA with Tukey's multiple comparisons test was performed in d, f, l–n . Two-tailed Student's t-test was performed in h, i, k .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Coagulation, Staining, Western Blot, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: C3aR blockade rescues dendritic spine loss in diabetic mice, and C3 may be a biomarker to predict the progression of DACD in humans. (a) Schematic representing chronological order of STZ injection, C3aR antagonist, and behavioral testing. We used C3aR antagonists to clarify whether C3aR blockade could mimic the protective effect of NDRG2 overexpression on DACD. (b) Y-maze alternation triplet (%) was improved in the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗∗∗ p = 0.0006 between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 between STZ + C3aRA and STZ + PBS). (c – d) Y-maze total distance ( c ) and total arm entries ( d ) ( n = 7/group, n = 8 vehicle + PBS). (e) Escape latency of MWM test was shorten at the STZ + C3aRA group compared with STZ + PBS mice ( n = 7/group, n = 8 vehicle + PBS) (∗ p = 0.0211 (day 1) , ∗ p = 0.0336 (day 2) , ∗∗ p = 0.0099 (day 3) , ∗∗ p = 0.0033 (day 4) between vehicle + PBS and STZ + PBS) ( ## p = 0.0021 (day 1) , # p = 0.0392 (day 3) between STZ + C3aRA and STZ + PBS). (f) Platform crossover of MWM test was upregulated at the STZ + C3aRA mice compared to STZ + PBS group ( n = 7/group, n = 8 vehicle + PBS) (∗∗ p = 0.0025 between vehicle + PBS and STZ + PBS) (∗∗ p = 0.0066 between STZ + C3aRA and STZ + PBS). (g) Representative images of 3D reconstruction of dendritic spines. Scale bar, 5 μm. (h – l) The densities of total spines ( h ), stubby spines ( i ), mushroom spines ( j ), long thin spines ( k ), and filopodia spines ( l ) ( n = 20/group) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0008 ( i ), ∗ p = 0.0191 ( j ), ∗∗∗ p = 0.0006 ( k ) between vehicle + PBS and STZ + PBS) (∗∗∗∗ p < 0.0001 ( h ), ∗∗∗ p = 0.0005 ( i ), ∗ p = 0.0133 ( j ), ∗ p = 0.0153 ( l ) between STZ + C3aRA and STZ + PBS). (m) The DSST scores of diabetic patients ( n = 27) and non-diabetic peers ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (n) Fasting plasma glucose levels for diabetic patients ( n = 27) compared to non-diabetic patients ( n = 13) (∗∗∗∗ p < 0.0001 between Normal and Diabetes). (o) The serum levels of complement C3 in diabetic patients ( n = 27) and normal peers ( n = 13) (∗∗ p = 0.0072 between Normal and Diabetes). (p) The correlations between DSST score and increased C3 levels in diabetic patients ( n = 27) and normal peers ( n = 13). Correlations were found using linear regression, with r = −0.3315 and p = 0.0366. Values presented as mean ± SEM. Two-way ANOVA with Tukey's multiple comparisons test was performed in e . One-way ANOVA with Tukey's multiple comparisons test was performed in b, f–l . Two-tailed Student's t-test was performed in m–o .

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: Biomarker Discovery, Injection, Over Expression, Clinical Proteomics, Two Tailed Test

Journal: eBioMedicine

Article Title: Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction

doi: 10.1016/j.ebiom.2023.104653

Figure Lengend Snippet: Characteristics of the study population.

Article Snippet: The Human C3 (Complement Component 3) ELISA Kit (E-EL-H6054) and Human INS (Insulin) ELISA Kit (E-EL-H2665c) were purchased from Elabscience, Wuhan, China.

Techniques: