murine recombinant c5a  (R&D Systems)

Journal: Molecular Genetics and Metabolism Reports

Article Title: Empagliflozin in GSD-Ib: Long-term safety and sustained recovery of neutrophil function including NET formation

doi: 10.1016/j.ymgmr.2026.101345

Figure Lengend Snippet: Empagliflozin restores neutrophil function in a GSD-Ib infant (P1) by lowering 1,5-AG and enhancing cell survival, NET formation, ROS activity, and bacterial killing. Highly purified human blood neutrophils were isolated from healthy donors and from P1, both before and after empagliflozin (empa) treatment. If required for the experiments, cells were primed with GM-CSF for 20 min and subsequently activated with 10 −8 M C5a, or stimulated with 25 nM PMA without prior priming. (A) Neutrophil viability. Cell death was assessed by propidium iodide (PI) uptake at the indicated time points ( n = 7). (B) Plasma concentrations of 1,5-AG were measured using liquid chromatography mass spectrometry (LC-MS). Box plots indicate the 5th–95th percentile. Differences between groups were assessed using the Mann-Whitney U test. (C) NET formation was assessed by confocal microscopy. Extracellular DNA fibers were stained with MitoSOX Red, and nuclei were counterstained with Hoechst 33342, followed by analysis using confocal microscopy ( n = 8). Scale bars, 10 μm. (D) Cells releasing DNA fibers were quantified in 10 randomly selected images for each condition (n = 8). (E) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye ( n = 5). (F) Total ROS activity was measured by flow cytometry (n = 8). (G) Bacterial killing of opsonized GFP- E.coli was assessed by flow cytometry. After incubation, supernatants were collected, and the reduction in viable bacteria was determined (n = 7). All data are presented as means ± SEM, with individual measurements depicted as dots. Difference between groups were assessed using unpaired t -test, unless stated otherwise. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. 1,5-AG, 1,5-anhydroglucitol.

Article Snippet: Human C5a was purchased from Hycult Biotech (Uden, The Netherlands).

Techniques: Activity Assay, Purification, Isolation, Clinical Proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY, Confocal Microscopy, Staining, Flow Cytometry, Incubation, Bacteria

Journal: Molecular Genetics and Metabolism Reports

Article Title: Empagliflozin in GSD-Ib: Long-term safety and sustained recovery of neutrophil function including NET formation

doi: 10.1016/j.ymgmr.2026.101345

Figure Lengend Snippet: Marked neutrophil dysfunction and elevated cell death in an untreated GSD-Ib patient (P2). Highly purified human blood neutrophils were isolated from healthy donors and from P2, who was not receiving empagliflozin treatment. When required for the experiments, cells were primed with GM-CSF for 20 min and subsequently activated with 10 −8 M C5a, or stimulated with 25 nM PMA without prior priming. Experimental conditions and stimulation protocols were as described in . (A) Neutrophil viability assessed by propidium iodide uptake. (B) Plasma 1,5-AG concentrations measured by LC-MS. (C) Representative confocal microscopy images of NET formation. Scale bars, 10 μm. (D) Quantification of DNA-releasing neutrophils. (E) Quantification of dsDNA release in culture supernatants. (F) Total ROS activity measured by flow cytometry. (G) Bacterial killing of opsonized GFP- E. coli assessed by flow cytometry. Data are presented as means ± SEM, with individual measurements depicted as dots. Difference between groups were assessed using unpaired t -test, unless stated otherwise. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. 1,5-AG, 1,5-anhydroglucitol.

Article Snippet: Human C5a was purchased from Hycult Biotech (Uden, The Netherlands).

Techniques: Purification, Isolation, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Activity Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: TNF primes neutrophils and an allosteric FFA2R modulator turns C5a into a more potent activator of the NADPH oxidase. For priming, neutrophils were incubated with TNF (10 ng/mL) at 37 °C for 20 min and then stored on ice until used. The NADPH oxidase activity (O 2 - production) induced by C5a was measured continuously and expressed in mega couts per minute (Mcpm). (A) The response induced in naïve (non-primed; dashed line) and TNF primed neutrophils (solid line) by C5a (2 nM, added at the time point marked with an arrow) was measured. Inset: The priming effect of TNF on the NADPH oxidase activity expressed as peak values (Mcpm; mean ± SEM) of O 2 - production induced by C5a in naïve (n = 5) and TNF-primed neutrophils (n = 14). (B) Inhibition of the C5a-induced response by the specific C5aR1 antagonist avacopan. TNF-primed neutrophils were incubated without (solid line) or with avacopan (50 nM, dashed line) for 5 min before addition of C5a (2 nM; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The inhibitory effect of avacopan on the neutrophil NADPH oxidase activity expressed as peak values (Mcpm) of O 2 - production induced by C5a in absence and presence of the antagonist, respectively (mean ± SEM, n = 6). (C) Effects of the allosteric FFA2R modulator Cmp58 on the response induced by different concentrations of C5a in TNF-primed neutrophils. Neutrophils were incubated without (dashed lines) or with Cmp58 (1 µM, solid lines) for 5 min before addition of C5a (0.1 nM, grey lines, or 2 nM, black lines; added at the time point marked with an arrow) and determination of the NADPH oxidase activity. Inset: The effect of Cmp58 on the NADPH oxidase activity expressed as fold increase of O 2 - production induced by different C5a concentrations (0.1, 0.25 or 2 nM) in the absence and presence of Cmp58, respectively (mean ± SEM, n = 6). The horizontal dotted line in the bar graph represents a ratio of 1. (D) TNF-primed neutrophils were pre-incubated with and without Cmp58 (1 µM) for 5 min and activated with different concentrations of C5a as indicated. Superoxide production was recorded continuously and expressed as the peak value obtained in terms of percent of the activity induced by C5a alone (2 nM, mean ± SEM, n = 3).One representative experiment is shown in each subset (A–C) . Statistically significant differences in the insets were evaluated by an unpaired Student’s t -test (A) , a paired Student’s t -test (B) , or a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) and are denoted as *( p ≤ 0.05), ***( p ≤ 0.001), and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation, Activity Assay, Inhibition, Comparison

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Antagonists selective for FFA2R and C5aR1, respectively, have inhibiting profiles beyond the expected receptor specificity. TNF-primed neutrophils were incubated with Cmp58 (1 µM; 5 min at 37 °C) and to determine the inhibitory effects of receptor specific antagonist, these cells were incubated without or with an antagonist (CATPB, 100 nM; avacopan, 50 nM) and the O 2 - production was measured continuously following an activation by different concentrations of C5a. (A-C) Inhibition by the antagonists (CATPB and avacopan) of the response in neutrophils incubated with Cmp58 and induced by C5a, at different concentrations, i.e., 2 nM (A; n = 3-8), 0.25 nM (B; n = 7), and 0.1 nM (C; n = 6), respectively. Inhibition is expressed as the remaining activity (peak value in percent) of neutrophils after activation with C5a in the presence of the respective antagonist. Bar graphs are presented as mean ± SEM. The statistical determinations are based on the difference between the C5a response without and with the antagonists. Statistically significant differences were evaluated by a repeated measures one-way ANOVA followed by Šίdák’s multiple comparison (C) or by a mixed effects analysis followed by Šίdák’s multiple comparison (A, B) and are denoted as *( p ≤ 0.05), **( p ≤ 0.01), ***( p ≤ 0.001), and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation, Activation Assay, Inhibition, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Differential NADPH oxidase activity triggered by Cmp58 in neutrophils pretreated with C5a or propionate. (A) TNF-primed neutrophils pre-incubated with Cmp58 (1 µM, 5 min at 37 °C) were activated by C5a (0.1 nM, solid line) or propionate (25 µM, dashed line). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by the ligands C5a and propionate, in neutrophils pre-incubated (abbreviation Pre-inc.) with Cmp58, expressed as the peak values of O 2 - production (Mcpm; mean ± SEM, n = 5). (B) TNF-primed neutrophils incubated for 5 min at 37 °C with non-activating concentrations of C5a (solid line; 0.1 nM) or propionate (dashed line; 25 µM) were activated by Cmp58 (1 µM). The result obtained in one representative experiment is shown. Inset: The NADPH oxidase induced by Cmp58, in neutrophils pre-incubated (abbreviation Pre-inc.) with C5a and propionate, respectively, expressed as the peak values of O 2 - production (mean ± SEM, n = 5). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05) and ns, not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Activity Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: C5a triggers a transient increase in the cytosolic concentration of free calcium ions ([Ca 2+ ] i ) in neutrophils, which is not affected by the allosteric FFA2R modulator Cmp58. Fura-2 loaded neutrophils were activated by propionate (25 µM) or different concentrations of C5a (2, 0.25, 0.1, and 0.05 nM) in the absence (upper panel) or presence (lower panel) of the allosteric FFA2R modulator Cmp58 (1 µM, pre-incubated with the cells for 10 min at 37 °C prior addition of the agonist). Results obtained are shown as one representative experiment out of 3. The arrows show the time point for the addition of the agonists to the Fura-2 labelled neutrophils.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Concentration Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: Activation of FFA2R inhibits (heterologously desensitizes) the neutrophil response induced by C5a. TNF-primed neutrophils incubated with Cmp58 (1 µM), were either left in a resting state (dashed lines) or activated by an FFA2R activating/transactivating ligand (solid lines) and the O 2 - production was measured continuously and expressed in Mcpm. When the response induced by the FFA2R activating agonist was terminated, the two cell samples were activated by an addition of C5a (2 nM). The results obtained in one representative experiment is shown together with an inset showing the peak activities induced by C5a (mean ± SEM, n = 3). (A) Propionate (25 µM), (B) ATP (25µM), and (C) AZ1729 (1µM) were used as the FFA2R activating ligand (the time point for addition is marked by arrow 1). The time point for addition of C5a to the cells is marked by arrow 2. Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as *( p ≤ 0.05).

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Activation Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: C5a-induced desensitization in Cmp58-sensitized neutrophils. (A–C) TNF-primed neutrophils incubated with Cmp58 (1 µM, 5 min at 37 °C) were either left in a resting state (dashed lines) or activated by C5a (2 nM, solid line; the time point for addition is marked by arrow 1), and the O 2 - production was measured continuously and expressed as Mcpm. When the response, induced by the C5aR1 activating ligand was terminated, the two cell samples were activated by the addition of (A) propionate (25 µM, an FFA2R activating ligand), (B) ATP (50 µM, a P2Y 2 R activating ligand), or (C) AZ1729 (1 µM, an FFA2R activating ligand), and the time point for their addition is marked by arrow 2 (D–F) . The experimental setup differs from that described above, in that Cmp58 (1 µM), was added first when the response induced by C5a (time for addition marked by arrow 1), had settled. The neutrophils were then activated with either propionate [ (D) ; 25 µM)], ATP [ (E) ; 50 µM)], or AZ1729 [ (F) ; 1 µM)]. The time point for addition of the FFA2R activating/transactivating ligand is marked by arrow 2, and one representative experiment is depicted. The results obtained are shown together with an inset showing the peak activities induced by the FFA2R activating/transactivating ligands (mean ± SEM, n = 3). Statistically significant differences in the insets were evaluated by a paired Student’s t -test and are denoted as * ( p ≤ 0.05) and ns = not significant.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Incubation

Journal: Frontiers in Immunology

Article Title: The free fatty acid 2 receptor regulates C5a induced neutrophil superoxide production and its sensitivity to inhibition by the antagonist avacopan

doi: 10.3389/fimmu.2026.1795719

Figure Lengend Snippet: A proposed model for how the neutrophil NADPH oxidase is turned to an active state by the C5aR1 agonist C5a in the absence and presence of the allosteric FFA2R modulator Cmp58. (A) Left: The neutrophil response induced by a 2 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating NADPH oxidase and have also the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 directly activate the O 2 -- generating oxidase and transactivate the allosterically modulated FFA2R; the transactivated FFA2R does not directly potentiate the NADPH oxidase response but reduces the inhibitory effect on this response, of the C5aR1 specific antagonist avacopan. (B) Left: The neutrophil response induced by a 0.1 nM concentration of C5a. In the absence of Cmp58, the signals generated by the activated C5aR1 have no NADPH oxidase activating effect. The signals have, however, the capacity to transactivate FFA2R, but the naïve fatty acid receptor is not receptive to these signals. Right: In the presence of Cmp58, the signals generated by the activated C5aR1 activate the O 2 -- generating NADPH oxidase, and this activation is totally dependent of the signals generated by the allosterically modulated FFA2R, a receptor made receptive to the transactivating signals generated by C5aR1.

Article Snippet: Recombinant human C5a (rhC5a) and AZ1729 was purchased from R&D Systems (Minneapolis, MN, USA) and avacopan was purchased from MedChemExpress (Princeton, NJ, USA).

Techniques: Concentration Assay, Generated, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: P-Rex1 controls phagocytosis and the killing of bacteria by murine neutrophils independently of its catalytic activity

doi: 10.3389/fimmu.2025.1591006

Figure Lengend Snippet: P-Rex1 mediates the killing of S. aureus by neutrophils independently of its catalytic Rac-GEF activity, whereas chemotaxis, ROS, and NETs require its Rac-GEF activity. (A) Bactericidal activity. Purified neutrophils from Prex1 –/– (red squares), Prex1 GD (green triangles), and wild type mice (grey circles) were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before incubation with serum-opsonized S. aureus for 90 min at a ratio of 1.5 bacteria per neutrophil. Heat-killed neutrophils were used as negative controls. Surviving bacteria were grown overnight and CFU enumerated. The % killing of bacteria by live neutrophils compared to heat-killed controls is plotted. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are one-way ANOVA with Tukey’s multiple comparisons test on log-transformed raw data; black p-values are significant, grey p-values non-significant. (B) Chemotaxis. Bone marrow cells from Prex1 –/– , Prex1 GD , and wild type mice were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before being stimulated with 3 nM C5a in transwell filters for 40 min, or mock stimulated. Transmigrated cells were analyzed by flow cytometry in parallel to control cells, using Ly6G hi /Mac1 hi staining to identify neutrophils. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw data. (C) fMLP-stimulated ROS production. Purified neutrophils as in (A) were primed with 1 μg/ml LPS for 90 min and then stimulated with 3 µM fMLP (filled symbols), or mock-stimulated (open symbols). ROS production was measured by real-time chemiluminescence assay with luminol and HRP for extra- and intracellular ROS. Left-hand panel shows luminometer traces from one representative experiment; right-hand panel shows the quantification as AUC over 2 min. Data are mean ± SEM of 3–5 independent experiments; each symbol represents the mean AUC from one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on log-transformed raw data. (D) S. aureus -stimulated intracellular ROS. Neutrophils were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min in the presence of 50 units/ml SOD and 2000 units/ml catalase to scavenge extracellular ROS and were then stimulated with S. aureus at a ratio of 10 bacteria per neutrophil (filled symbols), or mock-stimulated (open symbols). ROS production was measured as in (C) except without HRP and in the presence of SOD and catalase, and quantification was done over 60 min. Data are mean ± SEM of 4 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons tests. (E) Formation of NETs. Neutrophils were seeded onto glass slides and allowed to adhere for 30 min before stimulation with serum-opsonised S. aureus at a ratio of 10 bacteria per neutrophil (closed symbols), or mock stimulation (open symbols). Non-cell permeable Sytox Green and cell-permeable Hoechst 33342 DNA dyes were added to samples 15 min before the end of the incubation, and cells were live-imaged by wide-field microscopy. Left-hand panel shows representative images from one experiment after 120 min stimulation or mock stimulation. Insets are magnifications of the indicated areas. Red arrows highlight NETs, white arrows dead cells without NETs. Right-hand panel shows quantification of NETs by ImageJ. Data are mean ± SEM of 3–4 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on raw data; significant p-values between and Prex1 –/– and wild type are indicated in red, and between Prex1 GD and wild type in green. For all panels, closed symbols show stimulated cells, open symbols mock-treated cells.

Article Snippet: Cells were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min at 37°C, pipetted into transwell filters (400 μl/filter) in a 24-well plate containing HBSS ++++ (600 μl/well) in the presence or absence of 3 nM complement factor C5a anaphylatoxin (C5a, R&D Systems, 2037-C5-025), and incubated for 40 min at 37°C.

Techniques: Activity Assay, Chemotaxis Assay, Purification, Incubation, Bacteria, Transformation Assay, Flow Cytometry, Control, Staining, Chemiluminescence Immunoassay, Microscopy