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: bioRxiv

Article Title: SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

doi: 10.64898/2026.04.07.716973

Figure Lengend Snippet: ( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Article Snippet: Additional media with or without recombinant mouse complement component C5a (R&D system, #2150-C5/CF) was added after incubation.

Techniques: Live Cell Imaging, Wound Healing Assay, Invasion Assay, Staining