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
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![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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2905/pmc13012905/pmc13012905__fimmu-17-1795719-g004.jpg)
![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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2905/pmc13012905/pmc13012905__fimmu-17-1795719-g006.jpg)
