complement factor c5a anaphylatoxin  (R&D Systems)

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