formyl peptide receptor 2 fpr2 antagonist wrw4  (MedChemExpress)

Journal: Frontiers in Immunology

Article Title: Fumarate inhibits the formation of neutrophil extracellular traps (NETs) in a Nrf2-controlled and Annexin-A1-dependent manner associated with mitochondrial fusion

doi: 10.3389/fimmu.2026.1770063

Figure Lengend Snippet: Expression of Nrf2 and ANXA1 in relation to NET release by neutrophils treated with dimethyl fumarate. Neutrophils isolated from mice were either pre-treated with DMF (25 µM), ML385 (Nrf2 inhibitor), WRW4 (Fpr2 antagonist) for 1 h or their combination: DMF→ML385, WRW4→DMF incubated with lipopolysaccharide (LPS) at a concentration of 50 μg/mL (for 6 h) or left unstimulated (CTR). (A) Quantification of Nrf2 expression and (B) NETs (extDNA and citH3) quantification after Nrf2 inhibition. (Ci) Representative images and (Cii) area of NETs formed upon the above treatments with WRW4 and DMF. ExtDNA is shown in green and citH3 is shown in red. To visualize co-localization of extDNA with citH3 expression, the images from each channel were overlaid. (D) Levels of ANXA1 were measured in neutrophils supernatants upon the above treatments with DMF and ML385 by ELISA. The results are expressed as the mean values ± SD; n≥3. Values significantly different between the groups (p < 0.05) according to one-way ANOVA ( post hoc Bonferroni test) are designated by letters, where the same letter indicates no differences between groups (different letters indicate statistical differences). Explanation of abbreviations: Nrf2, nuclear factor erythroid 2-related factor 2; ANXA1, Annexin A1; extDNA, extracellular DNA; citH3, citrullinated histone H3.

Article Snippet: The following inhibitors were used: nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor – ML385 (10 μM, MedChemExpress), formyl peptide receptor 2 (Fpr2) antagonist - WRW4 (10 μM, MedChemExpress), NADPH oxidase (NOX) inhibitor - Diphenyleneiodonium chloride (DPI, 10 μM, Merck), necroptosis inhibitor - Necrostatin-1 (50 μM, Torcis Bioscence, Bristol, UK), pyroptosis inhibitor - Disulfiram (30 μM, MedChemExpress) and mitochondrial complex I inhibitor - Rotenone (5 μM, Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Innate Immunity

Article Title: Antimicrobial peptide LL-37 ameliorates a murine sepsis model via the induction of microvesicle release from neutrophils

doi: 10.1177/1753425920936754

Figure Lengend Snippet: Effect of LL-37 on the release of antibacterial ectosomes from mouse bone-marrow neutrophils, and effect of CXCR2 and FPR2 antagonists on the LL-37-induced ectosome release. (a) Mouse bone-marrow neutrophils (2 × 10 6 cells) were stimulated with N -formyl-Met-Leu-Phe (fMLF) (10 μM) or LL-37 (1, 3, or 10 μg/ml) for 45 min at 37°C, and the released ectosomes in the supernatant were analyzed by flow cytometry. Data are shown as the mean ± SD . Values were compared and analyzed between the incubation with vehicle (PBS) and fMLF (10 μM) or LL-37 (1, 3, or 10 μg/ml), and 1, 3, and 10 μg/ml of LL-37 by one-way ANOVA followed by Holm-Sidak’s multiple comparison test. An inlet shows a representative cytogram of microvesicles released from LL-37 (10 μg/ml)-stimulated neutrophils. (b) Microvesicles (containing 1.5 × 10 5 or 3.0 × 10 5 ectosomes) isolated from LL-37 (10 μg/ml) or fMLF (10 μM)-stimulated neutrophils were incubated with E. coli for 20 min at 37°C, and the relative bacteria viability was calculated and expressed as percentage. Data are shown as the mean ± SD ( n = 3 each). Values were compared and analyzed between microvesicles containing 1.5 × 10 5 and 3.0 × 10 5 ectosomes isolated from LL-37-stimulated (LL37-MV) or fMLF-stimulated neutrophils (fMLF-MV) by two-tailed unpaired Student’s t -test. (c) Microvesicles (1.6 μg) isolated from the supernatant of vehicle (Hank’s balanced salt solution; HBSS + ) or LL-37 (10 μg/ml) stimulated neutrophils were subjected to 8% or 13% SDS-PAGE, followed by Western blotting using anti-lactoferrin and CRAMP Abs. (d) Mouse bone-marrow neutrophils (2 × 10 6 cells) were pre-treated with SB225002 (CXCR2 antagonist; 3 μM), WRW4 (FPR2 antagonist; 0.3 μM), or vehicle (DMSO) for 20 min at 37°C, and then stimulated with LL-37 (10 μg/ml) for 45 min at 37°C. Released ectosomes in the supernatant were analyzed by flow cytometry. Values were compared and analyzed between the incubation with vehicle (DMSO) ( n = 6) and LL-37 without antagonists ( n = 8), and between the incubation with LL-37 alone and LL-37 with an antagonist (SB225002 or WRW4) ( n = 7 each) by one-way ANOVA followed by Holm-Sidak’s multiple comparison test. * P < 0.05, ** P < 0.01.

Article Snippet: WRW4 (formyl peptide receptor 2 (FPR2, a mouse ortholog of FPRL1) antagonist), normal rabbit IgG, 4% paraformaldehyde (PFA) phosphate buffer solution, and isoflurane were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan).

Techniques: Flow Cytometry, Incubation, Comparison, Isolation, Bacteria, Two Tailed Test, SDS Page, Western Blot