Zymosan

Adult male Balb/c mice (aged 6–8 weeks, PBS n = 5; vehicle n = 8; UCB-9260 10, 30, 100 and 300 mg/kg n = 8; Ab501 n = 3 mice, from Charles River UK) were dosed intravenously with 30 mg/kg of Ab501 (in-house anti-mouse TNF antibody) diluted in PBS 1 h prior to zymosan challenge. Vehicle (30% cyclodextrin) or UCB-9260 at 10, 30, 100 and 300 mg/kg was dosed orally at the time of zymosan challenge. zymosan (R&D Systems) was administered by intraperitoneal injection at a concentration of 1 µg per mouse in PBS (100 µL). One hour post oral administration of UCB-9260, mice were bled via the tail to analyse exposure levels of the compound. Four hours after the zymosan challenge, mice were sacrificed and the peritoneal cavity lavaged with 3 mL of FACS buffer. An aliquot (0.3 mL) of lavage fluid was used to assess the number of CD45⁺GR1⁺ cells by flow cytometry. Briefly, the cells were treated with ACK lysis buffer, washed and suspended in FACS buffer prior to staining with FITC anti-mouse CD45 and PE anti-mouse GR1 (BD Biosciences). Data are shown as mean ± standard error of the number of CD45⁺GR-1⁺ cells. Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001) was determined using one-way ANOVA with Dunnett’s multiple comparison test.

Male C57BL/6N mice (6–8 weeks) were purchased from Janvier (Le Genest, France) and treated according to the International Association for the Study of Pain guidelines. For all experiments the ethics guidelines for investigations in conscious animals were obeyed and the procedures were approved by the local ethics committee (Regierungspräsidium Darmstadt). The animals had free access to food and water and were maintained in climate‐ (23 ± 0.5°C) and light‐controlled rooms (light from 6.00 a.m. to 6.00 p.m.).
Inflammation was induced by injection of 10 μl zymosan (3 mg/ml in PBS; Merck, Darmstadt, Germany) subcutaneously into the plantar side of one hind paw. Eosinophil depletion was achieved by intraperitoneal (i.p.) injection of anti‐Siglec F antibody (0.883 mg/kg; clone 238,047; R&D Systems, Minneapolis, MN) 24 h prior zymosan injection. As control purified rat IgG2a (Biolegend, San Diego, USA) was used. IL‐4c was prepared using IL‐4 (Peprotech, Hamburg, Germany) and anti‐IL4 antibody (Biolegend, San Diego, USA) (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)) and administered i.p. (0.166 mg/kg IL‐4 and 0.883 mg/kg anti‐IL4 antibody) 24 h prior zymosan injection. (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)).