Fig. 3: Yellow-related salivary proteins act as chemoattractants for neutrophils in vivo.

a–d Flow cytometric analysis of neutrophil (Ly6G^high/CD11b⁺/Ly6C^low/−) recruitment in C57BL/6 mouse ears. Mice ears were injected with HBSS as negative control (Ctrl) or with a rPduM10 (0.25 μM), b rPduM35 (0.25 μM), c rPduM34, rPduM35 or rPduM10 (0.25 μM). Neutrophil recruitment was analyzed at different time points as shown in (a, b) or at 12 h for (c). d Mice ears were injected with salivary gland homogenate (SGH) treated or not treated with antibodies against PduM10 or PduM35. Neutrophil recruitment was analyzed after 12 h. e, f Intravital multiphoton imaging of neutrophil migration in eGFP-LysM C57BL/6 mouse ears. Ears were injected with HBSS as a negative control, rPduM10 + rPduM35 (0.25 μM of each) or SGH (0.5 pair). Neutrophil migration was recorded overtime. e Still images from a representative experiment show neutrophil migration (green) at different time points. f Neutrophil accumulation overtime is shown as GFP MFI. g Ribbon depictions of predicted three dimensional structures of rPduM10 (light blue) and rPduM35 (magenta) obtained by modeling using I-TASSER, compared with the structures of C5a (cyan, https://doi.org/10.2210/pdb4P3A/pdb) determined by X-ray crystallography, CXCL1 (blue and magenta, https://doi.org/10.2210/pdb1MGS/pdb) determined by NMR and CXCL8 determined by X-ray crystallography (green and cyan, https://doi.org/10.2210/pdb1IL8/pdb). Cumulative results of two (a–d) or three (e, f) independent experiments are presented as means ± SEM; a n = 13, 14, 16, 18, 8, 9, 10, 10, 10, 10; *P = 0.0136, *P = 0.0360, **P < 0.0089. b n = 11, 13, 10, 12, 10, 10, 8, 4, 4; *P = 0.0237, **P = 0.0021. c n = 12, 10, 9, 5; *P = 0.0120, ***P = 0.0009. d n = 6, 8, 10; **P = 0.0030, **P = 0.0011. f n = 3, 4, 3. *P < 0.0462, **P < 0.0223, calculated by a, b two-tailed Mann–Whitney test, c, d one-way ANOVA and f two-way ANOVA test.