Kinetic profile of mucosal proinflammatory cytokine responses after zebrafish gill, mouse, and human nasal stimulation with R848. A-C, Schematics showing mucosal administration of TLR agonists and sampling of mucosal tissue/fluids to assess responses. D, G, J, M, and P, qRT-PCR analysis of zebrafish gills (n = 5, representative of 3 experiments). Values are presented as means ± SEMs. Two-way ANOVA followed by the Sidak multiple comparison test was used. E, H, K, N, and Q, qRT-PCR analysis of mouse nasal mucosa (n = 4-10 pooled from 2 independent experiments). Values are presented as means ± SEMs. Two-way ANOVA followed by the Sidak multiple comparison test was used. F, I, L, O, and R, Soluble protein mediator analysis of human nasal samples (n = 9). Values are presented as geometric means and 95% CIs. Paired t tests on log₁₀-transformed area under the curve values were used. *P < .05, **P < .01, and ***P < .001.

R848 induces lymphocyte migration in zebrafish gills. A and C, Maximum z-stack projections of Tg(lyz:GFP; neutrophils; Fig 2, A) and Tg(lck:GFP; lymphocytes; Fig 2, C) gills after treatment with water or R848 for 3 (Fig 2, A) and 8 (Fig 2, C) hours (n = 7). Scale bars = 100 μm. B and D, Average number of GFP⁺ cells in the first 20 lamellae of each filament. Each dot indicates average counts per individual fish (n ≥ 6). Values are presented as means ± SEMs. Two-way ANOVA followed by the Sidak multiple comparison test was used.