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Clinical Trial
. 2019 Jul;144(1):342-345.e7.
doi: 10.1016/j.jaci.2019.04.003. Epub 2019 Apr 16.

Induction of innate cytokine responses by respiratory mucosal challenge with R848 in zebrafish, mice, and humans

Fränze Progatzky  1 , Akhilesh Jha  2 , Madina Wane  1 , Ryan S Thwaites  2 , Spyridon Makris  2 , Robin J Shattock  3 , Cecilia Johansson  2 , Peter J Openshaw  2 , Laurence Bugeon  1 , Trevor T Hansel  4 , Margaret J Dallman  5
Affiliations
Clinical Trial

Induction of innate cytokine responses by respiratory mucosal challenge with R848 in zebrafish, mice, and humans

Fränze Progatzky et al. J Allergy Clin Immunol. 2019 Jul.
No abstract available

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Figures

Fig 1
Fig 1
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.
Fig 2
Fig 2
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.
Fig E1
Fig E1
Kinetic immune response of nasal mediators induced by R848 nasal challenge in individual subjects. A-I, Immune response of individual subjects (n = 9) after saline (left panels) and R848 (middle panels) challenge. Of interest, the 3 volunteers with atopy had relatively enhanced type 1 interferon responses, although this study was not specifically powered to address this question. Grouped data (right panels) are displayed as geometric means and 95% CIs. Blue line, Saline; red line, R848. Paired t test on AUC of log10-transformed values between 0 and 8 hours in individual subjects after nasal challenge was used. IFN-β values: n = 7. A, Volunteer with allergic rhinitis; H, healthy.
Fig E2
Fig E2
Kinetic profile of mucosal proinflammatory cytokine responses after zebrafish gill, mouse, and human nasal stimulation with poly(I:C). qRT-PCR analysis of tnfa(A), il1b(B), il6(C), ifnphi1(D), and ifng1.1(E) transcripts in zebrafish gills (n = 4, representative of 2 experiments) after gill treatment. Values are presented as means ± SEMs. Two-way ANOVA followed by the Sidak multiple comparison test was used. qRT-PCR analysis of Tnfa(F), Il1b(G), Il6(H), Ifna2(I), and Ifng(J) transcripts in mouse nasal mucosa (n = 4-10 pooled from 2 independent experiments) sampled with SAM after intranasal stimulation. Values are presented as means ± SEMs. Two-way ANOVA followed by the Sidak multiple comparison test was used. Soluble protein mediator analysis of TNF-α (K), IL-1β (L), IL-6 (M), IFN-α2α (N), and IFN-γ (O) in human nasal mucosal lining fluid (n = 8), as measured by using the nasosorption technique after challenge. Values are presented as geometric means and 95% CIs, and AUC analyses were used. *P < .05 and ***P < .001.
Fig E3
Fig E3
Gillsorption as a noninvasive sampling tool to monitor mucosal proinflammatory cytokine and interferon responses after zebrafish gill stimulation with R848. A-D, qRT-PCR analysis of adult zebrafish gills sampled with SAM 1 hour after gill treatment with water (control, blue bars) or R848 (0.5 mg/mL, red bars) for 5 minutes. Dot plots show relative expression values obtained for individual fish (n = 8), which were normalized to 18S and expressed as fold change relative to the median control sample. Values are presented as means ± SEMs. *P < .05 and ***P < .001, Mann-Whitney test. E, Representative brightfield image of SAM corresponding to Fig E3, J. F-J, Representative images (maximum projection of confocal z-stack) of cells absorbed by SAM after application on gill tissue of live WT zebrafish stained with an L-plastin antibody (red; Fig E3, F) or the transgenic zebrafish Tg(mpx:GFP) (Fig E3, G), Tg(lck:GFP) (Fig E3, H), and Tg(fli:GFP) (Fig E3, I) stained with an anti-GFP antibody (green) or a cytokeratin antibody (red; Fig E3, J). SAM was costained with DRAQ5 (cyan).
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