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. Author manuscript; available in PMC: 2021 Nov 6.
Published in final edited form as: Sci Transl Med. 2020 Sep 9;12(560):eaaz8631. doi: 10.1126/scitranslmed.aaz8631

Fig. 7. R. mucosa activity depends on TLR5 signaling.

Fig. 7.

(A) Area of coverage for cultured human fibroblasts stimulated with diluent, RmHV1, or RmAD1, with or without addition of the lipid fraction of RmHV1 to RmHV3 in the cellular scratch assay. (B) Mean ear thickness for mice treated with R. mucosa isolates from RmHV1 to RmHV3 or RmAD is shown. Each dot represents an individual mouse. (C) Area of coverage in the cellular scratch assay for human keratinocytes stimulated with RmHV1 to RmHV3 after 2 hours of preincubation with either anti-TLR5 (αTLR5) or isotype control antibody. (D) RNA-seq normalized read counts for flagellin expression in R. mucosa isolates are depicted. (E) Area of growth of RmHV and RmAD in the motility agar assay is shown; open circle indicates RmHV3. (F) Area of coverage in the cellular scratch assay for human keratinocytes stimulated with RmHV1 to RmHV3 after 2 hours of preincubation with either anti-TLR2 (αTLR2), anti-TLR4 (αTLR4), or isotype control antibodies. (G) Area of coverage is shown for fibroblasts stimulated overnight with recombinant flagellin before scratch induction in the cellular scratch assay. (H) Area of coverage is shown for fibroblasts stimulated overnight with recombinant flagellin and anti-TNFR1, anti-TNFR2, or isotype control antibodies in the cellular scratch assay. (I) Mean mouse ear thickness in the MC903 mouse model after treatment of wild-type (WT), TLR5−/−, TLR4−/− (C3H), or TLR2−/− mice with MC903. Dots represent individual mice. (J) Representative H&E-stained sections of MC903-treated mouse ears from the WT, TLR5−/−, TLR4−/− (C3H), or TLR2−/− mice shown in (I). Scale bars, indicate 50 μm. (K) Mean mouse ear thickness after treatment of WT or MyD88-deficient (M88; MyD88−/−) mice with MC903. (L) Shown is mean mouse ear thickness in the MC903 mouse model after treatment of WT mice with either R. mucosa or a mixture of 1 nM carbachol (CCh), RmHV lipid extracts (Lipid), or 100 ng per ear of recombinant flagellin (Flag). (M to O) Mean mouse ear thickness in MC903-treated mice (M) and representative images (O) of mouse ears treated with diluent control (Dil) or RmHV2 or RmHV3 with or without 1% lipase (+L) or 5 μM mecamylamine (+M). Dots represent individual mice (B, I, and K to M) or replicate wells (A, C, and F to H). Data are representative of two (D and E and I to K) or three or more independent experiments and displayed as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus enrollment value by paired ANOVA with Dunnett correction.