Fig. 6. Lipid mediators of potential R. mucosa activity are dependent on cholinergic signaling.

(A) Pathway analysis on all annotated features from metabolomics comparison of RmHV and RmAD as determined by MetaboAnalyst. (B) Total phospholipid and phosphatidylcholine quantitation by enzymatic assay are presented. tRNA, transfer RNA. (C) Shown is coverage area in the cellular scratch assay for fibroblasts treated with an aqueous (Aq) fraction or a lipid fraction extract of R. mucosa from healthy volunteers. Fractions were treated with anti-TNFR1 (αR1), anti-TNFR2 (αR2), or isotype control (Iso) antibodies. (D) Thickness of mouse ears in the MC903 mouse model treated with RmHV1 or RmHV2 in the presence or absence of 1% lipase from Candida spp. (E) Production of acetylcholine in response to RmHV and RmAD isolates measured by enzymatic assay. (F) Area of coverage for cultured fibroblasts in the cellular scratch assay in the presence or absence of 1 nM carbachol or anti-TNFR2 (αTNFR2) antibody. (G) Area of coverage for cultured fibroblasts in the cellular scratch assay treated with lipid fraction extracts of RmHV or RmAD and 5 μM atropine or mecamylamine. (H) Mean coverage area for fibroblasts and representative images with digitally added white dashed lines to indicate scratch demarcation after stimulation with R. mucosa from healthy volunteers (RmHV) and incubation with 5 μM atropine or mecamylamine. Individual dots represent experimental replicates. (I and J) Mean mouse ear thickness (I) and representative images (J) for MC903-treated mice incubated with R. mucosa or 5 μM mecamylamine. (K) Mean mouse ear thickness after treatment of MC903-treated wild-type or nAChRα7^−/− mice (lacking the nicotinic acetylcholine receptor) with RmHV. (L) Volcano plot presents metabolite differences in antecubital tape strips taken from patients with AD before and after treatment with R. mucosa. Positive log₂ fold change (FC) represents increased detection of a given metabolite in posttreatment samples. Metabolite differences are shown as nonsignificant (blue) or significant by unpaired analysis (red) or paired analysis (green, annotated). (M) MetaboAnalyst pathways for all metabolites taken from tape strips showing differences before and after treatment with R. mucosa. Data are representative of two (B to H and K) or three or more (I and J) independent experiments and are 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.