Fig. 9

L. reuteri metabolites are ligands for the aryl hydrocarbon receptor and sufficient to elicit a Th17 immune response. Selected metabolites identified in L. reuteri monoculture or serum UPLC-MS/MS were analyzed in a cell-based luciferase assay for capacity to activate the AhR at A 10μM and B 100μM treatment of each individual metabolite. Percent inhibition of AhR activation by each metabolite was calculated between maximal luciferase response at 100μM and activity following pre-treatment for 4h with the AhR antagonist, CH-223191 (C). Splenocytes were differentiated under Th17 conditions with or without IGoxA, IAA, and/or AhR antagonist treatment followed by intracellular cytokine staining and flow cytometry. Major effects of metabolite treatment included frequency of CD4⁺ T cells of the total CD45⁺Live population (D), percent IL-17 production in CD4⁺ T cells (E), frequency of CD8⁺ T cells of the total CD45⁺Live population (F), and the percent IL-17 production in CD8⁺ T cells (G). Symbols indicate a significant difference between 0μM treatment and the indicated metabolite concentration as follows, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Results include at least (3) technical replicates per condition