Fig. 3. NGMs significantly differ in CD4⁺ cell differentiation.

Dendritic cells and autologously purified naïve CD4⁺ cells from serum of two healthy adult donors (biological replicates), were incubated with sterile fecal water from NGM1 (n = 7; three biological replicates per sample) or NGM3 (n = 5; three biological replicates per sample) participants. NGM3 induced significantly increased (a) proportions of CD4⁺IL–4⁺ (LME, P < 0.001; center line represents mean) and (b) expression of IL–4 (LME; P = 0.045). (c) Both NGMs expressed significantly increased proportions of CD4⁺CD25⁺Foxp3⁺ cells (LME; P < 0.001 for NGM1 and P = 0.017 for NGM3) compared to control. (d) Weighted correlation network analysis identified a metabolic module that differentiated NGM3 from NGM 2 and NGM1 participants (n = 28; ANOVA; P = 0.038). Boxplots define the 25^th and 75^th percentiles, median represented by centerline. IQR (75^th–25^th percentile) represented by whiskers. (e) Scatterplot of metabolite significance versus module membership (MM) of the 12 metabolites in the NGM3 discriminating metabolic module. Metabolites with a value of P < 0.05, significantly discriminate NGM3 from other NGMs. MM value indicates the degree of inter-connectedness of a specific metabolite to other metabolites in the module (higher MM value indicates greater inter-connectedness). (f) Using the same ex vivo assay as performed in 3a–c, 12, 13 DiHOME significantly reduced the proportion of CD4⁺CD25⁺Foxp3⁺ cells at three different concentrations (LME; P = 0.04, P < 0.001, P = 0.001 for concentrations of 75, 130 and 200 μM respectively).