Trajectories of microbiome-derived bile acids in early life - insights into the progression to islet autoimmunity

Abstract

Recent studies reveal that gut microbes produce diverse bile acid conjugates, termed microbially conjugated bile acids (MCBAs). However, their regulation and health effects remain unclear. Here, we analyzed early-life MCBA patterns and their link to islet autoimmunity. We quantified 110 MCBAs in 303 stool samples collected longitudinally (3-36 months) from children who developed one or more islet autoantibodies and controls who remained autoantibody-negative. Stool MCBAs showed distinct age-dependent trajectories and correlated with gut microbiome composition. Altered levels of ursodeoxycholic and deoxycholic acid conjugates were linked to islet autoimmunity as well as modulated monocyte activation in response to immunostimulatory lipopolysaccharide and Th17/Treg cell balance. These findings suggest MCBAs influence immune development and type 1 diabetes risk.

Figure 1.. Outline of the study.

We analyzed microbially conjugated bile acids in a longitudinal series of stool samples collected at 3, 6, 12, 18, 24, and 36 months of age from children at HLA-conferred risk for TD, who later developed (i) multiple islet autoantibodies (P2Ab), (ii) single islet autoantibody (P1Ab), or (iii) remained autoantibody negative (CTRs) during the follow-up.

Figure 2.. Presence of microbial conjugated bile acids (MCBAs) in fecal DIABIMMUNE samples.

The bar in the color map corresponds to the number of fecal samples in which MCBAs were detected.

Figure 3.. Trajectories of MCBA in early life.

The loess curve plot of MCBAs over time (3, 6, 12, 18, 24 and 36 months) for CA-conjugates, CDCA-conjugates, DCA-conjugates and UDCA-conjugates.

Figure 4.. MCBAs in progression to islet autoimmunity.

Forest plot illustrating the coefficient estimate of a linear mixed-effects model for individual MCBAs species, with fixed covariates of Case (CTR vs. P1Ab, CTR vs P2AB), Age, Sex, and length of breast feeding accounting for random effects within individual samples. Filled circles with corresponding confidence intervals represent significant MCBA species. Faded circles depict non-significant species. The loess curve plot of MCBAs over time for significant MCBA species obtained in the linear mixed-effects model. The p-values shown are nominal; adjusted p-values (corrected for multiple comparisons using the Benjamini-Hochberg method) are available in Supplementary Table S2.

Figure 5.

Cross-correlation between the microbes and stool levels of selected MCBAs. Heatmap showing the correlation coefficients of association between microbes and stool levels of MCBAs that were found altered in progression to islet autoimmunity, in a subset of the sample with available metagenomics data (n = 110). Red color represents positive correlations, while blue represent negative correlations, as determined by spearman rank correlation.

Figure 6.. Effects of secondary bile acids on LPS-induced signaling in human monocytes.

a. Time-course analysis of LPS-stimulated phosphorylation of p38 (pp38) and ERK (pERK), as well as degradation of IkBa, markers of canonical NF-kB activation, in human monocytes. Stimulation with LPS induces robust activation of pp38 and pERK and IkBa degradation at 30 minutes. Geometric Mean Fluorescent Intensity (MFI) is measured by flow cytometry, and the average between replicates is plotted in time curves. Quantitative differences among treatments were shown by area under the curve (AUC) analysis. Geometric MFI values were used for AUC calculations (n=6). “Inhibitors” serve as a positive control for pathway inhibition, and “No treatment” represents the baseline LPS response. b. UDCA and its conjugates (Trp-UDCA, Asp-UDCA, Glu-UDCA, Asn-UDCA, Cit-UDCA) show limited or no inhibition of these pathways. c. DCA markedly inhibits signaling across all three pathways. DCA demonstrates significant suppression of LPS-induced signaling. ANOVA and subsequent pairwise comparisons were performed to analyse the impact of treatments for multiple proteins across time. Statistical significance was determined using FDR-corrected p-values. Adjusted p-values are denoted as follows: ***p < 0.001, **p< 0.01, and *p < 0.05.

Figure 7.. Microbial conjugated bile acids modulate Th17 and Treg cell differentiation.

a. Schematic of the Th17 and iTreg differentiation protocol for primary human naïve CD4⁺CD25⁻ T cells isolated from the umbilical cord blood of healthy neonates. CD4⁺CD25⁻ T cells were activated with anti-CD3/anti-CD28 and differentiated into Th17 or iTreg cells in the presence of corresponding cytokines for 3 days. DMSO control or conjugated bile acids (Asn-UDCA, Ser-CDCA, Tyr-CDCA, and unconjugated UDCA) at 100 μM were added on day 0 of differentiation. b-e. IL-17a secretion in the supernatant of Th17 cultures treated with Asn-UDCA (b), Ser-CDCA (c), Tyr-CDCA (d), and UDCA (e) was quantified on day 3 of differentiation from four biological replicates using ELISA. f-i. Intracellular Foxp3 protein expression in iTregs cultured with Asn-UDCA (f), Ser-CDCA (g), Tyr-CDCA (h), or UDCA (i) was assessed on day 3 of differentiation by flow cytometry. Geometric mean fluorescence intensity (MFI) values are shown for four biological replicates. Statistical significance was determined using paired, two-tailed Student’s t-test.