A Multi-omic Approach to Understand the Development of the Gut-Liver Axis Joseph L. Dempsey, MPH, PhD Candidate Department of Environmental and Occupational Health Sciences
Gut microbiota and development > During the neonatal period, human gut microbiota is high in Lactobacillus, Bifidobacterium, Staphylococcus, and Enterococcus genera (Kundu et al. 2017, Cell ). Adult gut microbiota composition was high in the phylum Firmicutes with > increases in Proteobacteria and Bacteroidetes after age 70 (Odamaki et al., 2016, BMC Microbiol ). Modified from Kundu et al. (2017) Cell Yatsunenko et al. (2012) Nature > Knowing the core microbiota during development may help improve chronic diseases associated with the gut microbiota. 2
Gut microbiota and host diseases > Gut microbiome is associated with health and human disease, but there is limited research on how the developing gut microbiome may contribute to xenobiotic metabolism and disease risk. 3
Central hypothesis > Age-dependent gut microbiome modulates the metabolome within the gut-liver axis, altering the expression and function of xenobiotic processing genes. 4
Experimental Designs I. Mice Collect tissues at days: 1 5 10 15 25 60 120 Conventional (CV) and germ-free (GF) male and female Young Adult Neonate C57BL/6 adolescent Multi-omics approach 16S rDNA sequencing of Targeted metabolomics Liver transcriptomics Liver targeted large intestine (Qiime 1.9.1 of liver (UPLC-MS/MS; (RNA-Seq; STAR aligner quantitative proteomics and 2.0; PICRUSt; FishTaco) bile acids and aqueous v2.5.2b to mm10 and activity-based metabolites) gencodeV11 and cufflinks) proteomics (LC-MS) II. Humans • In collaboration with Dr. Sheela Sathyanarayana at Seattle Children’s Hospital, 60 healthy pediatric human subjects recruited at 4 developmental age groups for fresh fecal microbiome collection metagenomics and metabolomics 5
Gut microbiota diversity increases through adulthood α -Diversity β -Diversity 12 Phylogenetic Diversity Day 1 Day 10 10 Female Day 1 Male 8 Day 1 Faith's Day 25, 60, and 120 6 Day 15 4 Day 5 and Day 10 2 0 Days 15, 25, 0 1000 2000 3000 4000 Day 5 60, and 120 Rarefied Sequences > Alpha diversity is highest at Day 1, lowest at early developmental ages, and then increases through adulthood. For species heterogeneity ( β -diversity), there are 4 distinct clusters, > separating pre-weaning ages and adults. 6
Ontogeny of mouse gut microbiota Male Female 100 % Composition 80 Acinetobacter Lactobacillus Lactobacillus 60 Acinetobacter S24-7 40 20 0 1 5 10 15 25 60 120 1 5 10 15 25 60 120 Age (Days) Age (Days) o_Clostridiales;f_Lachnospiraceae;g_uncultured;s f_Staphylococcaceae;g_Staphylococcus f_Lactobacillaceae;g_Lactobacillus f_Streptococcaceae;g_Lactococcus f_Bacteroidales S24-7 group;g_uncultured bacterium;s_uncultured bacterium o_Erysipelotrichales;f_Erysipelotrichaceae f_Moraxellaceae;g_Acinetobacter g_Oscillibacter;s_uncultured bacterium f_Bacillaceae;g_Bacillus f_Enterococcaceae;g_Enterococcus g_Lachnospiraceae NK4A136 group;s_uncultured bacterium o_Bacteroidales;f_Bacteroidales S24-7 group f_Bacteroidales S24-7 group;Ambiguous_taxa;Ambiguous_taxa g_Ruminiclostridium;s_uncultured bacterium o_Clostridiales;f_Lachnospiraceae Other Taxa (188) o_Clostridiales;f_Lachnospiraceae;g_Lachnospiraceae NK4A136 group o_Clostridiales;f_Lachnospiraceae;g_uncultured;s_uncultured bacterium 7 f_Staphylococcaceae;g_Staphylococcus
Age-specific differences in gut microbiota BA metabolism Bacteria that drive 30000 Secondary Bile Acid Synthesis secondary BA synthesis Male * 25000 * * * 20000 15000 Gene counts Day 1 10000 5000 * 0 Day 120 1 5 0 5 5 0 0 35000 1 1 2 6 2 Secondary Bile Acid Synthesis * 30000 Female 25000 * * 20000 * 0 1 2 3 4 5 6 15000 Wilcoxon Test Statistic 10000 f_Lachnospiraceae-OTU352559 5000 * f_S24-7-OTU276802 f_Ruminococcaceae-OTU338796 0 f_S24-7-OTU392511 f_Ruminococcaceae-OTU190500 1 5 0 5 5 0 0 1 1 2 6 2 y y 1 f_S24-7-OTU183446 f_Enterobacteriaceae-OTU1100639 a a y y y y D D a a a a y D D D D a f_S24-7-OTU400466 f_Bacillaceae-OTU1042600 D f_Streptococcaceae-OTU516115 o_Clostridiales-OTU387435 * p < 0.05 (one-way ANOVA with o_Clostridiales-348336 Other (2 unique taxa) Dunnet’s post hoc) Other (85 unique taxa) o_Clostridiales-OTU768294 f_Lachnospiraceae-OTU352559 8 f_Ruminococcaceae-OTU338796
BAs in the gut-liver axis > Bile acids are endogenous detergents and signaling molecules that regulate lipid and glucose homeostasis, energy expenditure, inflammation, bacterial CA Primary BAs CDCA MCAs proliferation and Conjugation with taurine and glycine gastrointestinal motility. Gut microbiota produces > T-CA T-CDCA secondary bile acids from primary bile acids, which are synthesized from cholesterol in the liver. Secondary BAs Modified from Wahlstr ӧ m et al. (2016) Cell Metabolism 9
BA concentrations are correlated (Pearson) with distinct bacteria in large intestine -1 -0.5 0 0.5 1 2 Value 5 1 4 Cluster 1 3 b a Top 5 most abundant unconjugated 2 ° BAs in LI: Top 2 most abundant conjugated 1 ° BAs in LI (others are minimally present) 1. DCA ωMCA 2. a. T- αMCA 3α -OH-12keto-LCA 3. b. T-CA 8(14),(5β) -Cholenic Acid- 3α,12α -diol 4. 10 5. LCA
BAs and bacteria in large intestine of Cluster 1 T- / MCA M Ruminococcus 1 1500 Lachnospiraceae 3 S24-7 (2 taxa) Clostridiale Family 0.21 0.3 1000 0.4 F XIII UCG-001 2 500 0.14 0.2 0 0.2 1 1000 T-CA 0.07 0.1 500 0 0.0 0.0 0.00 Ruminiclostridium 5 Ruminiclostridium 9 Lachnoclostridium 0 Intestinimonas 0.6 nmol/g tissue 4 4 0.04 DCA 200 3 0.4 100 2 2 0.02 0 0.2 % OTU MCA 1 160 0.00 0 0.0 0 80 0.24 Erysipelotrichaceae Bifidobacterium Bacteroides Enterorhabdus 0 1.0 3.0 1.0 3- OH-12keto- 0.16 20 LCA 10 1.5 0.5 0.5 0.08 0 8(14),(5 )-Cholenic Acid- 20 0.0 0.00 0.0 0.0 3 ,12 -diol 8 Coriobacteriaceae Dorea 10 Akkermansia L. reuteri 0.4 0.8 UCG-002 0 6 LCA 20 0.2 4 0.2 0.4 10 2 0 0.0 0.0 0.0 0 1 5 10 15 120 1 5 10 15 25 60 120 1 5 10 15 25 60 120 1 5 10 15 25 60 120 1 5 10 15 25 60 120 Age (Days) Age (Days) > BA-metabolizing bacteria from Cluster 1 increase with age, corresponding to the increase in secondary BAs. 11
Xenobiotic metabolism and PXR > The xenobiotic-sensing nuclear receptor PXR modulates the expression of xenobiotic metabolizing genes. > PXR is known to be endogenously activated by the secondary bile acid LCA. Staudinger et al., 2001, Proc Cyp3a Natl Acad Sci USA Modified from Kakizaki et al., 2011, Front Biosci 12
Age-specificity of secondary BAs between CV and GF mice in liver Female Male 10 T-DCA (nmol/g) CV 8 3 GF 2 1 * * 0 1 5 10 15 120 1 5 10 15 120 Age (Days) Age (Days) Female Female Male Male 9.0 0.20 -MCA (nmol/g) T-LCA (nmol/g) 6.0 0.15 1.5 0.10 1.0 0.05 * * 0.5 * * 0.0 0.00 1 5 10 15 120 1 5 10 15 120 1 5 10 15 120 1 5 10 15 120 Age (Days) Age (Days) Age (Days) Age (Days) * p < 0.05 by enterotype; no gender differences (GLM; Tukey’s HSD) 13
Expression of important host xenobiotic biotransformation genes in liver is age- and gut microbiome-dependent Male Male Female Cyp3a11 2000 FPKM 1500 RNA-seq 1000 * 500 * * * 0 CV (normalized to total protein) Relative protein expression 1 5 10 15 25 60 120 1 5 10 15 25 60 120 GF Cyp3a11 500 400 Targeted 300 proteomics 200 * * 100 * * * p < 0.05 by enterotype * 0 1 5 10 15 25 60 120 1 5 10 15 25 60 120 14
Gender- and age-specific differential expression of xenobiotic processing genes between CV and GF mice Male Female Number of DE DPGs 120 100 Phase I Phase II 80 Transporters 60 Antioxidant components 40 Nuclear receptors 20 Bile acid synthesis 0 1 5 10 15 25 60 120 1 5 10 15 25 60 120 Age (Days) Age (Days) > Phase 1 genes are the top differentially expressed xenobiotic processing genes in both males and females. > Male mice tend to have more differentially expressed than females. 15
Activity-based protein profiling (ABPP) to identify functionally active proteins Modified from Sadler and Wright (2015) Curr Opin Chem Biol 16
Fold change between Day 120 male CV and GF mice of detected cytochrome P450s (Cyps) using RNA expression and ABPP 2 fold change (GF/CV) * * * Relative LOG2 1 * * 0 -1 -2 -3 -4 * 2 1 2 5 9 0 0 0 6 9 2 5 1 3 5 0 a 4 4 a a 1 a 2 4 7 1 2 d f j 1 1 2 1 2 1 1 2 2 1 7 a 2 c c c d d a a a a 1 a f 2 p p 4 p 2 2 p 2 2 2 3 3 3 4 a 4 2 2 p y y p y p p y p p p p p p p p p 4 p y C C y C y y C y y y y y y y p y y y C C C C C C C C C C C y C C C C For RNA expression: * FDR-BH < 0.05 by enterotype RNA expression (FPKM) For ABP abundance: ± 30% Activity-based protein abundance (LFQ Intensity) > Cyp3a11 RNA expression and ABP abundance are both markedly decreased by lack of gut microbiota. 17
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