PennCHOP MICROBIOME PROGRAM Physiologic implications of - PowerPoint PPT Presentation

PennCHOP MICROBIOME PROGRAM Physiologic implications of co-metabolism between the gut microbiome and its host David Shen, MD, PhD Division of Gastroenterology Perelman School of Medicine University of Pennsylvania

• Atherosclerosis • Asthma • Colon cancer • Inflammatory bowel diseases • Obesity and metabolic syndrome

Gut Microbiome and Liver Diseases Liver is first portal that emerges from intestinal mucosal surface: Receives approximately 75% of blood supply from splanchnic circulation Potential liver diseases/processes affected by gut microbiome • Cholestatic liver disease (PBC and PSC) • NASH and NAFLD • Cirrhosis • Hyperammonemia and hepatic encephalopathy • Hepatic drug metabolism

The Human World Urea Bile Acids Hydrolysis into ammonia Biotransformation and and its use by both the host alteration of receptor- and the gut microbiota as a ligand interactions via source of nitrogen FXR and TGR5 The Microbial World

Agenda • FXR-dependent modulation of the human small intestinal microbiome by the bile acid derivative obeticholic acid • Engineering the gut microbiota to treat hyperammonemia

Agenda • FXR-dependent modulation of the human small intestinal microbiome by the bile acid derivative obeticholic acid • Engineering the gut microbiota to treat hyperammonemia

Enterohepatic circulation of bile acids

A Bidirectional Relationship Between Bacteria and Bile Acids Bacteria Bile Acids Bacteria Bile Acids • Gram-positive bacteria are more sensitive to the toxic effects of bile than Gram-negative bacteria (MacConkey agar contains bile) Bile Salt Hydrolase • Bile acid toxicity to bacteria is (Deconjugation) multifactorial with membrane effects, DNA damage, oxidative stress, alterations in RNA structure, and protein denaturation • Bile salt hydrolases, found primarily 7α -Dehydroxylation in bacteria that inhabit the intestinal tract of mammals, enhance colonization efficiency M. Begley et al. FEMS Microbiology Reviews 2005; 29: 625 – 651

Bile acids are ligands for Farnesoid X Receptor (FXR) Schaap FG, et al. Nat Rev Gastroenterol Hepatol. 2014;11:55-67.

OCA (6 α -ethyl chenodeoxycholic acid) - Selective FXR agonist - Derived from CDCA, which is the strongest endogenous FXR ligand - Approximately 100 times more potent than CDCA in activating FXR Erlinger S. 2017

Obeticholic Acid for the Treatment of Primary Biliary Cholangitis

Plasma Levels of C4 as a Biomarker of OCA-Dependent Inhibition of Bile Acid Synthesis • Controlled human subject study examining the effect of OCA treatment (n=8 per group): • 5 mg OCA • 10 mg OCA • 25 mg OCA 7α -Hydroxy-4-cholesten-3-one (C4): intermediate in the biochemical synthesis of bile acids from cholesterol Friedman, Li, Shen, et al. 0 On OCA Off OCA 37 16 Gastroenterology 2018

Bacterial Taxonomic Associations with Bile Acid Synthesis-Specific Effects of Obeticholic Acid (10 mg/day): A general increase in Gram-positive bacteria and decrease in Gram-negative bacteria S. thermophilus Plasma C4

Bacterial Taxonomic Associations with Bile Acid Synthesis-Specific Effects of Obeticholic Acid (10 mg/day): A general increase in Gram-positive bacteria and decrease in Gram-negative bacteria Table 1: GEE model identified 15 species significantly associated with C4 change over time Phylum Species P value of C4 FDR of C4 OCA Response Gram Firmicutes Streptococcus_thermophilus 1 .87e-07 2 .30e-05 Increase pos Actinobacteria Bifidobacterium_breve 4 .46e-04 0.023 Increase pos Firmicutes Streptococcus_salivarius 0.001 0.023 Decrease pos Firmicutes Lactobacillus_casei_paracasei 0.001 0.03 Increase pos Firmicutes Lachnospiraceae_bacterium_5_1_63FAA 0.001 0.03 Increase pos Bacteroidetes Alistipes_putredinis 0.003 0.053 Decrease neg Firmicutes Lactococcus_lactis 0.01 0.172 Increase pos Bacteroidetes Bacteroidales_bacterium_ph8 0.022 0.316 Decrease neg Firmicutes Subdoligranulum_unclassified 0.024 0.316 Equivocal pos Firmicutes Dorea_longicatena 0.026 0.316 Increase pos Actinobacteria Bifidobacterium_longum 0.03 0.316 Increase pos Firmicutes Dialister_invisus 0.031 0.316 Decrease pos Bacteroidetes Bacteroides_plebeius 0.037 0.347 Decrease neg Firmicutes Ruminococcus_obeum 0.045 0.389 Decrease pos Bacteroidetes Paraprevotella_unclassified 0.049 0.389 Decrease neg

Bacterial Gene Associations with OCA Administration (time effect FDR <0.05)

Uniref 90 Genomic Pathway Analysis casei/paracasei Lactobacillus 135 pathways with significant association with time (Repeated Measure ANOVA, FDR <0.01) * * * Lactococcus lactis Top Metabolic Pathways in common across taxa: Streptococcus Thermophillus • Nucleotide synthesis • Amino Acid Biosynthesis

Hypothesis FXR activation by OCA decreases endogenous bile acid synthesis, leading to the outgrowth of bile-sensitive gram positive organisms in the small intestine

Minimal Inhibitory Concentrations of Two Conjugated Bile Acids on the Growth of Gram-Positive Bacteria that Increase in Abundance with OCA Treatment Blue=Physiologically relevant concentrations in the human small intestine GCDCA (uM) GCA (uM)

Obeticholic Acid Has Minimal Effects on Bacterial Growth at Physiologically-Relevant Concentrations in Humans Physiologically-relevant concentration of OCA* in the human small intestine (1-40 m M**) *Unconjugated OCA equivalents (i.e., summation of unconjugated OCA, glyco-OCA, and tauro-OCA) **Concentrations based on estimates of: calculation of OCA dose distributed in small intestine; simulated steady-state total OCA concentrations by physiological compartment for 10 mg OCA daily administration 1 . 1 From Intercept Pharmaceuticals.

The Bacterial Taxonomic Signature in Response to OCA is due to Small Intestinal Bacteria Streptococcus spp. accounts for 19% of 454-pyrosequencing reads in the human small intestine Pereira and Berry. Environ Microbiol 2017 Dlugosz A. et al. Sci Rep . 2015;5:8508

T o t a l p r o x i ma l sma l l i n t e st i n a l T o t a l f e c a l T o t a l d i st a l sma l l i n t e st i n a l OCA treatment inhibits endogenous luminal bile acid levels and leads to an T o t a l p r o x i ma l sma T l l o i t n a l t e p st r o i n x a i ma l l sma l l i n t e st i n a l T o t a l d i st a l sma l l i n T t o e t st a l i n d a i st l a l sma l l i n t e st i n a l T o t a l f e c a l T o t a l f e c a l increase in Gram-positive bacteria specifically in the small intestine of mice bile acid levels 600,000 600,000 *** *** *** ** ** ** * * *** 500,000 500,000 Concentration (nM) Concentration (nM) Control C Methylcellulose MC 400,000 400,000 OCA OC 20 0 , 0 0 0 20 0 , 0 0 0 300,000 300,000 2, 0 0 0 20 0 , 0 0 0 20 0 , 0 0 0 20 0 , 0 0 0 20 0 , 0 0 0 *p<0.05 2, 0 0 0 2, 0 0 0 **p<0.01 100,000 100,000 ***p<0.001 T o t a l p r o x i ma l sma l l i n t e st i n a l T o t a l d i st a l sma l l i n t e st i n a l T o t a l f e c a l bile acid levels 0 0 T o t a l T o t a l 7,000 T o t a l T o t a l T o t a l T o t a l Endogenous Primary Secondary Endogenous Primary Secondary Bile Acids Bile Acids Bile Acids Bile Acids Bile Acids Bile Acids Bile Acids Bile Acids Concentration (nmol/g stool) Proximal SI Distal SI 6,000 T o t a l T o t a l T o t a l d d 5,000 4,000 T T T T 3,000 T T T T T T T T T T T T d d 20 0 , 0 0 0 20 0 , 0 0 0 66NP 66NP 66NP 66NP 2, 0 0 0 67 NP 67 NP 67 NP 67 NP ) ) 1,000 SI SI 63NP 63NP 63NP 63NP P P ( ( e 65NP e 65NP 65NP 65NP 0 T o t a l T o t a l T o t a l n n Endogenous Primary Secondary i i st st 9 3NP 9 3NP 9 3NP 9 3NP Bile Acids Bile Acids Bile Acids Bile Acids e e > 25, 0 0 0 n M > 25, 0 0 0 n M t t > 25, 0 0 0 n M > 25, 0 0 0 n M n n I I 9 5NP 9 5NP 9 5NP 9 5NP Feces l l l l Sma Sma T T T T T T T T d d 9 6NP 9 6NP 9 6NP 9 6NP ! l l ma 0 0 NP ma 0 0 NP 0 0 NP 0 0 NP 66NP 66NP i i x x 0 2NP 0 2NP 0 2NP 0 2NP o o r r 0 2R1 0 2R1 0 2R1 0 2R1 P P 9 7 NP 9 7 NP 9 7 NP 9 7 NP T T T T T T T T 67 NP 67 NP 9 8 NP 9 8 NP 9 8 NP 9 8 NP 66NP 66NP 9 8 R1 9 8 R1 9 8 R1 9 8 R1 ) SI 9 9 NP 9 9 NP 9 9 NP 9 9 NP 67 NP 67 NP 63NP 63NP ) SI P 63NP 63NP ( P ( 65NP 65NP e 65NP 65NP e n i st n 9 3NP 9 3NP i e > 25, 0 0 0 n M st t > 2 5 , 0 0 0 n M n I 9 3NP 9 3NP 9 5NP 9 5NP l e l Sma > 25, 0 0 0 n M > 25, 0 0 0 n M t 9 6NP 9 6NP n I 9 5NP l 9 5NP ma 0 0 NP 0 0 NP l l i Sma x 0 2NP 0 2NP o r 0 2R1 0 2R1 9 6NP P 9 6NP 9 7 NP 9 7 NP 9 8 NP 9 8 NP l 9 8 R1 9 8 R1 ma 0 0 NP 0 0 NP 9 9 NP 9 9 NP i x 0 2NP 0 2NP o r 0 2R 1 0 2R 1 P 9 7 NP 9 7 NP 9 8 NP 9 8 NP 9 8 R 1 9 8 R 1 9 9 NP 9 9 NP