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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

  1. 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

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

  3. 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

  4. 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

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

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

  7. Enterohepatic circulation of bile acids

  8. 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

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

  10. 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

  11. Obeticholic Acid for the Treatment of Primary Biliary Cholangitis

  12. 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

  13. 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

  14. 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

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

  16. 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

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

  18. 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)

  19. 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.

  20. 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

  21. 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

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